Protein Targeting and Translocation [Core Textbook ed.] 9781400865017

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Protein Targeting and Translocation

Protein Targeting and Translocation Edited by D. A. Phoenix

PRINCETON UNIVERSITY PRESS PRINCETON, NEW JERSEY

Published in NorthAmerica by Princeton University Press, 41 William Street, Princeton, New Jersey 08540 Published in the United Kingdom by Portland Press Ltd, 59 Portland Place, LondonWlN 3AJ.U.K. Tel: (+44) 171 580 5530; e-mail: [email protected]

© 1998 Portland Press LtdlLondon

ISBN 0-691-00901-5

All rights reserved

Although, at the time of going to press, the information contained in this publication is believed to be correct, neither the authors nor the publisher assume any responsibility for any errors or omissions herein contained. Opinions expressed in this book are those of the authors and are not necessarily held by the editor or the publishers.

Typeset by Portland Press Ltd Printed in Great Britain by Information Press Ltd, Eynsham, U.K. http://pup.princeton.edu 1 3 5 7 9 10 8 6 4 2

PRINCETON UNIVERSITY LIBRARY

PAIR

32101 032262774

Contents

Preface

vii

Abbreviations

xi

Membrane interactions Biophysics of the membrane interface and its involvement in protein targeting and translocation

I

A.Watts andT.J.T. Pinheiro

Amphiphilic α-helices and lipid interactions

19

D.A. Phoenix and F. Harris

Signal sequences: initiators of protein translocation

37

N. NouwenJ.Tommassen and B. de Kruijff

Determinants of membrane protein topology and membrane anchoring

49

L. Hashemzadeh-Bonehi, J.P. Jacob1C. Mitsopoulos and J.K. Broome-Smith

Insertion of single- and multi-spanning proteins into the bacterial cytoplasmic membrane D. Kiefer and A. Kuhn

Prokaryotic protein targeting and translocation Prokaryotic protein translocation A.J.M. Driessen

67

Contents

Protein traffic from the cytosol to the outer membrane of Escherichia coli

105

H.Tokuda and S. Matsuyama

sec-dependent prokaryotic protein secretion

121

J.D.Thomas, S.D.Wharam, and G.P.C. Salmond

Targeting and assembly of fimbriae

143

C.J. Smyth, S.G.J. Smith and M.B. Marron

Eukaryotic protein targeting and translocation Targeting to and translocation across the endoplasmic reticulum membrane

169

J.L. Brodsky

Protein localization to the endoplasmic reticulum and Golgi complex

193

R. Qanbar and C.E. Machamer

Import and export of proteins at the nucleus

213

N. Imamoto.Y. Miyamoto and Y.Yoneda

Mitochondrial targeting and import

231

R. Hovius

Translocation of proteins into and across the thylakoid membrane

249

C. Robinson,A. Mant and S. Brink

Principles of peroxisomal protein sorting and assembly

259

J.A.K.W. Kiel, I.J. van der Klei and M.Veenhuis

Targeting of glyoxysomal proteins

273

A. Baker and B.Tugal

Subject index

287

Preface

Lipid bila/ers are flexible self-sealing boundaries that are used to allow subcellular compartmentalization of regions with different functions and hence different chemical compositions. Obvious examples are the mitochondria, where large electrochemical gradients can be generated to allow the production of ATP, or microbodies, which are involved in the metabolism of specific growth substrates such as fatty acids and C2 compounds. Indeed, microbodies are of special interest due to the fact that their exact role and number is a function of growth conditions and therefore varies throughout the organism's life cycle. Within a eukaryotic organism, up to 95% (w/w) of the cell's membranes are used in the formation of intracellular structures such as the nucleus, endoplasmic reticulum, Golgi, mitochondria, chloroplasts and microbodies (peroxisomes/glyoxysomes).To allow each membrane-bound compartment to carry out its role, there is considerable protein traffic between compartments, and proteins must be able to pass across these semi-permeable barriers and, in the case of membrane proteins, insert into the bilayer. Furthermore the cell must have a means of maintaining proteins at their final functional location against the bulk flow of protein, for example in the endoplasmic reticulum and Golgi (Chapter 12). The mechanism by which large amphiphilic proteins are able to undergo subcellular targeting and then cross a hydrophobic bilayer has been one that has puzzled scientists for a number of years, and a question that has been investigated by a wide range of techniques from biophysics to molecular biology. Nuclear targeting is perhaps an unusual case, since the protein passes through the nuclear pore and never leaves the cell's cytosolic phase. Furthermore the nucleus allows bi-directional transport, with material entering and exiting via the pore (Chapter 12). The remaining targeting systems described in this monograph, i.e. those of bacteria (Chapters 6 and 7), endoplasmic reticulum (Chapter 10), mitochondria (Chapter 13) and chloroplasts (Chapter 14), can be seen to have a number of common themes [I], For example, the targeted proteins appear to be synthesized with a targeting signal (Chapter 3).These signals allow recognition of the protein by the cellular machinery involved in the targeting and transport of the protein to the site of translocation at the membrane interface, but may have a variety of additional roles. For example, the signal that targets proteins to the endoplasmic reticulum may also determine whether transport occurs post- or cotranslationally. Both these and bacterial export signals also appear to affect protein

viii

Preface

folding, thus helping to maintain the protein in a translocation-competent form. The thylakoid targeting signal (Chapter 14) may determine if transport is ATP- or ΛρΗ-driven. The targeting signal can therefore be seen to have a level of complexity in itself that is not yet fully understood.This lack of knowledge may be emphasized in the case of mitochondria and chloroplast import signals. It is known that the basic, amphiphilic character of mitochondrial import signals is important and that chloroplast import is driven by signals that exhibit much lower levels of amphiphilicity and a smaller degree of basic character, yet some signals appear to transport proteins into both organelles. Hence, even though 25% of randomly generated targeting signals directed proteins to the endoplasmic reticulum, mitochondria and chloroplast, it may be that the full range of signal functions is depicted by as yet unrecognized motifs [I],thus explaining the low efficiency of these artificial targeting signals.The importance of secondary structure has been recognized for some years (Chapter 3), although it is much more recently that targeting patches and more complex motifs have been discussed. It is of interest to note that, in addition to their targeting role, if the bacterial export or endoplasmic reticulum targeting signals are not cleaved they can lead to the incorporation of the protein into the membrane (Chapter 4).The charge distribution and overall hydrophobicity of the signal can be key factors in determining how incorporation takes place (Chapter 5). Indeed, these two criteria are important when considering the interaction of many membrane proteins (Chapters I and 2) because of the amphiphilic nature of the bilayer. Once targeting has been initiated by the targeting signal, the protein appears to interact with one of a number of chaperones which help maintain the protein in a translocation-competent form, e.g. SecB in bacteria. While some of these chaperones act on a wide spectrum of proteins, some act only on a protein subclass. In some pathways chaperones may be required at a number of stages. For example, both mitochondria and chloroplasts contain homologues of the bacterial GroEL and GroES chaperones which may be involved in facilitating the folding of both imported proteins and proteins that are being exported from the matrix/stroma. In the case of co-translational translocation in the bacterial and endoplasmic reticulum systems, the signal mediates the interaction with the signal recognition particle, which functions to slow translocation until the protein reaches the site of translocation. Once at the membrane interface, the signal sequence mediates interaction with a membrane receptor.This receptor function appears to be regulated by NTP binding and hydrolysis, and these membrane proteins tend to mediate transfer of the targeted protein to a translocation channel.There appears to be a range of receptors (e.g. mitochondria contain Tom20p,Tom22p and Tom70p on the outer membrane), and this range may allow the cell to differentially regulate the targeting of different precursors. It now appears that the actual translocation of the protein through the bilayer occurs through a protein channel which is gated across the membrane by

ίχ

Preface

the targeting signal, but which is also gated in the plane of the bilayer by stoptransfer signals (Chapter 4) to allow the assembly of membrane proteins.The translocation process itself is envisaged to be driven in a number of ways. First, the cell may use a chaperone on the trans-side of the membrane which can act to pull the protein through the translocation channel. For example, Hsp70 (heat-shock protein of 70 kDa) is membrane-bound and effects transport into the endoplasmic reticulum, mitochondria and probably the chloroplasL Secondly, the cell can use an ATP-powered protein'motor' to push the protein through the translocation channel from the c/s-side of the membrane, such as in the case of bacterial SecA (Chapter 6). Finally, in the case of co-translational translocation, the ribosome may aid transport by pushing the protein into the channel during synthesis. It should also be remembered that, in many cases, the protonmotive force can play an important role in providing directionality to the translocation process, and only recently have data started to be produced that allow suggestions as to how this process occurs (Chapter 7). Finally, it has been suggested that targeting and uptake into microbodies occurs via a different system compared with other modes of translocation, but this is perhaps one of the less developed areas. As work progresses.it may be found that there are more similarities with other known systems; for example, targeted proteins do contain targeting signals and are likely to use a cytosolic targeting system coupled to a membrane receptor. Although a membrane channel has not yet been characterized, it may well exist (Chapters 15 and 16).

Reference 1.

Schatz, G. and Dobberstein, B. (1996) Science 271, 1519-1526

Abbreviations

ΔΨ

transmembrane electrical potential

*ο>%

internal membrane potentials surface membrane potentials

AO Azr mutant Azss mutant CAT CFP CGN

alcohol oxidase

CS

cold-sensitive mutant

CSP DWIH ER AG GAP GEF GSP Hsp IBV-M LHC Il Lpp LPS MAS mHsp70 MPP MSF MSS NAC NBS NEM NES NLS NPC NSF OMP

cleavable signal peptide

azide-resistant mutant azide-super-sensitive mutant chloramphenicol acetyltransferase channel-forming peptide c/s-Golgi network

depth weighted insertion hydrophobicity endoplasmic reticulum free energy change GTPase-activating protein guanine-nucleotide-exchange factor general secretion pathway heat-shock protein avian infectious bronchitis virus membrane protein M light-harvesting chlorophyll-binding protein of PSII major lipoprotein Iipopolysaccharide magic angle spinning mitochondrial Hsp70 matrix processing protein mitochondrial import stimulating factor membrane-spanning segment nascent-polypeptide-associated complex nucleotide-binding site N-ethylmaleimide nuclear export signal nuclear localization signal nuclear pore complex N-ethylmaleimide-sensitive fusion protein outer-membrane protein

xii

Abbreviations

PBP PC PE PG PhoA PKI PMB Pmp PSI and PSII PSI-N

protonmotive force penicillin-binding protein phosphatidylcholine phosphatidylethanolamine phosphatidylglycerol alkaline phosphatase inhibitor of cAMP-dependent protein kinase peroxisomal membrane protein involved in peroxisome biogenesis peroxisomal membrane protein photosystems I and Il respectively PSI subunit N

PTAC

nuclear-pore-targeting complex

PTS RBP

peroxisomal targeting signal retinol-binding protein

SNARE SRa, SRp

soluble NSF attachment protein receptor subunits of the SRP receptor

SRP SSR SST sequence ST sequence SV40

signal recognition particle signal sequence receptor start-stop-transfer sequence stop-transfer sequence simian virus 40

TCA cycle TGN

tricarboxylic acid cycle trans-Golgi network

TMS

transmembrane segment

TRAM TRAP tSNARE

translocating-chain-associating protein translocon-associated protein target membrane SNARE

USP vSNARE

uncleavable signal peptide vesicle SNARE

VSV-G

glycoprotein of the vesicular stomatitis virus

1 Biophysics of the membrane interface and its involvement in protein targeting and translocation A. Watts* andT.J.T. Pinheirof Department of Biochemistry, South Parks Road, University of Oxford, OxfordOXI 3QU, U.K.

Introduction The initial site of association for any component that may partition into and then, as one possibility, traverse a membrane is the polar/apolar interface of the membrane. Whether or not a protein or lipid acts as the target site, such associations are driven initially, and possibly subsequently, by electrostatic forces. These forces are important not only in ionic interactions and conductance effects, but also in determining the structure and activity of membrane proteins, including protein insertion and translocation [1-3]. Here, the relevant thermodynamic and electro­ static aspects of membrane protein association and insertion will be reviewed. As specific examples of such associations, the mode of interaction and kinetics of association of several peptides and proteins will be presented using a range of biophysical approaches, although it should be stated that the area is highly complex, and no simple explanations exist for any systems, and much information is piecemeal and incomplete. This short resume cannot hope to include every aspect of the topic, but some indication of contemporary methods and results will be given.

Thermodynamic and practical consideration of protein-membrane associations When considering membrane protein insertion and translocation, the thermo­ dynamics of the interactions are important, driven, as they are initially, by electrical

' To whom correspondence should be addressed. tPresent address: Department of Biological Sciences, University oj Warwick, Coventry CV4 7A L, U.K.

2

A.Watts andT.J.T. Pinheiro

forces. The free energy of peptide binding to a membrane can be approximated from [4]: AG = AG c l + AG i m m + AG f o b + AG p o l + AG l i p where AG c l denotes the electrostatic effects (see below), AG i m m is the positive binding energy caused by peptide immobilization, AG f o b is the energy gained from the hydrophobic effects, AG p o l is the energy contribution from backbone and sidechain hydrogen bonding, and AG l i p describes the lipid perturbation effects. Any configurational energy associated with peptide-membrane binding is included within the AG p o l term. Biophysical methods are able to allow some estimates of these various terms, although too little is known at present to permit complete descriptions of the association mechanisms. The value of AG i m m for the peptide on the membrane surface, when compared with isotropically moving peptide, is not really known. However, when compared with the more favourable disordered unfolded helixforming peptide, the folded and thus more ordered helix has an unfavourable energy of AG j m m -5.23 kj-mol" 1 (-1.25 kcal-mol" 1 ) per peptide bond, giving approx. 84-126 kj-mol" 1 (20-30 kcal-mol" 1 ) for a 20-amino-acid helix. Against this, a similar transmembrane helix would form 16 hydrogen bonds in the non-aqueous environment of the bilayer core, giving a AG p o l of approx. — 402 kj-mol" 1 ( — 96 kcal-mol"'), with only small contributions from van der Waals interactions between lipids and side chains [4], Estimates of AG i m m + AG f o b of at least 84 kj-mol" 1 (20 kcal-mol" 1 ) have been made for the desolvation/hydration of signal peptides [5]. However, it has been pointed out that the 'macroscopic' and 'microscopic' hydrophobic effects in membranes and protein binding sites are very different in magnitude and that highly curved surfaces can produce an anomalous, high, hydrophobic energy of binding [4]. Furthermore, for peptide insertion and folding, and again on energetic grounds, the insertion of an unfolded chain is extremely unfavourable [AG i m m +176 kj-mol" 1 (+42 kcal-mol" 1 )] when compared with insertion of a folded chain [AG i m m -126 kj-mol" 1 (-30 kcal-mol - 1 )] (20-22 residues) [4], From similar arguments, assembled β-structures are also unfavourably inserted into a bilayer. Based on such considerations, it has been argued that a polypeptide coil cannot be inserted into a bilayer and then fold, but rather a secondary structure must be formed either in the aqueous phase or, at the latest, at the membrane/water interface before insertion [6], The maximum energy from inserting the hydrophobic residues of the LamB wild-type peptide into the bilayer (AG t o b ) is approx. -381 kj-mol" 1 (-91 kcal· mol· 1 ), which is very substantial and favourable [7], Also, the free energy required for transfer of unbonded polar groups is much higher [OH, + 16.7 kj-mol" 1 ( + 4.0 kcal- mol - 1 ); -NH 2 , +20.9 kj-mol" 1 ( + 5.0 kcal -mol" 1 ); COOH, +20.1 kj-mol" 1 (+4.8 kcal-mol" 1 ); C = O, + 8.4 kj-mol· 1 (+2.0 kcal-mol" 1 )] than for bonded -NHO =C pairs [ + 2.3 kj-mol" 1 1 (+0.55 kcal-mol· )] [8].

3

Biophysics of the membrane interface

Lipid perturbation effects play an insignificant role in the energetics of peptide insertion and translocation [5]. Ordering of lipids around the peptide may be significant, but this is offset by the favourable interaction of acyl chains with hydrophobic peptide residues. Partially embedded peptides, or those on the bilayer surface, cannot be assessed so readily and lipid phase separation, cavity formation and lipid head-group perturbations are all relatively poorly described. However, as a general contribution to the entropic changes upon peptide binding, the restriction of a peptide on the bilayer surface, when compared with the isotropically moving peptide, may be countered by the disorder of the induced lipid perturbation effects, resulting in a balance (no gain or loss) of these two contributions to the overall energetics of the interaction. Other factors of importance in the energetics of transmembrane helix assembly are the stability of helix-helix interactions which overcome the entropies of helix separation [in the range 4.2—41.9 kj-mol

1

(1-10 kcal-mol" 1 )] [9], especially

when helix packing is better accommodated in the bilayer than lipid—helix packing lipids [10]. The major (-70%) contribution to the binding energy of signal peptides, from the studies reported to date, is therefore the hydrophobic (AG f o b ) interaction, although theory and experiment are still not within range of each other, and too little is known at present about the mechanism of peptide and protein interactions with biomernbranes to be able to describe the energetics of the process in detail.

Electrostatic contributions at the membrane interface The electrostatic interactions between a peptide or protein and a membrane interface are likely to be more important in the initial events of peptide associations than in the later stabilization of the peptide—membrane complex. Such interactions are known to involve positively charged amino acid side chains and negative charges in lipid head-groups at the membrane interface [1,2,5,7,11-14]. However, the dynamics and contributions of these multi-phase and multi-step interactions are not well understood. In particular, the way in which electrostatic interactions can induce gross conformational distortions of the peptides or proteins during contact with the lipid interface is only now being explored and recognized. These electro­ static contributions are significant and are the result of many complex factors, not least of these being the localization of very different chemical groups (carbonyls, methyls, methylene, phosphates, cholines, carboxvlic acids, primary amino groups, etc.) of the lipids within the structured bilayer (Figure 1). The electrical profile of a lipid bilayer is a complicated sum of multiple potentials (Figure 1) [14]. Although the transmembrane potential (ΔΨ) and surface potentials (Ψ 5 θ ; Ψ 5 Ι ) can be reasonably well described, the internal membrane potentials (^I f o ; Ψ,) can be quite varied, being made up predominantly of the membrane dipole potential and adsorption potentials. Typical values for the

_4

A . W a t t s andT.J.T. Pinheiro

Figure 1 Combine d representatio n o f the electrostati c an d structura l features o f a flui d lipi d bilaye r (a)

Aqueous

Membrane

Aqueous

® c B o Q.

(b)

(c)

>

15 ra -Q o CL

DISTANCE FROM HC CENTRE ( A ) (a) The total electrical potential profile as the sum of the transmembrane potential

, surface

potentials

An

and internal (for example, dipole or adsorption) potentials

intramembrane potential

may also exist Without surface

potentials, the intramembrane potential

or adsorption

is simply the transmembrane potential

but

generally it is necessary to define

in terms of the surface potentials

adsorption potentials

In many membrane phenomena, it is the intramembrane

potential

or

which is a major consideration.Also shown are the sizes of 27- and 20-amino-acid (aa)

helices in a transbilayer position (b) and on the bilayer surface (left-hand side of c) relative to the bilayer (a fluid dioleoyl PC bilayer) itself (c) whose structure was determined by joint X-ray and neutronscattering data. The time-averaged distributions of the principal structural groups are shown projected on to an axis normal to the bilayer plane, and represent the probability of finding a structural group at a particular location. Abbreviations: HC, hydrocarbon; Glyc., glycerol backbone. Adapted from [3,14],

Biophysics of the membrane interface

transmembrane potential are 10-100 mV, and in biological membranes the cytoplasmic side is negative relative to the outside of the membrane. The transmembrane potential can readily be calculated for equilibrium and steady-state conditions (Nernst and Goldman-Hodgkin-Katz equations) and is, itself, responsible for driving translocation of peptides across membranes [15]. The surface potentials (Ψ 5 0 ; r I i s ;) are the electrostatic potential at the membrane/aqueous interface relative to that in the corresponding bulk phase. In all biological systems, this potential is negative due to the existence of anionic phospholipids such as phosphatidylserine, but also cardiolipin and phosphatidylglycerol (PG), with a very small amount (

27.8

PmfD

28.5

PapD

Mass (kDa)

K88 fimbria

Fl 7 fimbria

CS3IA capsule-like protein

K99 fimbria

Type 2 and type 3 fimbriae

PEF

Fimbria

Long polar fimbria )

FIC fimbria

S-fimbria

Type I fimbria

28

30

29

31

31

32

32

33

33

33

33

34

34

35

f

Type 3 fimbria

47

-

PMF fimbria

MR/P-fimbria

-

assembled P-fimbria

Identity with PAPD (%)

Surface structure

Superfamily of iimmunoglobulin-like chaperones

MrpD

FGS subfamily

Chaperone

Table 1

Piglet scours

Diarrhoea

Diarrhoea

scours

Piglet, lamb and calf

Whooping cough

Gastroenteritis

meningitis

Otitis media;

>

Gastroenteritis

Cystitis

UTI

Cystitis

Pneumonia

Nosocomial UTI

Nosocomial UTI

Pyelonephritis

Disease

Table 1 (Contd.)

26

SefB

CafIM

Abbreviation: UTI, urinary tract infection.

27

27

CS3-I

25

Envelope antigen Fl

Y. pestis

Plague

Gastroenteritis

Traveller's diarrhoea 25 25

CS3 fimbria SEF14 fimbria

Diarrhoea

27

Antigen CS6

S. enteritidis

Ecoli

E coli

23

CssC

Diarrhoea Plague

29

Y. pestis

25

PsaB

pH6 antigen

Enterocolitis

Aggregative adherence fimbria I

E. coli

24.6

AggD

31

UTI

32 31

Non-fimbrial adhesin I Myf fimbria

E. coli

Y. enterocolitica

30.5

30.2

Diarrhoea

Disease

32

PapD (%)

AFA-III

Identity with

Surface structure assembled

NfaE

E. coli

Organism

30

Mass (kDa)

MyfB

AfaB

FGL subfamily

Chaperone

c.j. Smyth et al.

152

Figure 3

Ribbon model of the crystal structure of the PapD chaperone bound to the PapG peptide corresponding to the C-terminal 14 residues of the PapG adhesin mbrin

H2

Domain 1

Domain 2

The PapD chaperone comprises two globular domains that are oriented towards each other to give the molecule the overall shape of a boomerang. Each domain is a l3-barrel structure formed by two antiparalle/l3-p/eated sheets, and has a topology identical with that of an immunoglobulin fold. In each domain, the upper sheet comprises three [3-strands (E, 8 and A), and the lower sheet comprises four

13-

strands (G, F. C and D). In addition, the C-terminal domain of the PapD chaperone has a short eighth strand, H2, which is linked to l3-strand G2 by a disulphide bridge.The PapG peptide binds along the exposed edge of the G I l3-strand of the PapD chaperone. Highly conserved alternating hydrophobic residues make possible hydrophobic interactions with highly conserved hydrophobic residues in the Cterminal peptide. The PapG peptide binds along the exposed edge of the G I l3-strand on the PapD chaperone by hydrogen bonds (thin lines joining the PapG peptide to the G I [3-strand). The terminal carboxylate group of the PapG peptide also forms hydrogen bonds with two positively charged residues of the PapD chaperone, namely Arg-8 and Lys-I I 2 deep in the cleft Arrows indicate f)-strands. Adapted from Holmgren et al. [42] and the front cover of ASM News 61, Number 9, September 1995, which

accompanied Hultgren and Jones [6]. Reproduced with the permission of the author and the American Society for Microbiology.

[41] and the outer-membrane protein FaeD, which is the usher [43] (see below). The chaperone FaeE binds the fimbri al subunits FaeG, FacH and FaeI in the periplasm, but not the FaeC and FaeF subunits [44,45]. In contrast w ith other fimbrial chaperones, FaeE functions as a hom odimer. It forms trimeric complexes with the subunits FaeG, FaeH and Fad that consist of one subunit and two chaperone molecules [46]. Why the K88 fimbrial chaperone functions as a homodimer is unknown . Recently the N-terminal domain of the FaeE chaperone was shown to determine dimerization of the protein as well as subunit specificity [47].

Targeting and assembly of fimbriae

Chaperone subfamilies Although the entire chaperone family can be grouped together as one PapD-Iike superfamily sharing 30-40% amino acid sequence identity and up to 60% similarity [6,34], Hung et al. [48] have divided them into two subfamilies based on structural differences. This division of chaperones also defines the molecular architecture of the organelles that they assemble, namely (i) rod-like fimbrial structures and (ii) non-fimbrial adhesins, which are not thought to be components of any oligomeric structure, or thin fibres that tend to coil up into an amorphous mass on the surface of the bacterium. The amino acid sequences of the 26 chaperone proteins in the superfamily to date have been determined. Alignment with the sequence of the PapD chaperone revealed that gaps were only introduced in loop regions and never in the β-strands [48]. Ten residues were shown to be invariant in all 26 chaperones (Table 2). Residues Arg-8 and Lys-112 at the crevice of the cleft play a critical role in the subunit-binding site of the PapD chaperone. Mutations in these residues abolish the ability of the PapD protein to bind fimbrial subunits and to mediate their assembly into fimbriae [49,50], In PapD a buried interdomain charge-charge and hydrogenbond network is contributed by amino acids Glu-83, Arg-116 and Asp-196. The residues Asp-196 and Glu-83 are invariant, and residue Arg-116 is conserved in 25 of 26 members of the chaperone superfamily, the exception being the CssC chaperone, in which a Gln residue substitutes. An internal salt bridge is formed between the positively charged side chain of residue Arg-116 and the two negatively charged side chains of residues Asp-196 and Glu-83 (one from each domain), which is thought to orient the two globular domains of the chaperone towards one another, thereby stabilizing the subunit-binding cleft region between the domains [42], The other six invariant residues occupy critical points in loops or are involved in intramolecular interactions which orient loops. There are in addition 42 residues of a conserved character, 38 of which are hydrophobic, three polar and one positively charged. A further 11 residues can be regarded as consensus amino acids, i.e. present in >70% of the amino acid sequences of the superfamily. Most of the conserved hydrophobic amino acids form part of the hydrophobic core of the chaperone, with 30 of the hydrophobic side chains being buried. Eight of the surface-exposed conserved residues are hydrophobic; all except one of these are in domain 1, where they cluster in three regions. The amino acid sequences of the chaperones can be differentiated into two groups on the basis of the numbers of amino acids in the loop between β-strands Fl and Gl, termed the Fl-Gl loop (Figure 3) [48], The 17 chaperones that possess a short Fl-Gl loop, ranging in size from 10 to 20 residues (only the F17D chaperone possesses 20 residues) have been designated the FGS subfamily (Fl-Gl Short). The remaining nine chaperones have a longer Fl-Gl loop length of 21-29 residues (only the CS3-1 chaperone has 21 residues) and have been classified as the FGL subfamily (Fl-Gl Long). In addition, conserved structural differences are present in the β-

153

s

Critical role in subunit-binding site

Located at end of p-strand G l between hinge-connecting domains I and 2

Hydrogen-bonding t o T h r - 1 4 7 O H andTyr-149 O ; positioning of loop region between (3-strands

Domain interface

Back

Floor of cleft

Hinge

End of strand B2

Domain interface

F 2 - G 2 turn

Glu-83

Pro-94

Lys-I 12

Pro-117

Asn-145

Asp-196

Gly-198

Positioned at the reverse turn between (i-strands F2 and G 2

Hydrogen-bonding t o A r g - l 16 and t w o w a t e r molecules

B2 and C 2

Start of FI - G I loop; first residue after (3-strand F I , located on the surface of domain I

Hydrogen-bonding t o Arg-116

another

D l strand switch,positioned at bend in domain I, w h e r e (5-strand D shifts from o n e s h e e t t o

Hydrogen-bonding t o B I - C I loop; positioning of l o o p region

End of strand BI

Back

Pro-54

Critical role in subunit-binding site

Asn-24

Floor of cleft

n Function/role/characteristic

t amin o aci d residue s i n th e chaperon e superfamil y

Arg-8

Table 2 Invarian Amino aci d residue Locatio

£L

rt>

-s

3

i/>

n

VI -U

Targeting and assembly of fimbriae

strands A"l, Gl, Fl, Cl and D"1 of the conserved β-sheet in domain 1. In the FGS subfamily, residues Trp-36 and Asn-89 are invariant, and position IlOis always a positively charged residue. In the FGL subfamily, positions 110 and 89 are invariant cysteine residues, which are predicted by molecular modelling to form a disulphide bond. Position 38 in all of the FGS chaperones is a negatively charged residue, whereas in the FGL subfamily the amino acid in this position is variable. No FGL chaperone has a tryptophan residue at position 36; instead, a polar or charged residue is present. Structural basis for subunit recognition by FGS and FGL chaperones FGS chaperones assemble fimbrial subunits with the following features: (i) two cysteine residues, spaced approx. 30 amino acids apart in the N-terminal region, that form a disulphide bond; (ii) a conserved pattern of alternating hydrophobic residues at positions 4, 6 and 8 from the C-terminus; (iii) a penultimate tyrosine at the C-terminus; and (iv) a glycine at position 14 from the C-terminus [48]. The last three parameters define a conserved so-called β-zipper motif which is recognized by the chaperone. The FGL chaperones assemble subunits that display a variation of the βzipper motif at the C-terminus [48]. First, the majority of the subunits recognized by FGL chaperones have a tyrosine at position 12 from the C-terminus and a highly conserved glycine residue at position 14, except in the case of the PsaA and Nfa subunits. Secondly, six of the 10 subunits have a tyrosine at position 3 from the Cterminus. Thirdly, at least two alternating hydrophobic residues are present in each of the subunits recognized by FGL chaperones, usually at positions 6 and 8 from the C-terminus. The structural relatedness among the chaperone superfamily and the subunits they assemble was demonstrated by the ability of the PapD chaperone to substitute for the FimC chaperone in binding and assembling the FimA subunits of type 1 fimbriae [51]. This argues that the subunit-binding surfaces are conserved between these two chaperones, and probably within the entire chaperone superfamily. Part of the interaction of a chaperone with a subunit involves the Gl βstrand of the chaperone (Figure 3) and the C-terminus of the subunit [49]. Even Cterminal peptides encompassing the variation of the β-zipper motif present in the subunits recognized by FGL chaperones are also recognized by the FGS chaperone PapD [49]. From studies on mutation of the Arg-8 residue, the molecular anchoring interaction in the cleft appears also to be important in recognizing C-terminal peptides [49]. The affinity of binding of the C-terminal peptides of the different types of Pap subunits to the chaperone PapD also varies considerably [48]. However, the molecular basis for weak or strong binding is not known, although it may reflect (i) differences in the abilities of the C-terminal peptides of the subunits to adopt a conformation that fits into the cleft of the chaperone and along the exposed edge of the conserved sheet, and (ii) differences in the specific packing interactions between side chains in the peptide and the chaperone.

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Ushers Fimbrial biogenesis depends on the targeting of chaperone-subunit complexes to their assembly site and the conversion of the subunits into supramolecular surface appendages with a distinct architecture. While binding of the chaperone to fimbrial subunits occurs at the cytoplasmic membrane, thus maintaining the subunits in an assembly-competent conformation, the release of subunits takes place at the periplasmic face of the outer membrane following interaction of the chaperone-subunit bimolecular complex with an outer-membrane protein termed the usher [31]. Dissociation of the chaperone from the subunit uncaps the surface or surfaces on the subunit required for its assembly into the fimbria. In the simplest model, the usher may possess one site for the attachment of the growing fimbria and additional sites for interaction with the incoming chaperone-subunit complexes. While experimental data suggest that it is the subunits that contain the interactive sites that recognize and bind to the usher, the chaperone is required for this interaction, presumably to maintain an interactive conformation of the subunit [52], As yet the actual nature of the interactions between the chaperone-subunit complexes and the ushers remains unidentified. While the PapD and FimC chaperones are interchangeable, other chaperones are not able to assemble subunits from heterologous systems unless the chaperone and the usher are molecular partners from the same assembly system [53]. This strongly suggests that the usher may interact, at least in part, with non-conserved surfaces of a chaperone presented to the usher in a chaperone-subunit complex. When the PapC usher was deleted genetically, chaperone-subunit complexes accumulated in the periplasmic space, indicating that the usher is required to facilitate dissociation of the chaperone from subunits to allow their assembly. Hung et al. [48] have suggested that dissociation of the chaperone may be triggered by a rearrangement of the chaperone domains. Intriguingly, the binding of chaperone-Pap subunit complexes to the usher PapC in vitro did not initiate dissociation of the chaperone PapD. Others [51] have speculated that the dissociation of the chaperone may be triggered by the interactions of the trimolecular chaperone-subunit-usher complex, but how such a multicomponent interaction could facilitate such dissociation is unknown. Hung et al. [48] have hypothesized that interactions of the chaperone with the usher protein occur in the hinge region between the two domains of the chaperone (Figure 3), which could cause conformational changes that would be transmitted to the inter-domain region, possibly disrupting the charge-charge and hydrogen-bond network to trigger re-orientation of the domains and release of the chaperone via a conforma­ tional change. The association and dissociation of chaperone-subunit complexes appears to be independent of ATP, as no ATP-binding motif has been identified in the chaperone or in the fimbrial subunits. The processes of uncapping of the chaperone and assembly of the subunits into the nascent fimbria may derive energy from conformational changes in the proteins.

Targeting and assembly of fimbriae

Ordered assembly of a composite fimbria The differential affinities of the various fimbrial subunit proteins for the PapC usher and the PapD chaperone, the relative abundance of each of the subunit proteins and the complementary surfaces on each subunit type all appear to be factors that influence ordered assembly of the P-fimbria. The well choreographed assembly of the P-fimbria appears to proceed as a cascade of protein-protein interactions. Periplasmic concentrations of various chaperone-subunit complexes are determined by transcriptional control [33,54]. The usher appears to be able to distinguish between empty chaperone (PapD) and subunit-loaded chaperone (PapD-subunit complexes) in an in vitro binding assay [52]. The order of affinity of chaperone-fimbrin-subunit complexes for the usher PapC was shown to be PapD-PapG > PapD-PapF > PapD-PapE, whereas PapD-PapA and PapD-PapK complexes were unable to bind to the usher PapC. Thus incorporation of fimbrin subunits into the tip fibrillum (Figure 2) in the order PapG, PapF and PapE may reflect their own affinities for the PapC usher. The binding site for the PapD-PapK complex may be the polymerized tip fibrillum associated with the PapC usher. Incorporation of the PapK subunit is known to terminate the growth of the tip fibrillum and probably creates a binding site for the first PapD-PapA complex [6]. The subsequent targeting of PapD-PapA complexes to the assembly site allows polymerization of the fimbrial rods. Norgren et al. [55] suggested that the PapC protein is a porin-like molecule, forming a channel in the outer membrane through which fimbrial subunits are able to pass. Subsequent studies, however, indicated that the PapC usher does not merely form a passive channel, but plays an active role in the order of subunit passage through the outer membrane [52]. Because of this ordering function, the term usher was coined [31]. The usher protein may additionally serve as an anchor protein connecting the fimbria to the outer membrane. The PapC protein is but one member of a family of outer-membrane proteins specific for fimbrial assembly in Gram-negative bacteria; others include FanD, FaeD, FimD, MrkC, FhaA and Fl7C. These ushers have similar molecular masses of 86-96 kDa. From gene sequencing of the usher homologues, these proteins share about 25% identity and 40% similarity in amino acid sequence, especially in the N-terminal region. Thus it is likely that all of the proteins have similar ushering functions, acting in concert with their respective chaperone partners to ensure the correct interactions for the assembly of adhesive structures by ordered polymerization of the subunits and gradual transport of the polymerized structure to the bacterial cell surface. The domains of the usher involved in translocation of assembled subunits across the outer membrane have not as yet been identified. Residues in the β-zipper motif of the PapA subunit of the P-fimbria participate in subunit-subunit interactions after dissociation of the PapA subunits from the chaperone [48]. If the C-terminal amino acid residues of subunits assembled by FCL-chaperone-usher interaction also participate in

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subunit-subunit interactions, the structural differences in the β-zipper motif of such subunits compared with those assembled by FGS-chaperone-usher partners may have implications for the architecture of the fibres, resulting in the subunits being assembled into linear fibres as opposed to helical rods. Thus variation of the β-zipper motif in the subunits assembled by FGL-chaperone-usher partners may engender a different type of subunit-subunit interaction that affects the supramolecular architecture of the fibre formed [48], Alternatively, this variation in βzipper motif of subunits may preclude subunit-subunit interactions, resulting in afimbrial adhesins.

Role of protein disulphide isomerase (DsbA protein) in fimbrial biogenesis Disulphide bonds are critical to the folding and stability of many proteins. Bardwell et al. [56] and Kamitam et al. [57] independently identified a genetic locus dsbA in E. coll that encodes a periplasmic protein that catalyses disulphide-bond formation in vivo. In dsbA' mutants, the periplasmic protein alkaline phosphatase was translocated across the cytoplasmic membrane but not released into the periplasmic space, presumably because improper folding made the polypeptide associate with the outer surface of the cytoplasmic membrane. The DsbA protein and its homologues in other bacteria were shown subsequently to be required for many processes, including assembly of flagella and heat-labile enterotoxin, and secretion of cell envelope proteins, cellulase and the cholera toxin A subunit [58-61]. Jacob-Dubuisson et al. [62] investigated the role of the DsbA protein in the assembly of P-fimbriae and type 1 fimbriae, both of which are composite fibres. In contrast with PapD, the FimC chaperone does not contain cysteine residues [63], All of the P- and type 1 fimbrins contain an intramolecular disulphide bond between two cysteines about 30 amino acid residues apart, which is a characteristic feature of almost all fimbrins [64]. The respective adhesins PapG and FimH of Pand type 1 fimbriae possess four cysteines that are involved in intramolecular disulphide bonds [40]. , Type 1 but not P-fimbriae were assembled in a dsbA' background. For proper folding of the PapD chaperone in vivo, formation of the unusual intrasheet disulphide bond between the last two β-strands H2 and G2 of its C-terminal CD4Iike domain (Figure 3) was essential. The DsbA protein appears to maintain the nascently translocating PapD chaperone in a folding-competent conformation prior to disulphide-bond formation, thereby acting both as an oxidant and in a chaperone-like fashion. In contrast, the type 1 fimbrial chaperone FimC, which lacks cysteine, was stable in dsbA' cells, indicating that its functioning does not depend on protein disulphide isomerase. The DsbA protein also mediates disulphide-bond formation in fimbrial subunits, but, in order to achieve native-like, protease-resistant conformations, the chaperone is also necessary. Thus sequential interaction of the Pap fimbrial subunit proteins with the DsbA and PapD proteins is required for productive folding leading to chaperone-subunit complexes for assembly (Figure 2) [62]. Whether

Targeting and assembly of fimbriae

folding of the subunits is completed prior to interaction with the chaperone or occurs on the chaperone acting as a platform remains to be elucidated. The presence of two cysteine residues near the C-terminus is also a feature common to all type IV fimbrins. The resolution of the structure of gonococcal fimbrin by X-ray crystallography indicated that these cysteines form a disulphide bridge, thereby exposing the intervening residues at the surface of the assembled fimbriae [28], In the case of the biogenesis of type IV fimbriae, the DsbA protein has also been shown to be important for the stability of type IV fimbrins, namely the BfpA subunit of bundle-forming fimbriae of enteropathogenic E. coli [65] and the TcpA protein of Tcp fimbriae of cholerae [61,66,67], Moreover, the Cterminal disulphide-bond region otP. aeruginosa type IV fimbriae has been shown to be the adhesive domain at the tip of assembled fimbriae which recognizes asialoGM 1 receptors on epithelial cells [67], In the case of Bfp fimbriae, the function of the disulphide bond may be to maintain correct folding in order to prevent degradation by the DepP protease prior to assembly.

General secretion pathway Type IV fimbrial biogenesis is a variant of a much broader and widely distributed system for the assembly of surface-associated protein complexes in Gram-positive and Gram-negative bacteria, namely the general secretory pathway ([37]; Chapter 8). The evidence for this comes largely from amino acid sequence comparisons between gene products involved in type IV fimbrial biogenesis in P. aeruginosa (the pil genes) and in Tcp fimbriae biogenesis in V. cholerae (the tcp genes) and related proteins involved in protein secretion and DNA uptake systems [16,22,68,69]. Compelling evidence has accumulated from diverse prokaryotic systems that components required to translocate proteins to the cell surface are structurally and functionally related to proteins involved in the biogenesis of type IV fimbriae. At least four classes of genes, the products of which have significant sequence identity across diverse species boundaries, have been proposed to be part of similar systems for extracellular protein translocation. The genes include those encoding type IV prefimbrin-like polypeptides, peptidases processing prefimbrin homologues, proteins peripherally associated with the cytoplasmic membrane with highly conserved nucleoside triphosphate-binding sites, and outer-membrane proteins. These export systems include pullanase secretion in Klebsiella oxytoca, the secretion of elastase, exotoxin A, phospholipase C and alkaline phosphatase in P. aeruginosa, DNA uptake in Bacillus subtilis and Haemophilus influenzae, and filamentous phage morphogenesis and protein secretion in plant pathogens, e.g. Xanthomonas and Erwinia spp. [70]. In the case of filamentous phages, there is a parallel with type IV fimbriae. These phages have a structure reminiscent of fimbriae and are released from bacterial cells by extrusion rather than cell lysis [71]. Thus this assembly system has been adapted to the assembly of a variety of cellsurface-associated structures, of which one group is the fibrous organelles known as type IV fimbriae.

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Intensive study of the biogenesis of type IV fimbriae in a number of systems has revealed that fimbrial biogenesis requires a large number of genes, and that those involved specifically in fimbrial biogenesis represent only a small subset of the total number of genes, with the different organizations of genes in different species being largely idiosyncratic [72]. For example, in P. aeruginosa as many as 40 different genes are required for fimbrial biogenesis; these are in a number of different loci which are scattered in different parts of the genome. Components involved in the assembly of type IV fimbriae include: (i) a polytopic integral membrane protein located in the cytoplasmic membrane which possibly provides a platform for fimbrial assembly, (ii) a hydrophilic nucleotidebinding protein located in the cytoplasm or associated with the cytoplasmic face of the cytoplasmic membrane which possesses a conserved nucleotide-binding motif and energizes secretion, assembly and extrusion of fimbrial subunits by ATP hydrolysis, and (iii) an outer-membrane component which has been proposed to form a gated channel or pore through which translocation of assembling fimbrin or the assembled fimbria takes place [69,70]. Additional gene products may be involved in remodelling of the peptidoglycan layer to permit assembly of fimbriae. Chaperones and ushers have not as yet been identified in type IV fimbrial systems. The possibility that some sort of molecular chaperone may be involved in type IV fimbrial biogenesis has, however, been discussed [68]. Models for the assembly of type IV fimbriae in P. aeruginosa and V. cbolerae have been presented in a number of review articles [3,16,22,69]. Mattick and Aim [69] have proposed that the overall mechanism of export (and possibly of retraction) of fimbriae may be viewed as an energy-driven ratchet process involving rotation of the complex formed by proteins with type IV leader sequences.

Extracellular nudeation/precipitation pathway Polymerization of protein into polymeric assemblies of a single type of subunit is a common self-assembly process leading to the formation of tubular structures of helical or cylindrical conformation, e.g. tobacco mosaic virus, actin filaments and flagellar filaments. Such structures can be constituted in vitro from purified monomers (coat protein, G-actin and flagellin respectively) by the use of a nucleating agent to initiate polymerization. Diarrhoeagenic E. coli, Salmonella typhimurium and Salmonella enteritidis produce thin aggregative fimbriae, e.g. GWPQ fimbriae, SEFl 7 or Agf fimbriae and curli, which are long, surface-bound, flexible filaments [72-75]. These structures mediate plasminogen binding and activation, bacterial autoaggregation and binding to fibronectin [75-77], Curli are composed of a single protein termed curlin with a molecular mass of 15.3 kDa encoded by the csg/1 gene. SEFl7 fimbriae are assembled from AgfA subunits of 17 kDa which share 86% similarity in primary sequence with the CsgA protein. The genetic determinants encoding expression of these fimbriae share a high degree of similarity in their organization, suggesting that these fimbriae are assembled by analogous mechanisms [74],

Targeting and assembly of fimbriae

Hammar et al. [78,79] have shown that polymerization of curlin to fimbria-like structures differs from prevailing models of fimbrial assembly in that it occurs extracellularly through a self-assembly process which depends on a specific nucleator protein. In the absence of the CsgB protein, curlin is secreted into the medium in a soluble, polymerization-competent form. Hammar et al. [78,79] have proposed that curli polymers are formed as a result of a conformational change in soluble CsgA subunits which is initiated by interaction with CsgB, a nucleating cell-surface-bound protein. The conformational change may involve conversion of the CsgA protein from a disordered structure in the monomeric state into a readily ordered structure in the polymeric state, such as occurs with bacterial flagellar filament assembly. In the case of curli, computer-aided secondary structure predictions suggest that repeats in the CsgA and CsgB amino acid sequences reflect a tertiary structure made up from a basic unit consisting of 3~strand-turn-3strand-turn. The formation of curli fibres has been proposed to resemble the conversion of amyloidogenic proteins into fibrils, with extensive β-sheet structure [6,78,79], The addition of curlin monomers to the nascent filament at its free end appears to be driven by mass action and guided only by the diffusion gradient between the source of secreted monomers and the site of their condensation, unlike the biogenesis of P-fimbria or type IV fimbria, where the filament is growing from the base [80], The assembly of the flagellum in Salmonella depends on self-assembly of subunit proteins that form the hook and filament. The flagellin monomers are believed to be transported through a central channel in the filament to the assembly point at the distal end of the filament [81]. Although several genes have been found to be necessary for the formation of curli, their functions are largely unknown. Much remains to be learned about the export of curlin to the bacterial cell surface via the periplasmic space.

The CFA/I,CSI and CS2 fimbrial pathway Compared with the genetic determinants encoding P-, S- or type 1 fimbriae, the operons for the biogenesis of CFA/I, CSl and CS2 fimbriae produced by entero­ toxigenic E. coli are relatively simple, being composed of four functional genes in each instance. The subunits from which these fimbriae are assembled have been designated CfaB, CsoA and CotA respectively. These fimbrins share about 50% amino acid sequence identity and 65% similarity [13]. The genetic determinants lack genes encoding proteins with significant amino acid similarity to the chaperones and ushers of other fimbrial systems (Table 1 and above). Moreover, no additional genes encoding proteins with the properties of fimbrial subunits have been revealed by nucleotide sequencing of the respective genetic determinants. In the case of the CsoA and CfaB subunits, biochemical and molecular genetic studies have established that these subunits play both a structural role and the role of the adhesin in their respective fimbriae (Figure 1) [10,11], Alignments of the amino acid sequences of respective ancillary proteins, i.e. (i) CfaB, CsoB and CotB, (ii) CfaC,

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CsoC and CotC, and (iii) CfaE, CsoE and CotD, have demonstrated that these homologues each share 52-58% amino acid sequence identity and 67-73% similarity [13]. Indeed, their relatedness is attested to by the interchangeability between systems of genes encoding these ancillary proteins [11,13]. The CfaA, CsoB and CotB proteins appear to be necessary for assembly of their respective fimbrial subunits into intact fimbriae. The CfaC, CsoC and CotC proteins have similar molecular masses between 93 and 95 kDa, which are very similar to those of well characterized ushers. Each has properties indicative of an outer-membrane protein. Each set of homologues possesses signal sequences consistent with their targeting to the periplasmic space via the Sec pathway. Separate recombinant strains harbouring mutations in the csoE and csoC genes respectively expressed equivalent amounts of the CsoA subunit to the parent strain, indicating that the CsoA fimbrin was stable in the periplasm in the absence of these ancillary proteins [12]. The CfaB, CsoA and CotA subunits do not possess the general properties shared by fimbrial subunits that require a PapD-Iike chaperone for transperiplasmic transport. The CsoE protein was shown to be essential for CSl fimbrial biogenesis; in particular, the C-terminal region was important for either performing its function or promoting its stability [57]. Models for the assembly of CSl fimbriae consistent with the data available at the time have been proposed [3], These are illustrated in Figure 4. More recently, Sakellaris et al. [83] have proposed that the CsoE protein (termed CooD by these authors) is a minor intrinsic fimbrin (i.e. about one CsoE subunit to 2000 CsoA subunits) located at the tip of the CSl fimbria, and that it has a dual function in fimbrial assembly, which distinguishes it from tip adhesins such as the PapG and FimH proteins. These authors propose that the CsoE protein plays a role analogous to that of the PapK protein in P-fimbria biogenesis, i.e. nucleating or initiating assembly of the major structural CsoA subunits (cf. Figure 4c and Figure 1). Interestingly, these authors noted a conserved amino acid motif, AGxYxG(x) 6 T (where χ denotes a non-identical residue), at the same position near the C-terminus of the fimbrins CsoB, CsoA and CotA and the 'minor pilins' CfaE, CsoE and CotD, suggesting that this conserved sequence may be essential for interactions of fimbrin with the assembly machinery or other fimbrins. The major reservation with this model is that the CsoA protein, like the CfaB protein of CFA/I fimbriae, is the fimbrial adhesin of CSl fimbriae and performs this function at the tip of the fimbrial shaft (Figure 1) [3,10,11,82]. Whether the model of Sakellaris et al. [83] means that the adhesin-binding domain of the CsoA protein is exposed on the lateral aspect of the tip of the fimbria, or that the CsoE protein orients or exposes the adhesin domain of the tip CsoA subunit or indeed contributes itself to the adhesive interaction, which has implications for similar scenarios for the CfaB/CfaE and CotA/CotD homologues, requires further study.

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Figure 4

Models of the biogenesis of CSI fimbriae

Outer membrane Periplasm

Sec-dependent secretion

Inner membrane

Outer membrane Periplasm

Sec-dependent secretion

Inner membrane

Outer membrane Periplasm

Sec-dependent secretion

Inner membrane

The CsoA (C) protein is secreted into the periplasm via the general secretory pathway. In the periplasmic comportment it can interact with CsoB (B) or CsoE (E), which may protect it from proteolytic enzymes. The interaction of the CsoA subunit with the CsoE protein in conjunction with the CsoB protein induces a conformational change in the the CsoA subuniLThis allows the CsoA subunit to bind to the CsoB protein in an assembly E M B O J.

45. 46. 4".

Mol. O.. Yisschers. R A Y . de G r a a f . F.K. and O u d e g a . B. 11994 Mol. Microbiol. 11. 3 9 1 - C 2 Mol. O.. O u d . R.P.C.. de Graaf. F.K. and O u d e g a . B . • 1995 Microb . Pathog. 18. 115-12S Mol. O.. O u d h u i s . W.C.. Fokkeroa. H . and O u d e g a . B. ,1996 Mol. Microbiol. 22. 3 ~ 9 - 3 S S

4>.

53. 54.

H u n g . D . L . . K n i g h t . S . D . . W o o d s . R . M . . P i n k n e r . J . S . and H u l t g r e n . S . J . , 1996 > E M B O J . 15. 3-92-3SC5 Kuehn. M.J.. O g g . D.J.. K i h l b e r g . J . . S l o m m . L . X . . Flemmer. K.. Berglors. T. and Hultgren, S.J. 1993 Science262. 1 2 3 4 - 1 : 4 1 S l o m m . L . X . , P m k n e r . J . S . . B r i n d e n . C.-I. and Hultgren. S.J. 1 9 9 : : V b i > \ 11.4 4 " 4 > 6 J o n e s . C . H . . Pinkner. J . S . . X i c h o l e s . A . M . S l o n i m . L . X . . A b r a h a m . S . X . and H u l t g r e n . S.J. ' 1993 Proc. X a t l . Acad. Sci. U . S . A . 9 : . S39"-S4C 1 D o d s o n . K.W.. J a c o b - D u b u i s s o n . F.. Striker. R . T . and H u l g r e n . S.J. . 1993 Proc. X a t l . Acad. Sci. U . S . A . 9C. 3 6 - 3 - 3 6 - 4 K l e m m . P.. Joregense. B.J.. Kreft. B. and Christiansen. G . 119995 J. Bacteriol . 177 . 621-62 7 Smyth. C . J . and Smith. S.G.J. 11992 > S y m p . Soc. G e n . M i c r o b i o l 49. .N."

55. 56. 5".

X o r g r e n . M . B i g a . M.. Tennent. J . M . and X o r m a r k . S . . 19S~ Mol. Microbiol. 1. 1 6 9 - P S Bardwell. J . C . . M c G o v e r n . K. and B e c k w i t h . J . ( 1 9 9 1 ' Cell 67. 5 S 1 - 5 S 9 Kamitar.i. S.. A k i v a m a . Y. and h o . K . 11992^ E M B O J . 11. 5~-62

55. 59.

T o m b . J.-F. ( 1992 ) Proc. X a t l . Acad. Sci. U . S . A . 89. 12252-1C256 B o r t o l i - G e r m a n . I.. B r a n . E.. Py. B.. C h i p p a u x . M . and Barras. F. .1991 M o l . M i c r o b i o l . 11 .

bZ. 61. 6:.

Dailey. F.E. and Berg. H . C . .1993 . Proc. X a t l . Acad. Sci. U . S . A . 9C. i : 4 5 - i ; 4 " Yu. J.. Webb. H . and"Hirst. T.R. . 1 9 9 : . Mol. Microbiol. 6. 1949-195S J a c o b - D u b u i s s o n . F.. Pinkner. J . . X u . Z.. Striker, R.. P a d m a n h a b a n . A. and H u l t g r e n . S.J. 1994 . Proc. X a t l . Acad. Sci. U . S . A . 91. 11552-11556 K l e m m . P. 1992 > Res. Microbiol. 143. S31-S36 S i m o n s . B.. R a t h m a n . P.. Malii. C . . O u d e g a . B. and de G r a a f . F.K. 1 9 9 : F E M S M i c r o b i o l . Lett. 67. i : _ - n : Zhang. H . - Z . and Donnenberg. M.S. 1996 Mol. Microbiol. 21. " S " - ^ Peek. J .A. and Tavlor. R . K . • 1992' Proc. X a t l . Acad. Sci. U . S . A . 8 9 . 6 2 1 3 - 6 2 1 4 Lee. K . K . . Sheth. H . B . . W ong, Sherbourne. R.. P a n n c h v c h . H o d g e s . R.S.. Lmsr^'ood. _13 C . A . . K n v a n . H . and l m r . . R . T . 1994 M o l . Microbiol. 11. K o o m e y . M. . 1995 Trends Microbiol. 3. 4:9—111 Mattick. J.S. and Aim. R.A. .1995 Trends Microbiol. 3. 4] 1—413 H o b b s . M. and Mattick. J.S. ,1993 . Mol. Microbiol. 1C. 233-243 Russell. M. 1991 M o l . Microbiol. 5. 1 6 " - 1 6 1 3

49. 5:. 51. 52.

63. 64. 65. 66.

6S. 69. "1. 2. ~3. -

"4. ~5. ~b.

Collinson. S.K.. E m o d v . L.. Trust. T.J. and Kav. W W . . 1991 J, Bacteriol. 174.4493—1495 C o O i n s o n . S.K.. E m o d v . L . . Muller. K . - H . . Trust. T.J. and K a v . W W . 1991 > J . Bacteriol. 175, 4—3—4~S1 C o l l i n s o n . S . K . . C l o u t h i e r . S . C . . D o r a n . J . L . . B i n s e r . P.A. and K a v . WAX". ,1996 J . Bacteriol. 17S. 6 6 2 - 6 6 " Olsen. A.. J o n s s o n . A. and X o r m a r k . S. , 19S9 ! N a t u r e . L o n d o n 3 3 8 . 6 5 2 - 6 5 5 Olsen. A.. Arnquist. A.. H a m m a r . M.. Sukupolvi, S. and X o r m a r k . S. 1993 Mol. Microbiol. 7, 523-536

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77. 78. 79. 80. 81. 82. 83. 84. 85. 86.

Sjobring, U., Pohl 1 G. and Olsen, A. (1994) Mol. Microbiol. 14, 443-452 Hammar, M., Bian, Z. and Normark, S. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 6562-6566 Hammar, M., Arnquist, A., Bian, Z., Olsen, A. and Normark, S. (1995) Mol. Microbiol. 18, 661-670 Lowe, M.A., Holt, S.C. and Eisenstein, B.l. (1987) J. Bacteriol. 169, 157-163 Homma, M., lino, T., Kutsukake, K. and Yamaguchi, S. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6169-6173 Marron, M.B. (1995) Ph.D. Thesis, Trinity College, University of Dublin Sakellaris, H., Balding, D.P. and Scott, J.R. (1996) Mol. Microbiol. 21, 529-541 Pallesen, L. and Klemm, P. (1994) in Fimbriae: Adhesion, Genetics, Biogenesis, and Vaccines (Klemm, P., ed.), pp. 271-276, CRC Press, Boca Raton Gaastra, W. and Svennerholm, A.-M. (1996) Trends Microbiol. 4, 444^452 Smith, S.G.J. (1995) Ph.D. Thesis, Trinity College, University of Dublin

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10 Targeting to and translocation across the endoplasmic reticulum membrane Jeffrey L. Brodsky Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, U.S.A.

Introduction The biogenesis of secretory proteins begins with their insertion, or translocation, either into the lumen of the endoplasmic reticulum (ER) or into the lipid bilayer of the ER membrane. Consequently, protein translocation is the first committed step in the secretory pathway. Secreted proteins subsequently travel in vesicles that migrate sequentially from the ER to the Golgi complex, and finally to the plasma membrane, at which time they may be released from or retained in the plasma membrane. The secretory pathway also transports soluble and membrane proteins that may be withheld in the ERor Golgi complex (see Chapter 11). Early models for protein translocation hypothesized either that preproteins might insert spontaneously into and transit across lipid bilayers f 1—4], or that translocation is engineered by a pore in the ER membrane [5], It is now clear that translocation requires not only a specific pore but also sophisticated protein machines in the cytosol, in the ER membrane and in the lumen of the ER. Protein translocation can be divided schematically into three steps: (1) the recognition and targeting of a preprotein to the ER membrane; (2) the insertion of the preprotein into the translocation pore in the ER membrane; and (3) the energydependent import of the preprotein into the ER lumen. While protein processing in the ER may occur concomitantly with translocation, proteins attain their final conformations only after translocation is complete. Thus the biogenesis of secretory proteins is tightly coupled to translocation and may be regulated by the translocation machinery. This review will summarize much of our current knowledge about the factors comprising the translocation machines that direct preprotein targeting, membrane insertion and import into the ER. Pertinent questions that remain in this field will also be detailed. Particular attention will be placed on the knowledge gained from experiments conducted with the simple eukaryote Saccharomyces

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cerevisiae or 'Baker's yeast'. Finally, models that currently best describe how the translocation machine functions and how energy might be used to drive proteins into the ER will be discussed.

Signal peptides Proteins targeted to the secretory pathway almost always contain an N-terminal extension known as a signal peptide or signal sequence. Signal peptides are required to direct preproteins to the ER translocation machine, as they interact directly with components of the machinery. They also interact specifically with a cytosolic protein complex known as the signal recognition particle (SRP) (reviewed in [6]) and may insert spontaneously into lipid bilayers (reviewed in [7]). The preprotein forms a membrane-inserted hairpin loop, leaving the N-terminus of the signal peptide in the cytosol [8,9] (Chapter 3). Signal peptides contain a tripartite structure consisting of one to five amino acids at the extreme N-terminus displaying a net positive charge, a central hydrophobic core containing from seven t o 15 amino acids, and finally a more polar region comprising three to seven amino acids (reviewed in [7,10,11]). This final, polar region contains the recognition site for signal peptidase, the ER lumenal enzyme that catalyses the cleavage of the signal peptide from the preprotein ([12]; reviewed in [13,14]). Signal peptidase acts as the signal peptide emerges from the translocation channel on the lumenal side of the ER. The cleavage site recognized by signal peptidase is defined by amino acids closely following the hydrophobic core with small side chains residing at positions -1 and -3 relative to the cleavage site [10,11], Although signal peptides contain a hydrophobic core that may implant into membrane-like surfaces [11], experiments using ER-derived microsomes indicate that the signal peptides interact with many proteins during translocation. Most importantly, signal peptides bind to the SRP soon after their synthesis (reviewed in [6,15]), an event which slows down translation and helps target the ribosome to the ER membrane, where translation may re-initiate (see below). Thus the primary role for a signal peptide is to promote the early interaction of a preprotein with the membrane and to couple protein translation with translocation. The membrane environment in which the signal peptide resides was first probed by Gilmore and Blobel [16], who showed that a short preprotein containing its signal peptide could be extracted from the ER membrane with an aqueous perturbant, an unlikely event if the signal peptide were embedded in the lipid bilayer. Crowley et al. [17] then tagged the signal peptide of a preprotein with a fluorescent ligand and observed that the signal peptide exists exclusively in an aqueous environment, strongly suggesting that it is contained within a hydrophihc channcl.

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The interaction of the signal peptide with components of the translocation machinery might also regulate protein import into the ER. Protein translocation may occur either co-translationallv, where the ribosome remains associated with the nascent chain while protein import occurs, or post-translationally, where the completely synthesized preprotein is released from the ribosome and then translocates into the ER (see Figure 1). Recent evidence suggests that different signal peptides may target preproteins to subcomplexes of the translocation machinery in yeast that are committed to either the co- or post-translational pathways [18-20], It was also observed that the translocation complex itself can discriminate between a functional and non-functional signal peptide [21], and that pure signal peptides open a channel in reconstituted bacterial membranes [22]. Together, these results demonstrate that signal peptides are not simply lipophilic Figure 1

Translocation of preproteins into the ER

Preproteins can be translocated into the ER either co- or post-translauonally. During co-translavonal translocation (a), the ribosome-preprotein complex is targeted to the ER membrane (step A) before translation proceeds (step B).The nascent chain is driven directly into the ER lumen in this case. During post-translational trans/ocot/on (b), the preprotein is completely synthesized on a cytosolic ribosome.The preprotein may be released and maintained in a non-native state (step A), or may fold (step B).The nonnative preprotein can then be targeted to the translocation machinery in the ER membrane (step C) and

imported into the lumen. Details on both pathways are provided in the text For simplicity, the orientation of the signal peptide is not depicted.

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molecules, but contact many proteinaceous components and play an active role during protein translocation into the ER.

Preprotein targeting to the ER Co-translational protein translocation The SRP is an 11 S complex containing six equimolar polypeptides with molecular masses of 72, 68, 54, 19, 14 and 9 kDa [23], and a single 7 S RNA ('7SL' or 'SRP RNA') [24]. Pure SRP arrested the translation of a secreted but not of a cytosolic protein in a membrane-free wheatgerm extract, and bound loosely to ribosomes translating a cvtosolic protein, but tightly to ribosomes expressing a secreted protein [25], Because translation arrest exposes only the N-terminal signal peptide of the secreted protein to the cytosol, it was proposed that SRP recognizes signal peptides and thus binds selectively to ribosomes that are translating secretory proteins, inhibiting further protein synthesis [26], SRP-mediated translation arrest was alleviated when microsomes were added to the reaction [27], suggesting that a membrane-associated factor dissociates SRP from the nbosome, so that translation and translocation may proceed after SRP has ushered a preprotein-ribosome complex to the ER membrane. Co-translational protein translocation proceeds once SRP is released [28]. The secondary structure of 7SL RNA in SRP is highly conserved between most organisms and contains four domains. It has been proposed that the 7SL RNA provides the structural framework for SRP, and may mediate the interaction of the particle with the nbosome (reviewed in [6]). The interaction of the polypeptides in SRP with 7SL RNA has been studied extensively (see below). To perform a structure/function analysis of SRP, the protein components in the panicle have been dissected biochemically. It was first determined that SRP could be disassembled into subcomplexes and individual components [29,30]. The isolation of the genes encoding the polypeptides of mammalian SRP [31-35] permitted the expression of SRP subunits by in vitro transcription/translation reactions [31,33,34,36-38]. Thus the activities of the individual components or subcomplexes could be assayed for specific SRP-dependent activities, namelv signal sequence binding [38—43], membrane targeting [39] and translation arrest [30,34,44], The interaction of SRP proteins with each other and with the 7SL RNA has also been investigated extensively by in vitro binding assavs [31,36-38,40,45] and by nuclease and free-radical protection experiments [45,46]. The picture that emerges from these studies is that the 9 kDa and 14 kDa subunits (SRP9 and SRP14 respectively) form a tight complex, as do the 68 kDa (SRP68) and 72 kDa (SRP72) subunits (reviewed in [6,47]). The SRP9-SRP14 and SRP68-SRP72 complexes occupy unique positions on the 7SL RNA. In contrast, SRP54, the subunit of molecular mass 54 kDa, and SRP19 (the 19 kDa subunit) exist as monomers and

Endoplasmic reticulum targeting and translocation

bind to distinct regions on the 7SL RNA adjacent to SRP68-SRP72. SRPl9 is required, however, for the interaction of SRP54 with SRP RNA. Combining the results of the biochemical assays and mapping experiments described above indicates that the following functional domains are present in SRP: the domain containing the SRP9-SRP14 complex arrests translation, while the domain with the SRP68-SRP72 subunits is required for membrane targeting. When a cytosolic extract is incubated with ribosomes presenting photoactivatable probes on signal peptides, only SRP54 cross-links to the signal peptide [48,49]. Cross-linking between fragments of pure SRP54 and signal peptides [40,43], or proteolytic cleavage of cross-linked products [41,42], indicates that a specific domain in SRP54 recognizes signal peptides. This signal peptide-binding, methionine-rich domain of SRP54, known as the M domain, has been proposed to form an amphipathic α-helix that might accommodate the hydrophobic core of signal peptides [32]. It has recently been suggested that the SRP54-SRP-RNA complex functions as the core machinery of SRP, as this partial complex is sufficient to support the translocation of a translation-arrested preprotein [38], The cloning of the gene encoding SRP54 revealed a domain of the protein containing a GTP-binding site [32,33], now known as the G domain; a mutation in the G domain rendered SRP defective for ER-membrane and signal-peptide binding [50]. Because the dissociation of a signal peptide from SRP54 required GTP binding [51], Miller et al. [52] proposed that an ER-associated protein might act as a guanine nucleotide loading protein, causing the release of a signal peptide to the translocation complex. It is now clear that a component in the ribosome, and not an ERassociated factor, stimulates GTP binding to SRP [50]. A model for the GTP/SRP cycle arising from the work of Bacher et al. [50] is depicted in Figure 2. After the signal peptide is released, GTP hydrolysis frees SRP from the membrane-associated SRP receptor [53], permitting SRP to recycle for another round of preprotein targeting. Because this GTPase activity of SRP54 is catalysed by the SRP receptor [52], GTP is required both to trigger the release of a ligand (i.e. the signal peptide) and to recycle the complex when the reaction is complete. This cycle ensures the productive delivery of a nascent polypeptide to the ER membrane. Recent work by Rapiejko and GiImore [53a] indicates that some aspects of the model presented in Figure 2 remain uncertain (reviewed in [53b]). For example, it is no longer obvious if SRP is bound to nucleotide upon association with a signal sequence or upon docking at its receptor on the ER membrane. A new model incorporating these results and previous observations, known as the 'concerted switch model', has been proposed by Millman and Andrews and may be found elsewhere [53b].

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Figure 2

The SRP cycle

The SRP (hatched oval), bound to GDP, interacts with the emerging preprotein, and the ribosome

catalyses GTPIGDP exchange on SRP.The SRP-GTP-ribosome complex is then targeted to the ER membrane, and SRP binds to the SRP receptor (black rectangle), releasing the preprotein to the translocation complex and triggering GTP hydrolysis.The GDP-bound form of SRP is freed and may interaa with another emerging signal sequence. (Interpreted from Bacher et al. [50].)

'Nascent-polypeptide-associated complex' (NAC) Although SRP positively selects preproteins containing signal peptides, it was anticipated that there might be another complex that acts antagonistically to SRP, or that recognizes preproteins that ultimately reside in the cytosol. In fact, Wiedmann et al. [54] discovered that short nascent chains of both secreted and cytosolic proteins cross-linked to a dimeric protein complex they called NAC. NAC did not associate with proteins that were translated beyond a certain size, and the complex was extracted from ribosomes by salt, demonstrating that NAC was not composed of ribosomal proteins. NAC was then purified based on its ability to cross-link to the nascent chain of a cytosolic protein [54], revealing the NAC complcx to be composed of novel gene products with molecular masses of 33 and 21 kDa. Nascent chain cross-

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linking studies in cytosolic extracts depleted of NAC exposed a most intriguing result: cross-links between SRP54 and both secreted and cytosolic proteins were discovered. Furthermore, when NAC-depleted cytosol was assayed for preprotein translocation activity, it was observed that even SRP-bound cytosolic proteins could be imported into ER microsomes. When NAC was added back to the extract, cross-links between SRP54 and the cytosolic proteins were absent, and the aberrant translocation of the cytosolic protein was prevented. The conclusions from these studies were that: (1) NAC and SRP compete for preproteins at early stages during their synthesis; and (2) in the absence of NAC, SRP can bind to and target cytosolic proteins to the ER membrane for translocation. Thus NAC is a negative regulator of ER-membrane preprotein targeting. How, then, are preproteins targeted to the ER membrane? Both Lauring et al. [55,56] and Jungnickel and Rapoport [21] have now shown that, in NACdepleted extracts, the affinity of the ribosome for the protein translocation channel, Sec61p, may be sufficient to initiate protein translocation; therefore SRP-mediated translational pausing allows enough time for the ribosome to find Sec61p in the ER membrane, suggesting that the SRP receptor may not be required for ribosome-nascent-chain binding to the ER membrane, but might serve only to recycle SRP. This hypothesis is controversial, however, as the identity of the ribosome receptor in the ER membrane and whether SRP still positively selects secreted preproteins for ER targeting in vivo remain unsettled [56a]. The ribosome receptor It has generally been assumed that a ribosome receptor resides in the ER membrane. This conclusion is based on the work of Borgese et al. [57], who showed that an ER membrane protein mediates saturable, salt-dependent ribosome binding in vitro. The search for a ribosome receptor has continued in earnest since then, as it has remained unknown (1) whether the affinity of SRP for the SRP receptor is sufficient to target and dock the ribosome at the ER membrane, and (2) how and why the ribosome remains attached at the ER membrane once SRP has become dislodged during co-translational translocation. In extracts depleted of NAC, the ability of ribosome-tethered cytosolic proteins to be translocated into the ER, albeit at a low efficiency (see above), and of some preproteins to be targeted to the ER membrane in the absence of SRP [21,55,56], has only further stimulated the drive to identify the ribosome receptor. A strong candidate for this receptor, known as ρ 180, was identified by Savitz and Meyer [58]. Proteoliposomes reconstituted with pl80 supported ribosome binding and exhibited a K n identical with that obtained using native microsomes (-1.5 X IO 8 M) [57], Also, detergent-solubilized extracts specifically depleted of pl80 were unable to co-translationally translocate a preprotein or bind ribosomes when reconstituted into liposomes [59], while re-addition of pure ρ 180 restored preprotein translocation and ribosome-binding activity. Finally, anti-pl 80 monoclonal antibodies inhibited translocation and ribosome binding [59], The

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isolation of the gene encoding ρ 180 revealed that the protein contains an astounding 54 repeats of a basic 10-amino-acid repeat at its N-terminus, giving pl80 a predicted pi of approx. 10 [60]. Results contradicting the supposition that pi 80 is the receptor were obtained later [61-64], These groups either observed ribosome binding in reconstituted protein fractions lacking pl80 [61,62] or identified a novel protein, p34, as the ribosome receptor [63,64]. This controversy remains unresolved. While the function of ρ180 was being debated in the literature, Rapoport and colleagues took a direct approach to purifying the mammalian ribosome receptor. Gorlich et al. [65] fractionated detergent-solubilized microsomes to isolate ER-membrane-protein-ribosome complexes and uncovered an integral ER membrane protein homologous to Sec61p, the yeast translocation channel [66-69]. The mammalian Sec61p is -50% identical with the yeast protein and, like the yeast homologue, intimately associates with translocating nascent chains [65], Purified Sec61p bound to ribosomes with a K D in the nanomolar range, and selective proteolysis of Sec61p abrogated ribosome binding [70], The yeast Sec61p homologue could also be purified as a ribosome-bindmg protein [19]. The identity of Sec61p as a ribosome receptor is also consistent with the results of Crowlev et al. [17], who showed that, as nascent chains translocate into an aqueous channel, the ribosome forms a tight seal with the ER membrane. Thus, during co-translational protein translocation, the growing polypeptide chain may pass directly into the channel, Sec61p, and then into the ER lumen. This model is most easily envisaged if the ribosome contacts Sec61p directly.

Post-translational protein translocation Preproteins can be translocated into the mammalian and yeast ER both during and after translation (Figure 1). While the vast majority of preproteins are translocated into the mammalian ER co-translationally [6], there are many examples of small peptides that may be imported into mammalian microsomes post-translationally (reviewed in [71]). These molecules translocate into the ER without the aid of SRP, most likely because they are too short to interact with SRP when they emerge from the ribosome [26]. Furthermore, disruptions of the genes encoding the SRP components in yeast is not lethal, but results in the accumulation of some preproteins in the cvtosol and a reduced rate of growth [72-76]. The simplest conclusion from these studies is that the co-translational translocation pathway represents only one route for protein import into the ER. Post-translational translocation, the alternative route that handles short peptides in mammals and has been shown to operate both in vivo and in vitro in veast [77-81], is SRPindependent. Folded proteins are unable to translocate across membranes, most probably because the diameter of the translocation pore restricts the size of proteins that pass through it (reviewed in [82]). Probably the most vital role that SRP plays during co-translational translocation is to prevent a secreted protein from folding in

Endoplasmic reticulum targeting and translocation

the cytosol; by coupling translation to translocation, nascent proteins transit directly into the lumen of the ER. In contrast, post-translationally translocated proteins are synthesized fully before being targeted to the ER and could fold in the cytosol (Figure 1). Thus factors such as cytosolic molecular chaperones that may prevent premature protein folding are required to facilitate the targeting of posttranslationally translocated proteins to the ER membrane. Molecular chaperones are defined as molecules that prevent protein aggregation and may aid protein folding by maintaining polypeptides in productive folding pathways (reviewed in [83,84]). Many chaperones were first identified as heat-shock proteins (Hsps), factors whose synthesis is induced by cellular stress. To prevent unfolded proteins from aggregating under stress conditions, molecular chaperones shield hydrophobic amino acid motifs from the solvent [84]. Pelham [85] first suggested that chaperones might also be required during protein translo­ cation, because hydrophobic domains are similarly exposed. This problem is partic­ ularly relevant during post-translational translocation. It is now apparent from both biochemical and genetic studies that cytosolic molecular chaperones analogous to the bacterial DnaK and DnaJ proteins are absolutely required for post-translational translocation (reviewed in [82]). While DnaK and DnaJ were first identified because they are required for phage X DNA replication, these factors are also required for bacterial growth and cell division at elevated temperatures (reviewed in [86]), for protein translocation across the bacterial plasma membrane [87], and during protein folding and protein degradation (reviewed in [84]). Hsps with a molecular mass of -70 kDa (Hsp70s), such as DnaK, bind and release peptides concomitant with ATP hydrolysis to prevent protein aggregation [82], To determine the effect of Hsp70s on protein translocation in vivo, Deshaies et al. [88] used a yeast strain in which the genes encoding the major cytosolic Hsp70s were disrupted, but which was viable because one cytosolic Hsp70, Ssalp, was present on a galactose-regulated plasmid. When cells were grown in glucose to repress the transcription of SSA1, the cellular levels of Ssalp fell, and ER-targeted preproteins accumulated in the cytosol. To demonstrate directly that Ssalp is required for protein translocation, purified Ssalp was shown to restore translocation competence to a wheatgerm-synthesized preprotein [88-90], a result made possible because the Hsp70 in wheatgerm lysate is unable to support preprotein translocation into yeast microsomes [91], Chirico et al. [89] then showed that the Ssalp requirement for post-translational protein translocation in vitro could be bypassed if a preprotein was denatured in urea before being added to the translocation reaction. Thus cytosolic Hsp70s facilitate protein translocation by retaining preproteins in a non-native conformation, confirming the hypothesis presented previously by Pelham [85]. Zimmermann and colleagues [92] obtained data consistent with this hypothesis in studies using mammalian microsomes. Because Hsp70s sustain proteins in a non-native conformation, it was anticipated that any Hsp70 homologue might substitute functionally for Ssalp in

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the in vitro reaction. However, we found that only Ssalp, but neither a bacterial nor an ER lumenal Hsp70, was able to support preprotein translocation [90], suggesting either that cytosolic Hsp70s play a more elaborate role during post-translational translocation or that they interact specifically with other cytosolic factors during import. In accordance with these results, Wiech et al. [93] showed that a lumenal Hsp7Q could not substitute functionally for the cytosolic Hsp70 when post-translational translocation was measured in mammalian microsomes. The ATPase activity of Hsp70s is enhanced by DnaJ molecular chaperones. Escherichia coli DnaJ specifically stimulates the ATPase activity of DnaK, while a third chaperone, GrpE, facilitates the exchange of ADP for ATP on DnaK [94]. Optimal chaperone-dependent activities occur only when all three proteins are present (reviewed in [84]). A cytosolic DnaJ homologue in yeast, Ydjlp, was discovered independently by Caplan and Douglas [95] and Attencio and Yaffe [96], and is -32% identical to DnaJ. As expected, the ATPase activity of Ssalp is stimulated by Ydjlp [97], and the ability of Ssalp to bind to a permanently unfolded polypeptide [97] and to a yeast preprotein [98] is abolished in the presence of Ydjlp and ATP. Together, these results demonstrate that a DnaK-DnaJ interacting pair, Ssalp and Ydjlp, exists in yeast cytosol. To prove that Ydjlp is required for post-translational translocation, Caplan et al. [99] created a temperature-sensitive ydjl mutant strain that accumulates untranslocated preproteins in the cytosol at the non-permissive temperature. Because Ydjlp is prenylated and resides on the cytoplasmic face of the ER membrane [96,100], it is possible that Ssalp targets unfolded preproteins to the ER where, upon interaction with Ydj lp, the preprotein is released and translocates post-translationally into the ER lumen. The inability of the Ydjl temperaturesensitive protein to stimulate the ATPase activity of Ssalp [99], an event required for protein release [97,98], favours this model. Genetic evidence for the specific interaction between Ssalp and Ydjlp was obtained recently by Becker et al. [101].

Co-ordination between the co- and post-translational pathways in vivo As discussed above, disruption of the genes encoding the subunits of SRP and the SRP receptor in the yeast S. cerevisiae is not lethal [72-76], On the contrary, depletion of the gene products encoding these factors arrests cell growth and division [74,102,103]. Itwas suggested that yeast could adapt to the absence of the co-translational pathway, but when the proteins required for this pathway were depleted rapidly, the cells were unable to adapt. One could imagine that the adaptive response might induce the synthesis of factors required for the post-translational pathway. Indeed, this was shown by Arnold and Wittrup [104], who observed that the steady-state levels of both Ydjlp and Ssalp increased upon SRP54 depletion. This conclusion must be interpreted with caution, however, as the depletion of SRP54 and the resulting accumulation of some preproteins in the cytosol could trigger a cellular stress response.

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Ogg and Walter [103] also obtained evidence consistent with the model that disruption of the co-translational pathway activates the SRP-independent, or post-translational, pathway in yeast. Temperature-sensitive mutations in SEC65, the gene encoding an SRP19-related protein in yeast, destabilize yeast SRP [75,105], but the SEC65 translocation defect and temperature-sensitivity could be suppressed by low levels of cycloheximide. However, cycloheximide could not surpress the growth defects in strains containing SRP-deleted genes [103], suggesting that the SRP-independent pathway had supplanted the SRP-dependent pathway. The signal sequence dictates whether a preprotein translocates either coor post-translationally in yeast [20], and the translocation defects for a given preprotein vary depending upon whether components in the co- or post-transla­ tional translocation pathways are either absent or mutated [18,20,74,81,103]. Such in vivo analyses of the co- and post-translational pathways in yeast may be difficult to interpret, however, because the accumulation of one class of preproteins might block preproteins using the alternative pathway if a common component in the translocation machinery is required. One solution to this problem is to measure preprotein translocation in vttro in which the import of a single substrate is assayed. Although it is clear that yeast survive in the absence of SRP, only a few preproteins translocate post-translationally into yeast microsomes [80,106,107]. This result implies either that yeast SRP is not absolutely required for co-translational translocation or that the in vitro assay might be lacking components for the efficient post-translational translocation of some substrates. Although a functional assay for purified yeast SRP has not yet been developed, yeast cytosol depleted of SRP is unable to co-translationally translocate a preprotein in vitro [20].

ER membrane translocation machinery Yeast translocation complex To isolate temperature-sensitive mutations in yeast that are defective for preprotein translocation into the ER, Schekman and colleagues [66,81] initiated a selection that led to the identification of the membrane components of the translocation complex. The genes corresponding to these components were designated SEC61,SEC62 and SEC63, each of which is essential [66,81,108,109], Variations on the selection scheme either have yielded one of these genes [20,75,110] or have isolated new components of the translocation machinery [75,111]. Multicopy suppressor analyses [112,113] and biochemical methods [18,114,115] were also used to identify novel members of the yeast translocation complex. Chemical cross-linking and purification of the translocation complex from detergent-solubilized extracts indicated that many of these components associate physically with one another [19,114,116,117], and specific pairs of the temperature-sensitive mutants were synthetically lethal [81,112], a phenomenon in which the growth of a haploid cell

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containing two temperature-sensitive mutations in combination is more restrictive than in cells containing either one of the two mutations. In other cases, synthetic lethal interactions indicated that the respective gene products associate physically (reviewed in [82]). Overall, preprotein translocation in yeast requires a variety of integral membrane and membrane-associated factors that together comprise the translocation machine (Figure 3). This was demonstrated most conclusively by Panzner et al. [19], who purified the complete Sec protein complex in toto from detergent-solubilized yeast microsomes. In the initial attempt to identify which S E C gene product encodes the translocation pore, Musch et al. [68] and Sanders et al. [69] incubated microsomes with preproteins that were only partially translocated and were thus assumed to be stuck in the pore. After cross-linker was added, the membranes were solubilized and specific Sec proteins were immunoprecipitated to determine which factor associated with the arrested preprotein. Both groups discovered that Sec61p is the translocation pore. This result was somewhat anticipated, as Sec61p contains ten membranespanning segments [118] and thus has the capacity to form an aqueous pore in the ER membrane. Homologues of yeast Sec61p also function as translocation pores in the mammalian ER and in the bacterial plasma membrane (reviewed in [14,119]), and two small proteins associated with Sec61p (Sbhlp and Ssslp), whose functions are unknown, are conserved from yeast to mammals [19,120].

Figure 3

Members of the yeast translocation complex Cytosol Sec63p Sec71p

Sec61p

DnaJ BiP/Kar2p Lumen

The region ofSec63p homologous to DnaJ is highlighted.The cytosolic chaperones Ssalp andYdjlp, although required for the translocation of some precursors, have not been isolated as components of the complex, and are therefore not shown. See the text for details.

Endoplasmic reticulum targeting and translocation

It was also found that, in the absence of ATP (conditions under which preproteins bind to the ER membrane but do not translocate [121]), the preprotein cross-linked to Sec62p, suggesting that Sec62p may be a signal peptide binding protein [68]. Sec62p traverses the membrane twice, with both the N- and C-termini in the cytosol [122]. Because pairwise temperature-sensitive mutations in SEC61, SEC62 and SEC63 are synthetically lethal [81], it was anticipated that Sec61p, Sec62p and Sec63p might form a complex in the ER membrane. This hypothesis was confirmed by Deshaies et al. [114] by chemical cross-linking of detergent-solubilized microsomes. The predicted amino acid sequence and topology of Sec63p indicated that a lumenal domain of the protein shows marked identity with the DnaJ molecular chaperone from E. coli [109,123], As DnaJ forms a functional complex with DnaK (see above), it was expected that a DnaK homologue in the yeast ER might interact with Sec63p. A candidate for this interacting factor is BiP ('heavy chain binding protein'). BiP, a lumenal DnaK homologue, was first discovered because it binds to incompletely folded proteins in the mammalian ER [124]. A yeast homologue of BiP, Kar2p, was later identified by two groups [125,126]; it is -50% identical to DnaK and ~65% identical to the cytosolic Hsp70 in yeast, Ssalp (see above). A role for BiP during protein translocation in yeast was first demonstrated by Vogel et al. [127], who showed that strains containing temperature-sensitive mutations in KAR2, the gene encoding yeast BiP, accumulate preproteins in the cytosol at the non-permissive temperature. Cells depleted of BiP also accumulated preproteins [127,128]. Finally, microsomes prepared from strains containing these temperaturesensitive KAR2 mutations display temperature-sensitive co- and post-translational translocation defects in vitro [69,107], To demonstrate that Sec63p and BiP form a functional DnaK-DnaJ pair in the yeast ER, an active Sec63p-BiP complex was purified from octyl glucosidesolubilized microsomes that restored protein translocation activity to proteoliposomes prepared from a strain containing the sec63-l mutation [116]. Scidmore et al. [129] obtained genetic confirmation that Sec63p and BiP interact. Because the Sec63p-BiP complex was labile if purified in the presence of adenosine 5'-[γthio]triphosphate [116], and because Ydj lp, Ssalp and ATP are necessary for posttranslational protein translocation (see above), ATP-dependent DnaK-DnaJ pairs exist on both sides of the ER membrane. The Sec63p-BiP complex also contains two additional factors [116] that were observed previously as cross-linked members of the translocation complex containing Sec61p, Sec62p and Sec63p [114]. Disruptions in the genes encoding these proteins, known originally as SEC66 and SEC67, but now called SEC71 and SEC72 [111], indicated that they were not essential for growth [18,112,115,130]. Nonetheless, sec71 and sec72 strains accumulate some preproteins in the cytoplasm. Because the differing sensitivities of preproteins to the sec72 mutation lie in their

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signal peptides [18], it was suggested that Sec71p and Sec72p might comprise the signal peptide recognition complex. To define better how the components in the translocation machinery act, the protein translocation reaction has been reconstituted in vesicles prepared from detergent-solubilized yeast microsomes [19,90]. The isolation of subcomplexes of the translocation machinery [19,116], in conjunction with assays that measure partial reactions during translocation, will certainly facilitate the future dissection of this ornate protein complex.

Translocation machinery in the mammalian ER Co-translationally translocated preproteins interact with many ER components during their import into the mammalian ER. While most of these factors were identified through cross-linking studies, others were isolated using biochemical complementation assays (reviewed in [14]). Studies using reconstituted vesicles obtained from detergent-solubilized mammalian microsomes have served to elucidate which of these molecules are unnecessary, required or stimulatory for protein translocation in vitro [131-134], SRP receptor The SRP receptor is a heterodimeric protein complex consisting of a membraneassociated 69 kDa α-subunit (SRa) and an integral membrane 30 kDa β-subunit (SRP) [135]. To demonstrate that SRP and its receptor interact physically, Gilmore and Blobel [136] purified SRot from detergent-solubilized membrane extracts using an SRP affinity column. Isolation of the genes encoding SRa [51,137] and SR3 [138] revealed that they both contain consensus sites for GTP binding. The purified SRP receptor subunits bind GTP in vitro [138]. Mutations engineered into the GTPbinding site of mammalian SRa either impair or completely inactivate protein translocation and GTP binding [139]. Why SRP and both subunits of its receptor bind GTP is unknown, but it has been demonstrated that the bacterial SRP and SRP receptor homologues are able to reciprocally activate each other's GTPase activities [140], The GTP/SRP/SRP receptor cycle might increase the fidelity of translocation by introducing a timed proof-reading step, similar to the role played by elongation factors and GTP during protein translation [141]. Translocon-associated protein (TRAP) After the release of the signal peptide from the SRP, the preprotein interacts with the ER-membrane-associated translocation machinery. Initial attempts to identify a signal-peptide-binding protein in the ER membrane uncovered a glycoprotein with a molecular mass of 34 kDa that was named the signal sequence receptor (SSR). Although antibodies prepared against purified SSR inhibited protein translocation in an in vitro assay [142], Migliaccio et al. [133] later discovered that reconstituted vesicles lacking the SSR were translocation-competent, and it was found that both signal peptides and mature regions of preproteins cross-linked to the SSR [65,143].

Endoplasmic reticulum targeting and translocation

The SSR is now known to be one subunit of a tetrameric complex that has been renamed TRAP [144], While the function of TRAP during protein translo­ cation is unknown, its proximity to preproteins suggests that it might play a role in the import of only specific proteins, that it might chaperone the insertion of membrane proteins into the lipid bilayer [144], or that it might be required for the retention of ER membrane proteins [145]. Sec61 ρ complex Recent evidence indicates that the signal sequence of a preprotein interacts with Sec61p, and not SSR/TRAP, after it is released from the SRP (reviewed in [14]). As discussed above, Sec61p is the translocation pore that was identified initially from genetic selection in yeast [66,75], functionally characterized using translocationarrested precursor proteins [68,69] and later observed in the mammalian ER as a ribosome-associated membrane protein [65], The mammalian Sec61 protein (also called Sec61a) co-purifics with two additional proteins known as Sec6ip (homologous to the yeast Sbhl protein) and Sec6l7 (homologous to the yeast Sssl protein) [120,134]. Reconstituted vesicles containing only the mammalian Sec61p complex and the SRP receptor are able to co-translationally translocate some preproteins [134], emphasizing the central role of Sec61p in the translocation reaction. This observation led Jungnickel and Rapoport [21] to demonstrate that Sec61p determines whether a bona fide signal peptide has been presented. Thus Sec61p binds to ribosomes, proof-reads signal sequences and opens to allow the preprotein entry into the lumen of the ER. Because Sec61p plays an essential role at many stages of protein import, it was expected that preproteins would be found associated with it at all times during their translocation into the ER. Mothes et al. [146] confirmed that Sec61p contacts a soluble preprotein throughout its transit into the ER, although Nicchitta et al. [147] observed that the efficiency of cross-linking to Sec61p at later stages during protein translocation was reduced, a result interpreted as an indication that 'topological alterations' occur between Sec61p and the nascent chain. Such alterations may indicate that hydrophobic portions of the preprotein are diffusing laterally from Sec61p and into the lipid bilayer [148]. It is also clear that Sec61p does not interact equally with each portion of the signal sequence. The N-terminal hydrophilic region of the signal does not contact Sec61p, whereas the central hydrophobic domain and the C-terminal hydrophilic domains do associate with Sec61p [146,149]. The initial events during the co-translational translocation of a preprotein are as follows. The interaction of the nascent-chain-ribosome complex with the Sec61ρ complex triggers the oligomerization of Sec61ρ into a trimer or tetramer [149a]. The signal peptide resides initially in an aqueous environment, occluded within Sec61p [146,150]. After SRP is displaced and translation resumes, the affinity of the ribosome for Sec61p increases [21,151]. When the nascent chain has grown to approx. 70 amino acids, a gate opens in the translocation channel, allowing the

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preprotein to enter the lumen of the ER [150], and the signal peptide is cleaved by the lumenal enzyme signal peptidase. Longer segments of the nascent chain extending into the lumen continue to contact Sec61p [146], as lumenal proteins in the ER begin to associate with the elongating nascent chain (reviewed in [83]). During translocation, the tight association between the ribosome and Sec61p may loosen if 'pause transfer sequences' are encountered [151a], indicating that the translocon is quite dynamic. The translation of nascent chains containing transmembrane-spanning segments of integral membrane proteins presents a special problem, as these domains must be transferred from Sec61p or from a Sec61 p/lipid interface, in which they initially reside [148,152,153], to the lipid bilayer. It is unknown whether proteins translocate through individual Sec61p molecules or through a central pore formed at the junction of Sec61p monomers. If integral membrane-spanning domains translocate through individual Sec61p monomers, then each Sec61p molecule must open to allow the proteins to pass laterally into the ER membrane. If, however, the pore is formed at the interface of the Sec61p complex monomers, then the translocating membrane protein might slip between the monomers. Recent experiments have indicated that the membrane-spanning domains of polytopic membrane proteins can insert either sequentially into the lipid bilayer [153a] or en masse [153b] after their synthesis. Because fluorescence quenching experiments have determined that the diameter of the active translocation channel is between 40 and 60 A (4—6 nm) [153c], the pore is likely to be formed at the interface of multiple Sec61p complexes, and could certainly accommodate repeated membrane-spanning domains before their insertion into the lipid bilayer. Translocating-chain-associating protein (TRAM) In addition to Sec61p, a second mammalian ER membrane protein cross-links to translocating preproteins and is required for the translocation of some proteins. This protein, known as TRAM, is a 36 kDa polytopic glycoprotein that was purified based on its ability, when reconstituted into phospholipid vesicles, to cross­ link to the N-terminus of a preprotein [154]. Reconstituted vesicles depleted of TRAM were defective in the translocation of many, but not all, preproteins examined [154,155]. While TRAM-dependent translocation was dictated by a preprotein's signal sequence, no single characteristic of this sequence was found to be critical forTRAM-dependence [155]. However, it was observed that TRAM is not required to discriminate between functional and aberrant signal sequences, indicating that TRAM is required after the initial ER targeting event [21]. To define further the point at which TRAM acts during translocation, a protease protection assay was used by Voigt et al. [155] to indicate at which step preproteins become shielded within the translocation complex. It was concluded that TRAM is required once the preprotein has been received at the ER membrane, but before it becomes inserted into Sec61p. A critical event during this window of time is the insertion of the signal peptide as a loop, an event that precedes the import

Endoplasmic reticulum targeting and translocation

of the mature region of a preprotein. Because the mechanism that drives this process remains unknown, Mothes et al. [146] suggested that TRAM might serve to orient the signal sequence. This hypothesis is supported by the observation that TRAM associates with only the N-terminus of the signal peptide [146,149]. A putative role for TRAM during membrane protein biogenesis was elegantly detailed in a study by Do et al. [153], in which a photoreactive probe was engineered into the middle of the transmembrane domain of a preprotein. It was determined that the transmembrane domain interacted differentially with TRAM during translocation, suggesting that the complex between TRAM and the membrane-spanning segment is dynamic. One model is that TRAM associates with hydrophobic translocating segments and facilitates their insertion into the lipid bilayer, perhaps by altering the structure of Sec61p. This scenario is consistent with the observation that TRAM might play a role in signal peptide topogenesis (reviewed in [156]).

Lumenal components required for protein translocation Preproteins can translocate co-translationally into reconstituted vesicles devoid of lumenal contents [134]. The simplest interpretation of this result is that the energy required for co-translational translocation is derived exclusively from the ribosome 'pushing' or feeding the preprotein through Sec61p and into the lumen. By comparison, during post-translational translocation, translation is uncoupled from translocation and another force must exist to drive protein import. As a result of work on the mechanisms of post-translational protein translocation into the yeast ER and mitochondria, the picture that has emerged is that the preprotein is actually driven into the organelle by the action of Hsp70s (reviewed in [157]). Three models have been proposed to explain how the lumenal Hsp70 BiP might drive post-translational protein import into the ER lumen. The first model depicts BiP as a reversible 'glue' that prevents retrograde transport of the preprotein from the ER to the cytosol [158]. Such a role for lumenal Hsp70s was suggested because the translocation channel in the mammalian ER appears to be passive once opened [159,160]. Thus a polypeptide chain might slide bi-directionally across the membrane unless a lumenal factor locks on to the preprotein and prevents retrograde transport. Because Brownian motion might effect this bi-directional movement, the model has been termed the Brownian Ratchet [158]. Evidence supporting the Brownian Ratchet model has been obtained from studies on the mechanism of mitochondrial translocation [161]. The second model depicts the Hsp70 as a motor, grabbing and pulling the importing chain upon successive rounds of ATP binding and hydrolysis [162]. This model sprung from the observation that, during preprotcin translocation into isolated mitochondria (Chapter 13), cytosolic domains of proteins may unfold (reviewed in [162]), an event unlikely to occur spontaneously. Thus a folded domain

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may be unravelled as it is pulled through a channel and into the organelle. Inherent in this 'Motor Model' for Hsp70 function is that, to generate force, the Hsp70 must be anchored to an immobile substrate, such as a membrane protein. Indeed, Sec63p serves this role for BiP [116,129,163]. Definitive proof for the Motor Model awaits an in vitro system in which Hsp70-generated force may be observed. A final model to explain the action of BiP during post-translational protein translocation into the yeast ER envisions that BiP responds to the binding of a preprotein on the cvtosolic face of the ER and then conformational^ activates the translocation complex [157]. In this scenario, preprotein binding to its receptor, potentially composed of the Sec62, Sec71 and Sec72 proteins [18,68], activates the neighbouring Sec63 protein to stimulate ADP/ATP exchange on BiP, releasing BiP from the membrane [116]. Because it has now been shown that mammalian BiP gates Sec61p [162a]; freeing BiP from the translocation complex opens this gate and permits entry of a preprotein into the lumen. Although Scjlp, a soluble ER lumenal DnaJ homologue that interacts with BiP, has been proposed to catalyse ATP hydrolysis so that BiP may re-associate with Sec63p [164], Sec63p has recently been shown to stimulate the ATPase activity of BiP [165]. One prediction from this last model is that mutations in BiP might affect events on the cytosolic face of the ER during translocation. Three pieces of evidence support this hypothesis. First, in the absence of ATP, a preprotein does not interact with the translocation channel and remains associated with the receptor complex [68,163]. Because BiP is the only known ATP-requiring protein in the translocation complex, it must be required to facilitate the transfer of the preprotein to the channel. Secondly, Sanders et al. [69] characterized mutations in the gene encoding BiP, KAR2, that prevented the association of a preprotein with Sec61p, indicating that BiP must be required for early events during translocation that occur on the opposite face of the ER membrane. Thirdly, mutations in BiP prevented the import of both post- and co-translationally translocated substrates into ER-derived microsomes [107], As the energy required to drive co-translational translocation might derive from the ribosome (see above), BiP could be necessary to activate the translocation complex, a process that would be necessary for both co- and posttranslationallv translocated preproteins. In accordance with this view, the generation of translocation assays in solution indicates that BiP is necessary to both transfer and drive preproteins from a recognition complex to the channel, and through the channel into the lumen [166,167], Although Gorlich and Rapoport [134] reported that co-translational translocation into proteoliposomes containing pure mammalian proteins occurred in the absence of lumenal factors, another view holds that translocation into the mammalian ER is indeed stimulated by lumenal chaperones, but that the translo­ cation complex in this system was already in an 'activated', translocation-competent state. In addition, the preproteins examined in such defined systems may not represent the full range of translocating substrates encountered by the ER in vivo. In support of these later possibilities, Nicchitta and Blobel [160] showed that

Endoplasmic reticulum targeting and translocation

mammalian microsomes emptied of their lumenal contents were translocationdeficient, but that re-addition of 'reticuloplasm' restored preprotein translocation. Because these investigators demonstrated further that reticuloplasm-free vesicles could bind preproteins and cleave their signal sequences, but could not fully import the preprotein into the lumen, the role of lumenal factors must be to complete translocation [160,163]. Although BiP was a major component in the reticuloplasm, definitive proof that BiP is necessary and sufficient for translocation in this system is lacking.

Concluding remarks Protein transport across biological membranes is required for protein secretion, targeting and localization, and is essential. As described in this review, the membrane of the ER contains an elaborate machinery that facilitates this process. Cytosolic and lumenal proteins may also be required during translocation. The isolation and characterization of the components involved in translocation has utilized techniques ranging from genetic analyses to biophysical methods, and studies in this field have uncovered information on the function of molecular chaperones, GTP-binding proteins, proteinaceous pores and multiprotein complexes. Undoubtedly, the continued characterization of protein translocation will not only lead to a molecular mechanism for this process, but will also be likely to elucidate the roles played by a diverse spectrum of protein families.

Comments on the manuscript from Karin Romisch and Chris Niechitta were deeply appreciated. Work in the author's laboratory was funded by grant MCB-9506002 from the National Science Foundation.

References 1. 2. 3. 4. 5. 6.

von Heijne 1 G. and Blomberg, C. (1979) Eur. J. Biochem. 97, 175-181 Wickner, W. (1979) Annu. Rev. Biochem. 48, Tb-^b Inouye, M. and Halegoua, S. (1980) CRC Crit. Rev. Biochem. 7,339-371 Engleman, D.M. and Steitz, T.A. (1981) Cell 23,4X1—422 Blobel, G. and Dobberstein, B. (1975) J. Cell Biol. 67, 835-851 Walter, P. and Johnson, A.E. (1994) Annu. Rev. Cell Biol. 10, 87-119

7. 8. 9.

Gierasch, L.M. (1989) Biochemistn r 28,923-930 Kuhn 1 A. (1987) Science 238, 1413-1415 Shaw, A.S., Rottier, P.J.M. and Rose,J.K. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7592-7596

10. 11. 12.

von Heijne, G. (1990) J. Membr. Biol. 115,195-201 Izard^J.W. and Kendall, D-A. (1994) Mol. Microbiol. 13, 765-773 Evans, E.A., Gilmorc, R. and Blobel, G. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 581-585

13. 14. 15.

Gilmore, R. (1991) Curr. Opin. Cell Biol. 3, 580-584 Rapoport, T.A., Jungnickel, B. and Kutay, U. (1996) Annu. Rev. Biochem. 65,271-304 Walter, P. and Lingappa, V.R. (1986) Annu. Rev. Cell Biol. 2,499-516

16. 17. 18.

Gilmore, R. and Blobel, G. ¢1985) Cell 42,497-505 Crowley, K.S., Reinhart, G.D. and Johnson, A.E. (1993) Cell 73, 1101-1116 Feldheim, D. and Schekman, R. (1994) J. Cell Biol. 126,935-944

187

J.L Brodsky

188

19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.

Panzner, S., Dreier, L., Hartmann, E., Kostka, S. and Rapoport, T.A. (1995) Cell 81,561-570 Ng, D.T.W., Brown, J.D. and Walter, P. (1996) J. Cell Biol. 134,269-278 Jungnickel, B. and Rapoport, T.A. (1995) Cell 82,261-270 Simon, S.M. and Blobel, G. (1992) Cell 69, 677-684 Walter, P. and Blobel, G. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 7112-7116 Walter, P. and Blobel, G. (1982) Nature (London) 299,691-698 Walter, P., Ibrahimi, I. and Blobel, G. (1981) J. Cell Biol. 91,545-550 Walter, P. and Blobel, G. (1981) J. Cell Biol. 91,551-556 Walter, P. and Blobel, G. (1981) J. Cell Biol. 91, 557-561 Gilmore, R. and Blobel, G. (1983) Cell 35,677-685 Walter, P. and Blobel, G. (1983) Cell 34, 525-533 Siegel, V. and Walter, P. (1985) J. Cell Biol. 100,1913-1921 Lingelbach, K., Zwieb, C., Webb, J.R., Marshallsay, C., Hoben, P.J., Walter, P. and Dobberstein, B. (1988) Nucleic Acids Res. 16,9431-9432 32. Bernstein, H.D., Poritz, M.A., Strub, K., Hoben, P.J., Brenner, S. and Walter, P. (1989) Nature (London) 340,482—+86 33. Romisch, K., Webb 1 J., Herz,]., Prehn, S., Frank, R., Vingron, M. and Dobberstein, B. (1989) Nature (London) 340,478—482 34. Strub, K. and Walter, P. (1990) Mol. Cell. Biol. 10, 777-784 35. Herz, J., Flint, N., Stanley, K., Frank, R. and Dobberstein, B. (1990) FEBS Lett. 276, 103-107 36. Romisch, K., Webb, J., Lingelbach, K., Gausepohl, H. and Dobberstein, B. (1990) J. Cell Biol. 111,1793-1802 37. Lutcke, H., Prehn, S., Ashford, A.J., Remus, M., Franke, R. and Dobberstein, B. (1993)J. Cell Biol. 121,977-985 38. Hauser, S., Bacher, G., Dobberstein, B. and Lutcke, H. (1995) EMBO J. 14,5485-5493 39. Siegel, V. and Walter, P. (1988) Cell 52,39^t9 40. Zopf, D., Bernstein, H.D., Johnson, A.E. and Walter, P. (1990) EMBO J. 9, 4511—4517 41. High, S. and Dobberstein, B. (1991) J. Cell Biol. 113,229-233 42. Liitcke, H., High, S., Romisch, K., Ashford, A. and Dobberstein, B. (1992) EMBO J. 11, 1543-1551 43. Zopf, D., Bernstein, H.D. and Walter, P. (1993) J. Cell Biol. 120,1113-1121 44. Siegel, V. and Walter, P. (1986) Nature (London) 320, 81-84 45. Strub, K., Moss,J. and Walter, P. (1991) Mol. Cell. Biol. 11,3949-3959 46. Siegel, V. and Walter, P. (1988) Proc. Natl. Acad. Sci. U.S.A. 85,1801-1805 47. Siegel, V. and Walter, P. (1988) Trends Biochem. Sci. 13, 314—316 48. Krieg, U.C., Walter, P. and Johnson, A.E. (1986) Proc. Natl. Acad. Sci. U.S.A. 83,8604—8608 49. Kurzchalia, T.V., Wiedmann, M., Girshovich, A.S., Bochkareva, E.S., Bielka, H. and Rapoport, T.A. (1986) Nature (London) 320,634-636 50. Bacher, G., Liitcke, H., Jungnickel, B., Rapoport, T.A. and Dobberstein, B. (1996) Nature (London) 381,248-251 51. Connolly, T. and Gilmore, R. (1989) Cell 42,497-505 52. Miller, J.D., Wilhelm, H., Gierasch, L., Gilmore, R. and Walter, P. (1993) Nature (London) 366, 351-354 53. Connolly, T., Rapiejko, P.J. and Gilmore, R. (1991) Science 252,1171-1173 53a. Rapiejko, P.J. and Gilmore, R. (1997) Cell 89, 703-713 53b. Millman, J.S. and Andrews, D.W. (1997) Cell 89, 673-676 54. Wiedmann, B., Sakai, H., Davis, T.A. and Wiedmann, M. (1994) Nature (London) 370, 434-440 55. Lauring, B., Sikai, H., Kriebich, G. and Wiedmann, M. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 5411-5415 56. Lauring, B., Kriebich, G. and Wiedmann, M. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 9435-9439 56a. Raden, D. and Gilmore, R. (1998) Mol. Biol. Cell 8,117-130 57. Borgese, N., Mok, W., Kreibich, G. and Sabatini, D.D. (1974) J. Mol. Biol. 88, 559-580 58. Savitz, A.J. and Meyer, D.I. (1990) Nature (London) 346,540—544 59. Savitz, A.J. and Meyer, D.I. (1993) J. Cell Biol. 120, 853-863 60. Wanker, E.E., Sun, Y., Savitz, A. and Meyer, D.I. (1995) J. Cell Biol. 130,29-39

Endoplasmic reticulum targeting and translocation

61.

N u n n a r i , J . , Z i m m e r m a n , D . L . , O g g , S . C . and Walter, P. (1991) N a t u r e ( L o n d o n ) 3 5 2 , 6 3 8 - 6 4 0

62. 63.

Collins, P.G. and G i l m o r e , R. ( 1 9 9 1 ) J . Cell Biol. 1 1 4 , 6 3 9 - 6 4 9 T a z a w a , S . , U n u m a , M . , T o n d o k o r o , N . , A s a n o , Y., O h s u m i , T., I c h i m u r a , T. and S u g a n o , H . (1991) J . Biochem. ( T o k y o ) 109, 89-98 I c h i m u r a , T., O h s u m i , T., S h i n d o , Y., O h w a d a , T., Y a g a m e , H . , M o m o s e , Y., O m a t a , S. and Sugano, H . (1992) F E B S Lett. 2 9 6 , 7 - 1 0

64. 65. 66. 67.

Gorlich, D , Prehn, S., H a r t m a n n , E., Kalies, K . U . and R a p o p o r t , T.A. (1992) Cell 71, 489-503 Deshaies, R.J. and Schekman, R. (1987) J. Cell Biol. 1 0 5 , 6 3 3 - 6 4 5 Stirling, C . J . , R o t h b l a t t , J . , H o s o b u c h i , M . , D e s h a i e s , R . and S c h e k m a n , R . (1992) M o l . Biol. Cell 3 , 1 2 9 - 1 4 2

68. 69.

Miisch, A., Wiedmann, M . and R a p o p o r t , T.A. (1992) Cell 69, 3 4 3 - 3 5 2 S a n d e r s , S . L . , W h i t f i e l d , K . M . , Vogel, J.P., R o s e , M . D . a n d S c h e k m a n , R.W. (1992) Cell 69, 353-365

70. 71.

Kalies, K., Gorlich, D . and R a p o p o r t , T.A. (1994) J. Cell Biol. 126, 925-934 Wiech, H . and Z i m m e r m a n n , R . (1993) in P r o t e i n F o l d i n g and R e c o v e r y ( C l e l a n d , J . L . and K i n g , J., eds.), pp. 84-101, American Chemical Society B o o k s D e p a r t m e n t , Washington, D C

72.

Felici, F., Cesareni, G . and H u g h e s , J . M . X . (1989) M o l . Cell. Biol. 9, 3260-3268

73. 74. 75.

A m a y a , Y. and N a k a n o , A . (1991) F E B S Lett. 283, 325-328 H a n n , B . C . and Walter, P. (1991) Cell 6 7 , 1 3 1 - 1 4 3 Stirling, C . J . and Hewitt, E.W. (1992) N a t u r e ( L o n d o n ) 356, 5 3 4 - 5 3 7

76.

B r o w n , J . D . , H a n n , B . C . , M e d z i h r a d s z k y , K.F., N i w a , M . , B u r l i n g a m e , A . L . and Walter, P. (1994) E M B O J . 1 3 , 4 3 9 0 - 4 4 0 0 H a n s e n , W., Garcia, P.D. and Walter, P. (1986) Cell 45, 3 9 7 ^ 0 6

77. 78. 79. 80. 81. 82.

Rothblatt, J . A . and Meyer, D . I . (1986) Cell 44, 6 1 9 - 6 2 8 Waters, M . G . and Blobel, G . (1986) J. Cell Biol. 102, 1543-1550 H a n s e n , W. and Walter, P. (1988) J . Cell Biol. 106, 1075-1081 Rothblatt, J . A . , Deshaies, R.J., Sanders, S „ D a u m , G . and Schekman, R. (1989) J . Cell Biol. 109, 2641-265 2 B r o d s k y , J . L . and S c h e k m a n , R. (1994) in T h e B i o l o g y of H e a t S h o c k Proteins and M o l e c u l a r C h a p e r o n e s ( M o r i m o t o , R.I., Tessieres, A . and G e o r g o p o u l o s , C . , eds.), p p . 8 5 - 1 1 0 , C o l d Spring H a r b o r L a b o r a t o r y Press, C o l d Spring H a r b o r , N Y

83. 84.

Gething, M.J. and S a m b r o o k , J . (1992) N a t u r e ( L o n d o n ) 3 5 5 , 3 3 ^ 1 5 Hartl, F.U. (1996) N a t u r e ( L o n d o n ) 381, 571-580

85. 86.

Pelham, H . R . B . (1986) Cell 46, 959-961 G e o r g o p o u l o s , C . , L i b e r e k , K . , Zylicz, M . and A n g , D . (1994) in T h e B i o l o g y of H e a t S h o c k P r o t e i n s a n d M o l e c u l a r C h a p e r o n e s ( M o r i m o t o , R . I . , Tessieres, A . a n d G e o r g o p o u l o s , C . , eds.), pp. 2 0 9 - 2 4 9 , C o l d Spring H a r b o r L a b o r a t o r y Press, C o l d Spring Harbor, N Y Wild, J., A l t m a n , E., Yura, T. and G r o s s , C . A . (1992) G e n e s Dev. 6 , 1 1 6 5 - 1 1 7 2 D e s h a i e s , R . J . , K o c h , B . D . , Werner-Washburne, M . , C r a i g , E . A . and S c h e k m a n , R . (1988) N a t u r e ( L o n d o n ) 332, 800-805

87. 88. 89. 90. 91. 92.

Chirico, W.J., Waters, M . G . and Blobel, G . (1988) N a t u r e ( L o n d o n ) 3 3 2 , 8 0 5 - 8 1 0 B r o d s k y , J . L . , H a m a m o t o , S . , Feldheim, D . and Schekman, R. (1993) J. Cell Biol. 120, 95-102 Waters, M . G . , Chirico, W.J. and Blobel, G . (1986) J . Cell Biol. 1 0 3 , 2 6 2 9 - 2 6 3 6 Z i m m e r m a n n , R . , Sagstetter, M „ L e w i s , M . J . and Pelham, H . R . B . (1988) E M B O J . 7, 2875-2880

93.

W i e c h , H . , B u c h n e r , J . , Z i m m e r m a n n , M., Z i m m e r m a n n , R. and J a k o b , U . (1993) J . Biol. C h e m . 268, 7414-7421 L i b e r e k , K., Marszalek, J . , A n g , D . , G e o r g o p o u l o s , C . and Zylicz, M . (1991) Proc. N a t l . Acad. Sci. U . S . A . 8 8 , 2 8 7 4 - 2 8 7 8

94. 95. 96. 97.

C a p l a n , A J . and D o u g l a s , M . G . (1991) J. Cell Biol. 1 1 4 , 6 0 9 - 6 2 2 Attencio, D.P. and Yaffe, M.P. (1992) Mol. Cell. Biol. 12,283-291 C y r , D . M . , L u , X . and D o u g l a s , M . G . (1992) J. Biol. C h e m . 267,20927-20931

98. 99.

C h i r i c o , W. (1992) B i o c h e m . B i o p h y s . Res. C o m m u n . 189,1150-1156 C a p l a n , A.J., Cyr, D . M . and D o u g l a s , M . G . (1992) Cell 7 1 , 1 1 4 3 - 1 1 5 5

100. C a p l a n , A.J.,Tsai, J., Casey, P.J. and D o u g l a s , M . G . (1992) J . Biol. C h e m . 267, 18890-18895 101. Becker, J . , Walter, W„ Yan, W. and Craig, E . A . (1996) Mol. Cell. Biol. 16,4378^4386 102. O g g , S.C., Poritz, M . A . and Walter, P. (1992) Mol. Biol. Cell 3, 895-911

189

190

J . L Brodsky

103. 104. 105. 106. 107.

O g g , S . C . and Walter, P. (1995) Cell 81, 1075-1084 Arnold, C . E . and Wittrup, K . D . (1994) J. Biol. C h e m . 269, 30412-30418 H a n n , B . C . , Stirling, C . and Walter, P. (1992) N a t u r e ( L o n d o n ) 3 5 6 , 5 3 2 - 5 3 3 Rothblatt, J. A., Webb, J . R . , Ammerer, G . and Meyer, D.I. (1987) E M B O J. 6 , 3 4 5 5 - 3 4 6 3 B r o d s k y , J . L „ G o e c k e l e r , J . a n d S c h e k m a n , R . ( 1 9 9 5 ) P r o c . N a t l . A c a d . Sci. U . S . A . 92, 9643-9646

10S. Deshaies, R.J. and Schekman, R. (1989) J. Cell Biol. 1 0 9 , 2 6 5 3 - 2 6 6 4 109. Sadler, I., C h i a n g , A., K u n h a r a , T „ R o t h b l a t t , J . , Way, J . and Silver, P. (1989) J . Cell Biol. 109, 2665-2675 110. T o y n . J . , H i b b s , A.R., Sanz, P., C r o w e , J . and Meyer, D.I. (1988) E M B O J. 7 , 4 3 4 7 - 1 3 5 3 111. Green, N „ Fang, H. and Walter, P. (1992) J. Cell Biol. 116, 597-6C4 112. Kurihara, T. and Silver, P. (1993) M o l . Biol. Cell 4 , 9 1 9 - 9 3 0 113. E s n a u l t , Y „ B l o n d e l , M „ D e s a h i e s , R . J . , S c h e k m a n , R. and K e p e s , F. ( 1 9 9 3 ) E M B O J. 12, 4083-4093 114. D e s h a i e s , R . J . , S a n d e r s , S „ F e l d h e i m , D . A . and S c h e k m a n , R. (1991) N a t u r e ( L o n d o n ) 349, 806-80S 115. Feldheim, D . and Schekman, R. (1993) M o l . Biol. Cell 4, 9 3 1 - 9 3 9 116. B r o d s k y , J . L . and Schekman, R. (1993) J . Cell Biol. 1 2 3 , 1 3 5 5 - 1 3 6 3 117. Esnault, Y., F e l d h e i m , D „ B l o n d e l , M., S c h e k m a n , R. and K e p e s , F. (1994) J. Biol. C h e m . 269, 27478-27485 118. Wilkinson, B.M., Critchlev, A.J. and Stirling, C . J . (1996) J. Biol. C h e m . 2 7 1 , 2 5 5 9 0 - 2 5 5 9 7 119. R a p o p o r t , T.A. (1992) Science 2 5 8 , 9 3 1 - 9 3 6 120. H a r t m a n n , E., S o m m e r , T., P r e h n , S., G o r l i c h , D . , J e n t s c h , S. and R a p o p o r t , T . A . (1994) N a t u r e ( L o n d o n ) 367, 6 5 4 - 6 5 7 121. Sanz, P. and Meyer, D.I. (1989) J . Cell Biol. 1 0 8 , 2 1 0 1 - 2 1 0 6 122. Deshaies, R.J. and Schekman, R. (1990) M o l . Cell. Biol. 10, 6024-6035 123. Feldheim, D „ Rothblatt, J. and Schekman, R. (1992) M o l . Cell. Biol. 12, 3288-3296 124. Haas, I.G. and Wabl, M . (1983) N a t u r e ( L o n d o n ) 3 0 6 , 3 8 7 - 3 8 9 125. N o r m i n g t o n , K., K o h n o , K., K o z u t s u m i , Y., G e t h i n g , M . J . and S a m b r o o k , J . (1989) Cell 57, 1223-1236 126. R o s e . M . D . , M i s r a . L . M . and Vogel,J.P. (1989) Cell 57, 1211-1221 127. Vogel,J.P., Misra, L . M . and R o s e , M . D . (1990) J . Cell Biol. 1 1 0 , 1 8 8 5 - 1 8 9 5 128. N g u v e n , T . H . , Law, D . T . S . and Williams, D . B . ( 1 9 9 1 ) P r o c . N a t l . A c a d . Sci. U . S . A . 88, 1565-1569 129. Scidmore, M „ O k a m u r a , H . H . and Rose, M . D . (1993) M o l . Biol. Cell 4, 1145-1159 130. Fang, H . and Green, N . (1994) M o l . Biol. Cell 5, 9 3 3 - 9 4 2 131. Nicchitta, C.V. and Blobel, G . (1990) Cell 60, 2 5 9 - 2 6 9 132. Nicchitta, C.V., Migliaccio, G . and Blobel, G . (1991) Cell 65, 587-598 133. Migliaccio, G „ Nicchitta, C.V. and Blobel, G . (1992) J. Cell Biol. 1 1 7 , 1 5 - 2 5 134. Gorlich, D . and R a p o p o r t , T.A. (1993) Cell 75, 6 1 5 - 6 3 0 135. Tajima, S „ Lauffer, L., Rath, V.L. and Walter, P. (1986) J . Cell Biol. 103, 1167-1178 136. Gilmore, R. and Blobel, G . (1982) J. Cell Biol. 9 5 , 4 7 0 - 4 7 7 137. Lauffer, L., G a r c i a , P.D., H a r k i n s , R . N . , C o u s s e n s , L „ Ullrich, A . and Walter, P. (1985) N a t u r e ( L o n d o n ) 318, 3 3 4 - 3 4 0 138. 139. 140. 141.

Miller, J . D . , Tajima, S., Lauffer, L. and Walter, P. (1995) J. Cell Biol. 1 2 8 , 2 7 3 - 2 8 2 Rapiejko, P.J. and G i l m o r e , R. (1992) J. Cell Biol. 1 1 7 , 4 9 3 - 5 0 3 Powers, T. and Walter, P. (1995) Science 2 6 9 , 1 4 2 2 - 1 4 2 4 T h o m p s o n , R . C . (1988) Trends Biochem. Sci. 1 3 , 9 1 - 9 3

142. Hartmann, E., Wiedmann, M. and R a p o p o r t , T.A. (1989) E M B O J. 8 , 2 2 2 5 - 2 2 2 9 143. Krieg, U . C . , J o h n s o n , A . E . and Walter, P. (1989) J. Cell Biol. 1 0 9 , 2 0 3 3 - 2 0 4 3 144. H a r t m a n n , E., G o r l i c h , D . , K o s t k a , S., O t t o , A., K r a f t , R . , K n e s p e l , S., Burger, E. , R a p o p o r t , T.A. and Prehn, S. (1993) Eur. J. Biochem. 214, 375-381 145. Wada, I., R i n d r e s s , D . , C a m e r o n , P.H., O u , W.J., D o h e r t v , J . J . , L o u v a r d , D „ Bell, A.W., Dignard, D „ T h o m a s , D.Y. and Bergeron, J . J . M . (1991) J. Biol. C h e m . 266, 19599-19610 146. Mothes, W., Prehn, S. and R a p o p o r t , T.A. (1994) E M B O J. 13, 3973-3982 147. Nicchitta, C.V., Murphy, E . C . , H a y e s , R. and Shelness, G . S . (1995) J. Cell Biol. 129, 9 5 7 - 9 7 0 148. Martoglio, B., H o f m a n n , M.W., Brunner, J. and Dobberstein, B. (J 995) C d i 8 1 , 2 0 7 - 2 1 4

Endoplasmic reticulum targeting and translocation

149. H i g h , S., M a r t o g l i o , B., Gorlich, D . , Andersen, S.S.L., A s h f o r d , A.J., Giner, A., H a r t m a n n , E., P r e h n , S., R a p o p o r t , T . A . , D o b b e r s t e i n , B . and B r u n n e r , J. ( 1 9 9 3 ) J . B i o l . C h e m . 268, 26745-26751 149a. H a n e i n , D . , Matlack, K.E.S., J u n g n i c k e l , B., Plath, K., Miller, K . R . , R a p o p o r t , T . A . and Akev, C.W. (1996) Cell 87, 721-732 150. 151. 151a. 152.

C r o w l e y , K.S., Liao, S., Worrel, V.E., Reinhart, G . and J o h n s o n , A . E . (1994) Cell 78, 461-171 W o l i n , S . L . and Walter, P. (1989) J. Cell Biol. 1 0 9 , 2 6 1 7 - 2 6 2 2 H e g d e , R.S. and Lingappa, V.R. (1996) Cell 8 5 , 2 1 7 - 2 2 8 H i g h , S., G o r l i c h , D . , Wiedmann, M . , R a p o p o r t , T.A. and D o b b e r s t e i n , B. (1991) J . Cell Biol. 113,35-14 153. D o , H., Falcone, D . , Lin, J., A n d r e w s , D.W. and J o h n s o n , A . E . (1996) Cell 85, 3 6 9 - 3 7 8 153a. M o t h e s , W., H e i n r i c h , S . U . , G r a f , R . , N i l s s o n , I., von H e i j n e , G . , B r u n n e r , J . and R a p o p o r t , T.A. (1997) Cell 89, 523-533 153b.Borel, A . C . and Simon, S.M. (1996) Cell 85, 3 7 9 - 3 8 9

153c. 154. 155. 156. 157. 158. 159. 160. 161. 162. 162a. 163. 164. 165. 166. 167.

H a m m a n , B . D . , C h e n , J., J o h n s o n , E . E . and J o h n s o n , A . E . (1997) Cell 8 9 , 5 3 5 - 5 4 4 Gorlich, D., H a r t m a n n , E., Prehn, S. and R a p o p o r t , T. (1992) N a t u r e ( L o n d o n ) 357, 4 7 - 5 2 Voigt, S., Jungnickel, B., H a r t m a n n , E. and R a p o p o r t , T.A. (1996) J . Cell Biol. 134, 2 5 - 3 5 Martoglio, B. and Dobberstein, B. (1996) Trends Cell Biol. 6, 142-147 B r o d s k y , J . L . (1996) Trends Biochem. Sci. 21, 121-126 Simon, S.M., Peskin, C . S . and Oster, G.F. (1992) Proc. Natl. Acad. Sci. U . S . A . 8 9 , 3 7 7 0 - 3 7 7 4 O o i , C . E . and Weiss, J . (1992) Cell 71, 8 7 - 9 6 Nicchitta, C.V. and Blobel, G . (1993) Cell 73, 9 8 9 - 9 9 8 U n g e r m a n n , C . , N e u p e r t , W. and Cyr, D . M . (1994) Science 266, 1250-1253 Glick, B.S. (1995) Cell 80, 11-14 H a m m a n , B . D . , H e n d e n s h o t , L . M . and J o h n s o n , A . E . (1998) Cell 92, 747-758 Lyman, S . K . and Schekman, R. (1995) J. Cell Biol. 131, 1163-1171 Schlenstedt, G „ Harris, S., Risse, B „ Lill, R. and Silver, P. (1995) J. Cell Biol. 129, 979-988 C o r s i , A . K . and Schekman, R. (1997) J. Cell Biol. 1 3 7 , 1 4 8 3 - 1 4 9 3 Lyman, S.K. and Schekman, R. (1997) Cell 8 8 , 8 5 - 9 6 Matlack, K . E . , Plath, K., Misselwitz, B. and R a p o p o r t , T. A. (1997) Science 2 7 7 , 9 3 8 - 9 4 1

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11 Protein localization to the endoplasmic reticulum and Golgi complex R. Qanbar and C.E. Machamer* Department of Cell Biology and Anatomy1Johns Hopkins School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, U.S.A.

Introduction Many specialized reactions in the cell occur in membrane-bounded compartments. This compartmentalization may serve a number of purposes, including the temporal and spatial isolation of specific reactions, the enclosure of potentially harmful enzymes (e.g. the hydrolases of the lysozome), the creation of specific micro-environmental conditions in which certain processes can take place and the concentration of specialized components to a region, or a combination of these functions. The targeting of resident proteins that perform compartment-specific functions is a major area of interest in cell biology. Proteins destined for export follow either the classical [1-3] or the nonclassical [4] secretory pathway. The better understood of the two, the classical or vesicular secretory pathway, is characterized by the abundance of membranebounded compartments through which a protein must pass sequentially. Proteins destined for secretion, for the plasma membrane or for residency in one of the compartments of this pathway are sorted from all other proteins first by translo­ cation into the endoplasmic reticulum (ER) ([5,6], Chapter 10). Proteins to be translocated are usually recognized through the presence of a signal peptide or a hydrophobic translocation sequence (Chapter 3). The signal peptide is often cleaved, and the translocated protein may be membrane-associated or soluble in the ER lumen. In the ER, the protein is folded into the proper conformation, acquires its core glycosylation unit if N-glycosylated, and often associates with other subunits if part of a multimeric complex. The protein is then exported to the Golgi, where further post-translational modifications, particularly the processing of Nlinked sugars and the addition of O-Iinked sugars, may occur. As it exits the Golgi, the protein is sorted to its final destination. The orientation of the protein with respect to the cytosol is always maintained in this transport process. Resident

To whom correspondence should be addressed.

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proteins are localized specifically to their compartments, despite the rapid and continuous flow of membranes surrounding them.

Compartments of the classical secretory pathway The ER and the Golgi complex have long been identified as major components of the secretory pathway. The role of the ER as the site of synthesis of secretory and membrane-bound proteins, and that of the Golgi as a sorting station, are well established. However, in recent years it has become increasingly apparent that these two organelles encompass a number of subcompartments, which may be morpho­ logically or biochemically distinct. Depending on the criteria used for differen­ tiation, the number and description of these subcompartments varies, making it difficult to establish a universally accepted number and nomenclature for them. In general terms, it is recognized that transport from the ER occurs at an area devoid of ribosomes, known as the transitional ER. One or more transport steps take the protein to the Golgi complex [7,8], which is composed of a number of closely stacked flat saccules or cisternae in which sequential steps of sugar processing occur. These sugar processing stages are the basis for the subdivision of the Golgi into cis, medial and trans cisternae [9]. These 'distinct' entities are by no means static or isolated from one another. There is incessant communication and activity in the busy traffic pathways of the cell, making the morphological definition of subcompartments a difficult task. Biochemical analysis using pulse-chase and density-gradient studies has suggested the presence of an intermediate compartment between the ER and the Golgi [10-12]. If the localization of a protein or a set of proteins is taken as the determining criterion for the presence of other subcompartments, not only can an intermediate compartment between the ER and the Golgi be defined [13], but so also can several subcompartments within the ER itself (e.g. [14-16]). Also, a distinct compartment beyond the rrans-most cisterna of the Golgi may be identified [17], If the classification is to be based on morphology, two additional tubular/reticular regions at either end of the Golgi stack can be discerned: the as-Golgi [18] and trans-Golgi [19-21] networks (CGN and TGN respectively). If the number of energy-dependent transport stages is used as a criterion, there is evidence that there is only one energy-requiring transport step between the ER and the Golgi, at least in coronavirus-infected cells [22]. This would indicate that the intermediate compartment is continuous with the ER, or possibly a specialized area within it. Biochemical evidence from the effects of the fungal metabolite brefeldin A [23] clearly segregates the TGN as a separate compartment [24], On the other hand, the distinction between the CGN and the intermediate compartment is still the subject of debate [25-27]. Regardless of their number, however, subcompartments have to exhibit differences from one another. Some of the areas in which these differences may be

Localization to endoplasmic reticulum and Golgi

manifested are: protein and lipid composition, concentration and content of ions, biophysical properties of the membranes, pH, redox potential, and surfacearea/volume ratio. These differences, no matter how subtle, are required to maintain a functional distinction between the different subcompartments, possibly by determining binding affinities of receptors for their ligands. pH, one of the most obvious candidates for variability among compartments, is close to neutral in the ER, but becomes more acidic at the TGN [28]. Direct measurement of pH in the TGN of living skin fibroblasts estimated it to be around 6.2 [29]. pH and cation gradients have been shown to be required for the secretion of chorionic gonadotropin from human trophoblast cells in culture [30]. The presence of sodium and potassium ions has been shown to be required for transport out of the ER [31,32]. A similar requirement for lumenal calcium for ER exit has also been elucidated [33-35], but no such requirement was demonstrated for intra-Golgi traffic, even though evidence from laser scanning confocal microscopy and ion microscopy studies suggests that the calcium concentration in the Golgi may be the highest among intracellular organelles [36]. The free calcium concentration has been measured directly in the ER, and found to be 1-5 mM [37,38], several orders of magnitude higher than that of the cytosol. The ER has been found to have a considerably more oxidative lumen than the cytosol. The redox potential is apparently due to the ratio of reduced to oxidized glutathione (GSH/GSSG), which is between 1:1 and 3:1 in the lumen of the ER, in contrast with between 30:1 and 100:1 in the cell overall [39]. Thiol reagents, which disrupt disulphide-bond formation, impair the folding and traffic of proteins in the ER [40-^14], but not that in or past the Golgi [43,45]. Another 'gradient' has been indirectly implicated, based on the observation that there is much more cholesterol in the plasma membrane than in the ER [46]. The difference in cholesterol, and possibly other lipid species, may play an important role in defining the characteristics of a compartment. Of particular interest are the sphingolipids, whose synthesis does not even commence before the Golgi complex [47,48]. Though simple in principle, direct quantification of different lipid species in intracellular compartments has been hampered by the technical difficulties in obtaining any of the membrane fractions in a pure enough form for reliable biochemical analysis. This problem has been circumvented by the utilization of viruses that bud from different intracellular compartments to analyse the lipid composition of these compartments [49,49a]. These studies have also identified a lipid that is enriched in Golgi membranes [49]. The functional signif­ icance of this finding is under active investigation.

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Vesicular traffic Machinery The movement of membranes and proteins from one compartment to the next in the secretory pathway takes place via vesicle budding from membranes of one compartment, then detaching, diffusing, docking and fusing with the next. Dissection of the secretory pathway was made feasible by several instrumental contributions. Schekman's group followed a genetic approach, identifying yeast mutants defective in secretion [50,51]. A wealth of information resulted from the study and characterization of these mutants, complemented by the identification of other mutants by others (e.g. [52]). The second approach, followed by Rothman and his colleagues, was mainly a biochemical one that culminated in the development of an in vitro transport system [53-55]. Moreover, several systems use semi-permeabilized cells in which N-glycosylation [56-58] and/or O-glycosylation [59] are assessed as compartment markers. These systems offered the tools for the biochemical characterization of several of the factors involved in vesicular traffic, as well as the partial delineation of the sequence of events required [3], In vitro, purified components have been used for reconstituting the transport process. Thus the minimal complement of factors required for vesicular traffic has been charac­ terized [60]. With an outstanding conservation of machinery between yeast and mammals [61], the genetic and biochemical analyses resulted in the recognition of four events: first, the formation of a vesicle, a process that requires the binding of GTP and cvtosolic factors, including coat proteins and ADP-ribosylation factor [62]; secondly, the detachment of this vesicle from the parent membrane followed by uncoating, a process that requires fatty acylation [63] and GTP hydrolysis, and after which the vesicle can diffuse freely in the medium in vitro·, thirdly, the recognition of a target membrane and binding to it (docking), a process that requires recognition molecules or receptors, small GTP-binding proteins (rabs) and GTP; and finally the fusion of the vesicle with the membrane, with the concomitant hydrolysis of ATP, a process that involves an iY -ethylmaleimide-sensitive fusion protein (NSF). Our current understanding of the transport process has been comprehensively addressed in recent reviews [3,64], The specificity of the budding and fusion events is thought to be imparted, at least partially, by membrane proteins on the vesicle and on the target membrane [65,66], Specific proteins on the vesicles (vSNAREs, where SNARE stands for soluble NSF attachment protein receptor), interact specifically with proteins on the target membrane (tSNAREs), thus achieving specificity. The best understood example of this specificity comes from synaptic vesicle formation and fusion [67-71], Since transport vesicles are coated vesicles, the coat proteins constitute another contributor to specificity. The recruitment of specific coat proteins is dependent directly or indirectly on the cargo and its destination. Two coat protein

Localization to endoplasmic reticulum and Golgi

complexes have been shown to be involved in ER-to-Golgi and intra-Golgi traffic: coatomer (COP I) and COP II. The subject has been reviewed in detail [64,72,73]. The number of coats identified, however, is much smaller than the number of transport steps in the secretory pathway. This additional level of specificity may be conferred through the specific binding of small GTP-binding proteins (rabs [74]). In contrast with the vSNAREs, which are integral membrane proteins, isoprenylated rabs [75] associate peripherally with the cytosolic side of membranes. The number of different rabs identified to date further supports a potential role for these proteins in regulating vesicular traffic. Despite the fact that, in vitro, budded vesicles are free to diffuse in the medium [54], vesiele transport from one compartment to the next in the dense cytosol in vivo is likely to involve cytoskeletal elements. Microtubule motors are used by vesicles involved in transport from the Golgi to the plasma membrane [76]. A role for microtubule motor proteins in ER-to-Golgi traffic [77] and in the organi­ zation of the ER and the Golgi is recognized [78],

Anterograde and retrograde traffic To balance the constant flow of vesicles from the ER to the plasma membrane (forward or anterograde traffic), there must exist a mechanism that works in the opposite direction, retrieving lipids and proteins and delivering them back to the proper compartments. This is what is known as retrograde traffic [3,79], Retrograde traffic returns vSNAREs and other recycling proteins, lipids and compartmentspecific resident proteins to earlier compartments along the secretory pathway. Proteins active in retrograde traffic are at least partially different from those of anterograde traffic, with some level of cross-talk to allow regulation [64]. For anterograde transport, it is generally accepted that traffic proceeds in a vectorial, stepwise fashion, carrying lipids and proteins from one compartment to the next. The same mechanism may be in effect for retrograde traffic. Alternatively, retrograde transport could return lipids and proteins directly to the ER, followed by their redistribution to the proper compartments by anterograde traffic. The two possible pathways for retrograde traffic may co-exist in cells, but this remains to be demonstrated.

Default destination The sequence of a particular protein determines its destination. This is achieved through specific sequence motifs and/or conformational determinants. In the absence of such targeting information, a protein that is translocated into the ER follows a 'default' pathway, which is different in different organisms. In animal cells, the default pathway leads to the secretion of soluble proteins and to the expression of membrane proteins on the cell surface [80], whereas in yeast the vacuole appears to be the default compartment for membrane proteins [81].

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Proteins in the ER Quality control The ER is a major site of quality control in the secretory pathway [82]. After their synthesis and translocation into the ER, proteins are retained until they acquire the proper conformation. Proteins that are recognized as 'defective' are retained in the ER for degradation or rescue. Proteins that monitor the maturation of other proteins in the ER are referred to collectively as chaperones [83]. They include several families of proteins, many of which were identified previously as heat-shock or stress-induced proteins [84]. Proteins such as BiP [85] and calnexins [86] associate with specific subsets of proteins and detect different 'defects' (e.g. [87,88]). Mutations and deletion of a portion of a protein (e.g. [89]), failure to cleave a signal peptide in glycosylphosphatidylinositol-anchored proteins (e.g. [90,91]), incorrect disulphide-bond formation (e.g. [40,42,43]), limited processing of sugar chains (e.g. [92,93]) or certain amino acid changes (e.g. [94,95]) can all cause a protein that would otherwise leave the ER to be retained there. Protein degradation in the ER is a complex phenomenon that occurs in yeast [96] as well as animal cells [97], Proteins that are destined for degradation are processed at very different rates, thought to be determined by the protein itself. Degradation sequences have been identified in several proteins that are not normally resident in the ER [98-100], These sequences are thought to be masked when the protein is properly folded. It is not well understood whether degradation occurs in the ER itself [101] or a subcompartment thereof [102], Evidence for ER degradation comes from numerous studies in which proteins are degraded in the presence of inhibitors of lysosomal hydrolases or of vesicular traffic (e.g. [101,103-105]). Degradation is sensitive to redox potential and calcium [106], the depletion of which enhances protein degradation, possibly through the activation of calcium-sensitive proteases [35], Localization of the ER degradative compartment is further complicated by evidence for post-ER, non-lysosomal degradation (e.g. [107,108]). Similar to the variability in protein degradation rates, translocated proteins exit the ER at different rates [10]. Secretory and plasma membrane proteins take between 5 min and several hours to reach their final destination. Most of the delay is at the point of exit from the ER. Similar or co-expressed proteins may have different ER exit kinetics. For example, different glucose transporter isoforms exit the ER at different rates [109]. Also, the Gl and G2 glycoproteins of Uukuniemi virus, which are produced by cleavage of a single polypeptide, exit the ER with different kinetics [110]. A protein may exit the ER only when it is 'ready', i.e. properly folded and conjugated.

Localization to endoplasmic reticulum and Golgi

Exported proteins Proteins transported past the ER fall into three major categories: proteins that are transported at a bulk flow rate; proteins with export signals that make them traverse the secretory pathway at an enhanced rate; and proteins that are resident in a compartment of the secretory machinery. One measurement of the bulk flow rate suggested the half-time for the secretion of synthetic tripeptides lacking targeting signals to be approx. 10 min [80], These tripeptides are not detained in the ER for proper folding, as proteins are. Taking into account folding time, some proteins may actually move more rapidly than the synthetic tripeptides. In addition, some proteins are 5-10 times more concentrated than would be predicted for non-selective bulk flow, indicating that they are selected for exit from the ER. Immunolocalization studies have confirmed that the glycoprotein of the vesicular stomatitis virus (VSV-G) [111] and albumin [112] are concentrated in active budding regions of the ER.

ER resident proteins Mechanisms for steady-state localization of proteins Proteins resident in compartments of the secretory pathway can utilize two mechanisms to maintain a steady-state localization to one compartment in the face of the continuous flow of membranes. 'Retention', whereby a protein is excluded from transport vesicles, and 'retrieval', whereby a protein is constantly retrieved from compartments subsequent to the one it is localized to, are by no means mutually exclusive. Several ER proteins have been shown to make use of both mechanisms [113,114]. Calreticulin is one example [115]. ER resident proteins use a multitude of targeting motifs, some of which are better understood than others, to maintain their steady-state localization [116]. The different signals function at different stages. A short signal in the C-terminus of ER lumen proteins, or on the cytoplasmic tail of ER membrane proteins, returns these proteins to the ER if they escape this compartment. These sequences are sufficient to confer ER localization to reporter proteins. However, they are not always necessary, suggesting that retrieval is probably not the primary ER localization mechanism. When such retrieval signals are disrupted, proteins leak out of the ER only at a low rate. Also, some proteins (e.g. BiP [117]) have been shown to be excluded from secretory vesicles to start with. Whatever the primary mechanism of localization may be, proteins that escape the ER are retrieved mostly from the next compartment, the intermediate compartment or the c/s-Golgi. The retrieval capacity, nonetheless, extends to the trans-most cisterna of the Golgi [118]. The retrieval machinery is saturable, and can be overwhelmed by overexpression. Surprisingly, some overexpressed proteins that appear at the cell surface are rapidly internalized, using a mechanism dependent on the very motif that constitutes their retrieval signal for ER localization. Whether all of these retrieval stages are involved in the steady-state localization of all ER proteins is not known; this is probably

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unlikely. Nonetheless, these multiple mechanisms highlight the importance of maintaining proteins in their steady-state compartments. KDEL sequence signal in Iumenal proteins Many soluble ER proteins have a sequence at their C-terminus specifying their localization [119]. With numerous variations tolerated (e.g. [120]), this signal is LysAsp-Glu-Leu (KDEL) in mammals, His-Asp-Glu-Leu (HDEL) in yeast, and probably both in plants [121]. Chimaeric proteins containing either a KDEL or HDEL sequence were efficiently localized to the ER of plant cells [122]. In addition, a viral protein that bears a functional KDEL-Iike sequence has been identified [123]. Proteins can be localized in the ER indirectly by interacting with a KDEL-sequence-bearing protein. For example, β-glucuronidase remains in the ER through its association with esterase-22 (egasyn), which has an HTEL sequence [124]. This is also one mechanism by which misfolded proteins are retained in the ER. Proteins are often found associated with BiP, which has its own KDEL sequence [85]. KDEL specificity is conferred by a ds-Golgi receptor called the KDEL receptor in mammals [125,126] and Erd2p in yeast [127,128], This receptor recognizes proteins carrying the KDEL sequence at their C-termini and returns them to the ER by retrograde traffic. The recycling of the receptor to the ER is dependent on the binding of ligand [126]. Mutation or deletion of the KDEL signal results in the secretion of KDEL proteins, as does their overexpression. However, the most dramatic result of the disruption of Erd2p is not the secretion of KDELbearing proteins, but rather morphological changes to the Golgi apparatus [127]. This may be a result of the disruption of the normal flow of membranes between the ER and the Golgi [129]. The function of Erd2p in maintaining Golgi morphology could be uncoupled from its ability to retrieve KDEL proteins [127], since normal Golgi morphology was maintained in yeast cells harbouring Erd2p mutants defective in KDEL binding. Subsequent studies demonstrated that this uncoupling was due either to the presence of a redundant, inducible pathway that compensated for the loss of KDEL binding [130] or to the existence of minimal residual binding activity of the mutated receptors [131]. These results suggest that the retrieval of minimal levels of KDEL proteins is sufficient for maintaining Golgi morphology in the presence of a recycling Erd2p with compromised ligand binding. Even though the majority of KDEL proteins are soluble ER proteins, one exception where a membrane protein utilized a lumenal KDEL sequence is documented [132]. The KDEL sequence is not the final determinant of the steadystate compartment of a protein. Some KDEL proteins have been identified as residents of the intermediate compartment [133].

Localization to endoplasmic reticulum and Golgi

Dibasic amino acid motif for integral membrane proteins Some integral membrane proteins that reside in the ER carry a dibasic amino acid motif [134]. If the protein has type I topology (Chapter 4), with its C-terminus in the cytosol, the sequence is usually KKXX (where K is Lys and X is any amino acid). Proteins of type II topology, with their N-terminus facing the cytosol, usually have the N-terminal sequence XXRR (R is Arg) [135]. Like the KDEL motif, several variations in the amino acid composition and location of the dibasic amino acid signal are tolerated. Also, like KDEL proteins, proteins with a KKXX signal have been found in the intermediate compartment (e.g. ERGIC-53 [136]), and even the Golgi [137], Coatomer has been shown to interact with KKXX proteins [138] as an essential component for their retrieval to the ER [139], Similar to that for KDEL proteins, the KKXX retrieval machinery is saturable. Overexpression of ERGIC53, which contains a KKFF retrieval signal, results in its appearance at the plasma membrane. The KKFF sequence of overexpressed ERGIC-53 mediates its internal­ ization and endocytosis at the plasma membrane [140,141].

Other mechanisms Several ER resident proteins lack the general ER sequence motifs described above (e.g. type 2 1 Ιβ-hydroxysteroid dehydrogenase [142]), but have been shown to reside in the ER in a sequence-dependent manner. Sequence requirements could not be reduced to a common structural feature or a linear sequence motif. Nor were any of these sequences proven to be sufficient to localize a reporter protein to the ER. Such sequences have been recognized in the N-terminal (e.g. [143-145]) and Cterminal [146] regions of proteins, as well as in the transmembrane segments [147,148]. It is likely that other ER localization motifs remain to be discovered. In addition to retrieval and retention, another conceivable mechanism of restricting a protein to a given compartment would involve the degradation of any molecules that escape. This process of distal degradation has been reported for the neuroendocrine-specific 18 kDa protein RESP18 [149]. Identification of more examples of proteins that are degraded past their steady-state localization compartments will help to determine whether distal degradation is limited to a subset of resident proteins, and also whether it is present as a back-up mechanism for other localization signals. Proteins can associate with the ER transiently or permanently via interactions with membrane components at either leaflet of the ER membrane. Many proteins that are required for vesicular transport are recruited from the cytosol and thus associate transiently with the ER membrane. Lysyl hydrolase is an example of a protein that associates with the lumenal leaflet of the ER membrane, possibly via weak electrostatic interactions [150], If expressed independently, one or more of the subunits of multimeric proteins may be retained in the ER. Some of these subunits have classical ER localization signals that become inaccessible to the localization machinery when

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assembled. Other proteins are dependent on non-classical sequences for their ER localization. For example, a tyrosine, leucine and arginine have been shown to be important for the localization of unassembled e-subunit of the T-cell receptor to the ER [151]. Formation of heterodimers is important for the localization of some proteins. The unassembled subunits may appear at different locations, with one of the subunits commonly, but not necessarily (e.g. [152]), retained in the ER. When assembled, the protein complex can be localized at the cell surface (e.g. [ 153]) or the Golgi (e.g. [154,155]). Although the retention of one subunit of a heterodimeric protein in the ER may be considered as part of the ER quality control, this quality control obviously does not apply to the other subunit, which exhibits the same steady-state localization as the heterodimer when expressed independently. Some signals may dominate over others. For example, the addition of a glycosylphosphatidylinositol anchor (which directs proteins to the cell surface) to the rubella virus El protein (which is localized to the ER if expressed indepen­ dently) causes its expression at the cell surface [156]. In such cases, the final localization of a protein is often dependent on the overall interpretation of the multiple signals encoded in its subunits.

Golgi localization Localization of proteins to the Golgi complex is likely to be achieved through multiple signals, utilizing both retention and retrieval as localization mechanisms [114,157,158]. In a system dependent mainly on retrieval, overexpression of resident proteins would result in their appearance at the cell surface. This has been shown not to be the case for many Golgi proteins [157,158], suggesting the involvement of true retention as one localization mechanism. On the other hand, in a system dependent mainly on retention, none of the proteins would be found past their steady-state compartment. This again has been shown not to be the case, since many as- and medial-Golgi proteins do recycle through later Golgi compartments, even though several others do not seem to travel past their steady-state location (e.g. [159]). This clearly illustrates that the achievement of steady-state localization in the Golgi is dependent on a number of factors and may be protein-specific. Unlike the ER localization signals, which are usually exposed to soluble factors and are involved mainly in protein-protein interactions, Golgi localization involves membrane-spanning segments, lumenal sequences and cytoplasmic domains, and is thus more likely to involve lipid-protein interactions as well as protein-protein interactions [157,158],

Integral membrane proteins The majority of Golgi-resident proteins are type II integral membrane proteins containing localization information in their transmembrane domains [157,158,160].

Localization to endoplasmic reticulum and Golgi

When attached to reporter proteins, these transmembrane sequences have been shown to be sufficient for Golgi localization (e.g. [161]). However, additional targeting information has been found to reside in the flanking sequences of the transmembrane domains [157,158,162], as well as the cytoplasmic tails (e.g. [163-165]), of Golgi proteins. Mutations in the lumenal and transmembrane domains are usually tolerated in the context of the wild-type protein, thus supporting the hypothesis of the presence of redundant signals. A role for lipids

One way in which transmembrane domains may direct localization of a protein to a specific compartment is through preferred association with certain lipids. Such an association would direct a protein to or exclude it from budding-competent membrane domains. Close examination and analysis of the putative transmembrane domains of Golgi and plasma membrane proteins revealed an interesting correlation between the length of the putative transmembrane domain, its content of the amino acids phenylalanine and tyrosine, and localization [166-168]. This led to the development of a model [166] that correlates the length of transmembrane domains with earlier observations demonstrating the great enrichment of cholesterol in the plasma membrane over that in the ER. If cholesterol was gradually concentrated in transport vesicles throughout the secretory pathway, a cholesterol gradient would be predicted to exist. These transport vesicles would also be enriched in sphingohpids, which are synthesized in the Golgi. Cholesterol- and sphingolipidrich membranes tend to be thicker, and thus do not easily accommodate short transmembrane segments. Plasma membrane proteins, which have longer transmembrane segments on average, would be more likely to associate with these thicker membranes and thus be transported forward, whereas Golgi resident proteins, which have shorter transmembrane domains on average, would be excluded from these vesicles and thus be retained in the Golgi. This model offers a plausible mechanism for the non-saturable nature of Golgi localization and its requirement for the presence of transmembrane domains, yet its independence on their exact sequence. However, the model relies on the assumption that the prediction of transmembrane domains from the sequence of a protein reflects what portion of that protein is buried in a membrane bilayer in vivo. 'Deviations' from prediction may explain some of the many exceptions [168] to the general rule of membrane span length, but could also give rise to more such exceptions. One example where the length of the transmembrane domain did not seem to affect the localization of a protein spans a set of chimaeric A/-acetylglucosaminyltransferase I molecules. Even though the leucine content of the transmembrane domains had a profound effect on the morphology of the Golgi upon expression of the chimaeras, no effect of the length of the transmembrane segments on the localization of the chimaeric proteins was noted [169]. Increasing evidence points to the importance of the lipid composition of Golgi membranes in protein localization. The earliest clues came from the charac-

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terization of Secl4p as a phosphatidylinositol transfer protein [170,171], It has been hypothesized that this protein acts as a phosphatidylinositol sensor, since yeast cells harbouring the sec!4 mutant show high phosphatidylcholine levels [172]. Phosphoinositides (metabolites of phosphatidylinositol) are potential regulators of the secretory pathway through their activation of ADP-ribosylation factor [173], This factor activates an isoform of phospholipase D, which may modulate the lipid composition of the membranes that form transport vesicles. Phospholipase D activity has recently been identified in Golgi-enriched membranes and shown to be activated by ADP-ribosylation factor in a brefeldin Α-sensitive manner [174]. The role of phosphatidylinositol and phosphoinositides in the secretory pathway has recently been reviewed [175], Further support for lipid involvement comes from the recent demonstration of the relative enrichment of the unusual lipid semilysobisphosphatidic acid in the CGN and TGN in comparison with the Golgi stack [49a]. The structural characteristics of this lipid may be important for the establishment and possibly the maintenance of the distinctive characteristics of these two tubular/reticular compartments. Oligomerization In addition to lipid-protein interactions, protein-protein interactions may be important for Golgi localization. Two medial-Golgi enzymes (mannosidase II and N-acetylglucosaminyltransferase) have been shown indirectly to form heterodimers, since the mislocalization of one to the ER resulted in the mislocalization of the other [176]. This lent support to the 'kin recognition' hypothesis [177] for Golgi retention, whereby newly synthesized dimers associate with pre-existing oligomers at their destination and are thus excluded from transport vesicles. This model accounts for the observation that overexpressed Golgi proteins tend to 'back up' into the ER, presumably due to premature oligomerization [114]. A second line of evidence for protein-protein interactions comes from the dissection of the Golgi localization signal of the avian infectious bronchitis virus membrane protein M (IBV-M), which has three membrane-spanning domains. When transplanted on to a reporter protein, the first membrane-spanning segment has been demonstrated to be sufficient for Golgi localization [161]. Careful examination of the first membrane-spanning domain of IBV-M showed that mutations in polar but uncharged amino acids that line up on one face of a predicted α-helix were not tolerated in the context of reporter proteins [178]. This suggests a role for protein-protein interactions in the plane of the membrane possibly leading to the formation of aggregates that are inaccessible for transport. Oligomer formation can also be mediated or stabilized by the cytoplasmic tails of proteins. A chimaeric protein in which the transmembrane domain of VSVG was replaced with the first membrane segment of IBV-M resulted in the formation of a detergent-resistant oligomer. The formation of the oligomer correlated with the Golgi localization of the chimaera, was dependent on the

Localization to endoplasmic reticulum and Golgi

transmembrane domain sequence and was stabilized to detergent solubilization by the VSV-G cytoplasmic domain [179], In the context of this chimaera, neither covalent nor non-covalent association of the VSV-G cytoplasmic domain with other proteins has been observed [179a]. Hypotheses concerning Golgi localization by retention all involve restriction of Golgi proteins from entry into transport vesicles, whether by oligomerization, interaction with stationary proteins like those of the cytoskeleton, or limitation to budding-incompetent lipid domains. Restriction of movement would necessarily limit the ability of the protein to move in the plane of the membrane, and consequently reducc its diffusion coefficient. Using recovery of fluorescence in photobleaching experiments, diffusion coefficients have been measured for green-fluorescent-protein-tagged versions of native and mutant forms of the KDEL receptor, mannosidase II and a reporter protein with the trans­ membrane domain of jV-acetylglucosaminyltransferase [180], These proteins have been shown to have diffusion coefficients close to the highest known for any membrane protein (that of rhodopsin in rod outer segments), suggesting that they are free to diffuse in the plane of Golgi membranes without any significant hindrance. These values were 10-30-fold higher than those of proteins that are known to interact with other membrane or cytoskeletal proteins. The study also showed that these proteins diffused rapidly from one Golgi stack to the other, demonstrating the interconnectedness of Golgi stacks in the same cell. Diffusion between different cisternae of the same stack, however, has not been demonstrated. Assuming that the native proteins have diffusion coefficients similar to those of the chimaeras, these findings present an extra challenge to the existing hypotheses for Golgi localization: the maintenance of Golgi character in spite of the diffusibility of its resident proteins. Recycling of Golgi proteins Several Golgi residents have been shown to have sugar modifications that could only occur in compartments beyond their steady-state location [181-183]. Either these proteins are cycling through a later compartment or the localization of the sugar transferases may vary between cell types. Evidence supports a recycling hypothesis, even though no receptors have been identified to date. The extent to which the transmembrane domains of these proteins contribute to their steady-state localization, the nature of retrieval or recycling signals, and the mechanisms by which this information is interpreted remain unclear. Addressing the localization of Golgi proteins as a homogeneous group is an oversimplification. The fact remains that Golgi proteins often show specific localization to a subset of Golgi compartments even within the Golgi stack. This subcompartment specificity may be explicable by extending the principles of existing models, utilizing one or more of the proposed mechanisms. This, however, is not the only possibility. It is conceivable that independent targeting mechanisms

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exist for different types of Golgi proteins. Also, the presence of unique subcompartment-specific signals cannot be ruled out.

TGN proteins A specific subset of recycling Golgi proteins shows a steady-state localization to the TGN. Proteins such as TGN38 [17], furin-processing protease [184] and yeast Kex2 [185] recycle rapidly between the TGN and the plasma membrane. As with endocytosis signals, tyrosine residues in the cytoplasmic tails of these proteins are important for their steady-state localization [186,187]. This process has been shown to involve clathrin [188], either for rapid internalization or limited transport to the plasma membrane. In addition, another sequence rich in acidic amino acids seems to be involved in the maintenance of this steady-state localization to the TGN (e.g. [189]).

Lumenal Golgi proteins Until recently, all known Golgi proteins were found to be integral membrane proteins. The trend was broken by the identification of the first lumenal Golgi protein, Cab45 [190]. This protein has a sequence at its C-terminus (HEEF) reminiscent of the KDEL ER-Iocalization signal. However, the protein has been shown to be localized to the Golgi lumen at steady state. Whether this localization is dependent on the C-terminal sequence or involves a receptor similar to the KDEL receptor is under active investigation.

Conclusions The vesicular secretory pathway ensures the delivery of properly folded, newly synthesized proteins to the cell surface. Quality control, efficient post-translational modification and sorting take place within a series of membrane-bounded compartments. In these compartments, the synthesis of several lipid species, lipid sorting and lipid transport also take place. Transport in a vectorial manner from one compartment to the next is achieved via budding and fusion of membrane vesicles, a process requiring complex machinery and a multitude of components. In addition to modifying and sorting itinerant proteins and lipids, the secretory pathway machinery maintains a balanced flow of non-itinerant components back to where they are needed. To maintain the identity, and hence functionality, of the different compartments, their constituents have to be identified and specifically kept in place. Very little is known about how steady-state localization of lipids in the different compartments of the secretory pathway is attained, or about lipid sorting in general. Protein localization is achieved via two mechanisms: retrieval and retention. Several linear sequence signals that are recognized by other proteins capable of recycling between compartments have been identified for the retrieval of ER and TGN proteins. Localization to the Golgi stack involves the cytoplasmic tails as well as the transmembrane domains and flanking sequences of Golgi proteins. Retention is

Localization to endoplasmic reticulum and Golgi

favoured as a mechanism for Golgi localization, possibly through the exclusion of Golgi proteins from transport vesicles. Models that have been proposed to explain how retention is achieved invoke protein-lipid interactions, resulting in the association of Golgi proteins with budding-incompetent lipid domains, as well as protein-protein interactions, resulting in structures too large to fit in secretory vesicles. Association with self, the intracellular matrix or other Golgi proteins could produce such structures. There is no reason to assume that the localization mechanisms described for Golgi proteins do not apply to ER proteins and vice versa. Localization information may be found as multiple, redundant and possibly competing signals, in which case the final localization of a protein or protein complex will depend on the overall rendition of the information it contains. Work in recent years has shed some light on how retrieval and retention are accomplished for some ER and Golgi proteins, but neither process is close to being fully understood. Many of the players in the proposed retention and retrieval mechanisms have not been identified. Also, exceptions have been found for every proposed model. Additional information will be needed for the formulation of an integrative model that adequately explains and accounts for the localization of all the resident proteins in all subcompartments of the secretory pathway. Due to the breadth of the topic and space limitations, we had to limit the number of citations. Where possible, review articles have been cited. Where examples are given, preference is given to more recent articles that would not have been included in earlier reviews. We apologize to the many researchers whose work is not cited here, but whose findings contributed to our current understanding of the field.

References 1. 2. 3. 4.

8.

Hong, W. and Tang, B.L. (1993) Bioessavs 15,231-238 Pelham, H.R. and Munro, S. (1993) Cell 75,603-605 Rothman, J.E. and Wieland, F.T. (1996) Science 272,227-234 Muesch, A., Hartmann, E., Rohde, K., Rubartelli, A., Sana, R. and Rapoport, T.A. (1990) Trends Biochem. Sci. 15, 86-88 Walter, P. and Johnson, A.E. (1994) Annu. Rev. Cell Biol. 10, 87-119 Schatz, G. and Dobberstein, B. (1996) Science 271, 1519-1526 Beckers, C.J., Plunter, H., Davidson, H.W. and Balch, W.E. (1990) J. Biol. Chem. 265, 18298-18310 Hauri, H.P. and Schweizer, A. (1992) Curr. Opm. Cell Biol. 4, 600—608

9. 10. 11.

Dunphy, W.G. and Rothman,J.E. (1985) Cell 42, 13-21 Lodish, H.E, Kong, N., Hirani, S. and Rasmussen, J. (1987)J. Cell Biol. 104, 221-230 Schweizer, A., Matter, K., Ketcham, C.M. and Hauri, H.P. (1991) J. Cell Biol. 113,45-54

12. 13.

Schweizer, A., Fransen, J, A., Matter, K., Kreis, T.E., Ginsel, L. and Hauri, H.P. (1990) Eur. J. Cell Biol. 53, 183-196 Schweizer, A., Fransen, J.A., Bachi, T., Ginsel, L. and Hauri, H.P. (1988) J. Cell Biol. 107,

14. 15.

1643-1653 Arber, S., Krause, K.H. and Caroni, P. (1992) J. Cell Biol. 116,113-125 Orci, L., Perrelet, A., Ravazzola, M., Amherdt, M., Rothman, J.E. and Schekman,R. (1994)

16.

Proc. Natl. Acad. Sci. U.S.A. 91, 11924-11928 Krijnse-Locker 1 J., Parton, R.G., Fuller, S.D., Griffiths, G. and Dotti, C.G. (1995) Mol. Biol.

5. 6. 7.

Cell 6, 1315-1332

207

208

R. Q a n b a r and C.E. Machamer

17.

L u z i o , J.P., B r a k e , B „ B a n t i n g , G „ H o w e l l , K . E . , B r a g h e t t a , P. a n d Stanley, K . K . ( 1 9 9 0 ) B i o c h e m . J . 270, 9 7 - 1 0 2

18. 19. 20. 21.

H s u , V.W., Shah, N . and Klausner, R . D . (1991) Cell 6 9 , 6 2 5 - 6 3 5 Taatjes, D . J . and Roth, J . (1986) Eur. J. Cell Biol. 42, 3 4 4 - 3 5 0 Griffith, G . and S i m o n s , K . (1986) Science 234, 4 3 8 - 4 4 3 G e u z e , H . J . and M o r r e , D . J . (1991) J . Electron M i c r o s c . Tech. 1 7 , 2 4 - 3 4

22. 23. 24. 25. 26. 27. 28. 29.

Krijnse-Locker, J., Ericsson, M., Rottier, P.J. and Griffiths, G . (1994) J. Cell Biol. 1 2 4 , 5 5 - 7 0 D o m s , R.W., R u s s , G . and Yewdell, J.W. ( 1 9 8 9 ) J . Cell Biol. 1 0 9 , 6 1 - 7 2 C h e g e . N . W . and P f e f f e r , S . R . ( 1 9 9 0 ) J . Cell Biol. 1 1 1 , 8 9 3 - 8 9 9 Saraste, J . and K u i s m a n e n , E. (1992) Semin. Cell Biol. 3, 3 4 3 - 3 5 5 Bonatti, S. a n d T o r r i s i , M . R . (1993) Subcell. B i o c h e m . 2 1 , 1 2 1 - 1 4 2 H e n d r i k s , R.J. and Fuller, S . D . (1994) Subcell. B i o c h e m . 2 2 , 1 0 1 - 1 4 9 A n d e r s o n , R . G . and Pathak, R . K . (1985) Cell 4 0 , 6 3 5 - 6 4 3 Seksek, O . , Bewersi, J . and Verkman, A.S. (1995) J. Biol. C h e m . 2 7 0 , 4 9 6 7 ^ 1 9 7 0

30.

P e t e r s , B.P., B r o o k s , M „ H a r t l e , R . J . , K r z e s i c k i , R.F., Perini, F. and R u d d o n , R.W. ( 1 9 8 3 ) J . Biol. C h e m . 2 5 8 , 1 4 5 0 5 - 1 4 5 1 5

31. 32. 33. 34.

J u d a h . J . D . , H o w e l l , K . E . , T a v l o r , J . A . and Q u i n n , P.S. (1989) J. Cell Sci. 9 2 , 1 7 3 - 1 8 5 D o c h e r t y . P . A . and Snider, M . D . (1991) J. Cell. Physiol. 1 4 6 , 3 4 - 4 2 B o o t h , C . and K o c h , G . L . (1989) Cell 59, 7 2 9 - 7 3 7 L o d i s h , H.F. and K o n g , N . (1990) J. Biol. C h e m . 265, 10893-10899

35. 36. 37.

Wileman, T., K a n e , L.P., C a r s o n , G . R . a n d T e r h o r s t , C . (1991) J. Biol. C h e m . 266, 4 5 0 0 - 4 5 0 7 C h a n d r a , S., Kable, E.P. and M o r r i s o n , G . H . (1991) J. Cell Sci. 100, 747-752 K e n d a l l , J . M . , D o r m e r , R . L . and C a m p b e l l , A . K . ( 1 9 9 2 ) B i o c h e m . B i o p h y s . R e s . C o m m u n . 189,1008-1016

38. 39.

M o n t e r o , M . , Brini, M . , M a r s a u l t , R . , A l v a r e z , J . , Sitia, R . , P o z z a n , T. and R i z z u t o , R . (1995) E M B O J . 14, 5 4 6 7 - 5 4 7 5 H w a n g , C „ Sinskey, A . J . and L o d i s h , H . F . (1992) Science 257, 1496-1502

40. 41. 42.

B r a a k m a n , 1., H e l e n i u s , J. and H e l e n i u s , A . (1992) N a t u r e ( L o n d o n ) 3 5 6 , 2 6 0 - 2 6 2 Tatu, U . , B r a a k m a n , I. and Helenius, A . (1993) E M B O J. 12,2151 - 2 1 5 7 J a m s a , E., S i m o n e n , M . and M a r a r o w , M . (1994) Yeast 10, 3 5 5 - 3 7 0

43. 44.

Valetti, C . and Sitia, R. (1994) M o l . Biol. Cell 5 , 1 3 1 1 - 1 3 2 4 Verde, C . , Pascale, M . C . , M a r t i r e , G . , L o t t i , L.V., T o r r i s i , M . R . , H e l e n i u s , A . and B o n a t t i , S. (1995) Eur. J . Cell Biol. 67, 267-274

45. 46.

L o d i s h , H.F. and K o n g , N . (1993) J. Biol. C h e m . 268, 2 0 5 9 8 - 2 0 6 0 5 O r c i , L., M o n t e s a n o , R., M e d a , P., M a l a i s s e - L a g a e , F., B r o w n , D . , Perrelet, A . and Vassalli, P. (1981) Proc. N a t l . Acad. Sci. U . S . A . 78, 2 9 3 - 2 9 7 Allan, D . and Kallen, K.J. (1993) P r o g . Lipid Res. 32, 195-219

47.

48. van H e l v o o t , A. and van Meer, G . (1995) F E B S Lett. 369, 18-21 49. Cluett, E . B . and Machamer, C . E . (1996) J. Cell Sci. 1 0 9 , 2 1 2 1 - 2 1 3 1 49a. Cluett, E.B., K u i s m a n e n , E. and Machamer, C . E . (1997) M o l . Biol. Cell 8 , 2 2 3 3 - 2 2 4 0 50. N o v i c k , P., Field, M . C . and Schekman, R. (1980) Cell 21, 2 0 5 - 2 1 5 51. Schekman, R. (1992) Curr. O p i n . Cell Biol. 4 , 5 8 7 - 5 9 2 52. N e w m a n , A.P. and F e r r o - N o v i c k , S. (1987) J . Cell Biol. 1 0 5 , 1 5 8 7 - 1 5 9 4 53. Fries, E. and R o t h m a n , J . E . (1981) J. Cell Biol. 90, 6 9 7 - 7 0 4 54. R o t h m a n , J . E . , Miller, R . L . and U r b a n i , L.J. (1984) J . Cell Biol. 99, 260-271 55. 56. 57.

R o t h m a n , J . E . and O r c i , L. (1992) F A S E B J. 4, 1460-1468 Beckers, C.J., Keller, D.S. and Balch, W.E. (1987) J . Biol. C h e m . Cell 50, 5 2 3 - 5 3 4 H e l m s , J . B . , K a r r e n b a u e r , A . , W i r t z , K.W., R o t h m a n , J . E . and W i e l a n d , F.T. ( 1 9 9 0 ) J . B i o l . Chem. 265,20027-20032

58. 59.

Schwaninger, R „ Beckers, C . J . and Balch, W.E. (1991) J . Biol. C h e m . 2 6 6 , 1 3 0 5 5 - 1 3 0 6 3 W i l s o n , B . S . , P a l a d e , G . E . and F a r q u h a r , M . G . ( 1 9 9 3 ) P r o c . N a t l . A c a d . Sci. U . S . A . 90, 1681-1685

60.

O s t e r m a n n , J . , O r c i , L., Tani, K . , A m h e r d t , M . , R a v a z z o l a , M., Elazar, Z. and R o t h m a n , J . E . (1993) Cell 7 5 , 1 0 1 5 - 1 0 2 5

61. 62. 63.

N o t h w e h r , S.F. and Stevens, T . H . (1994) J . Biol. C h e m . 269, 10185-10188 B o m a n , A . L . and K a h n , R . A . (1995) Trends B i o c h e m . Sci. 20, 147-150 M u n d v , D . I . (1995) Biochem. Soc. Trans. 23, 5 7 2 - 5 7 6

Localization t o endoplasmic reticulum and Golgi

64. 65.

Schekman, R . and Orci, L . ( 1 9 % ) Science 271, 1526-1533 R o t h m a n , J . E . (1994) Adv. Second Messenger P h o s p h o p r o t e i n Res. 2 9 , 8 1 - 9 6

66. 67.

R o t h m a n , J . E . and Warren, G . (1994) Curr. Biol. 4 , 2 2 0 - 2 3 3 Kelly, R . B . (1993) Cell. 72 (suppl.), 43-53

68. 69. 70. 71.

Bennet, M . K . (1995) Curr. O p i n . Cell Biol. 7 , 5 8 1 - 5 8 6 Fried, G . ( 1995) A c t a Physiol. Scan. 154, 1-15 S u d h o f , T . C . (1995) N a t u r e ( L o n d o n ) 375, 645-653 C a l a k o s , N . and Scheller, R . H . (1996) Physiol. Rev. 7 6 , 1 - 2 9

72. 73. 74.

Pelham, H . R . (1994) Cell 79, 1125-1127 ' Harter, C . (1995) F E B S Lett. 369, 89-92 S c h w a n i n g e r , R., Plutner, H . , B o k o c h , G . M . and Balch, W.E. (1992) J . C e l l B i o l . 119, 1077-1096

75. 76. 77.

Simons, K . and Zerial, M . (1993) N e u r o n 11, 7 8 9 - 7 9 9 L a f o n t , F. and Simons, K. (1996) Semin. Cell Dev. Biol. 7 , 3 4 3 - 3 5 5 Lippincott-Schwartz, J . and C o l e , N . B . (1995) Biochem. Soc. Trans. 23, 544-548

78. 79. 80.

Allan, V. (1996) Semin. Cell Dev. Biol. 7, 3 3 5 - 3 4 2 Lippincott-Schwartz, J . (1993) Subcell. Biochem. 21, 9 5 - 1 1 9 Wieland, F.T., G l e a s o n , M . L . , Serafini, T.A. and R o t h m a n , J . E . (1987) Cell 5 0 , 2 8 9 - 3 0 0

81. 82. 83. 84.

Roberts, C.J., N o t h w e h r , S.F. and Stevens, T . H . (1992) J. Cell Biol. 1 1 9 , 6 9 - 8 3 H a m m o n d , C . and Helenius, A . (1995) Curr. O p i n . Cell Biol. 7, 5 2 3 - 5 2 9 McMillan, D . R . , G e t h m g , M.J. and S a m b r o o k , J. (1994) Curr. O p i n . Biotechnol. 5 , 5 4 0 - 5 4 5 Craig, E . A . , G a m b i l l , B . D . and N e l s o n , R.J. (1993) Microbiol. Rev. 57, 402-414

85. 86.

Haas, I.G. (1994) Experientia 5 0 , 1 0 1 2 - 1 0 2 0 Bergeron, J.J., Brenner, M . B . , T h o m a s , D.Y. and Williams, D . B . (1994) Trends B i o c h e m . Sci. 19, 124-128 H a m m o n d , C . and Helenius, A. (1994) Science 2 6 6 , 4 5 6 - 1 5 8

87. 88. 89. 90.

Hebert, D . N . , Foellmer, B . and Helenius, A . (1996) E M B O J . 1 5 , 2 9 6 1 - 2 9 6 8 L a n k f o r d , S.P., C o s s o n , P., B o n i f a c i n a , J . S . and K l a u n s e r , R . D . (1993) J . B i o l . C h e m . 268, 4814-4820 D e l a h u n t y , M . D . , S t a f f o r d , F.J., Y u a n , L . C . , S h a z , D . and B o n i f a c i n a o , J . S . ( 1 9 9 3 ) J . Biol. Chem. 268,12017-12027

91.

Field, M . C . , M o r a n , P., L i , W „ Keller, G . A . and C a r a s , I.W. ( 1 9 9 4 ) J . Biol . C h e m . 269, 10830-10837

92. 93. 94.

H a m m o n d , C , B r a a k m a n , I. and Helenius, A . (1994) Proc. N a t l . Acad. Sci. U . S . A . 91, 913-917 Helenius, A . (1994) M o l . Biol. Cell 5 , 2 5 3 - 2 6 5 Tsuji, E . , M i s u m i , Y., F u j i w a r a , T., Takami, N . , O g a t a , S. and Ikehara, Y. (1992) B i o c h e m i s t r y 31, 11921-11927

95.

Busca, R., Pujana, M . A . , P o g n o n e c , P., A u w e r x , J., D e e b , S.S., Reina, M . and Vilaro, S. (1995) J . Lipid Res. 3 6 , 9 3 9 - 9 5 1

96. 97.

Finger, A., K n o p , M . and Wolf, D . H . (1993) Eur. J. Biochem. 2 1 8 , 5 6 5 - 5 7 4 Fra, A. and Sitia, R. (1993) Subcell. Biochem. 21, 143-168

98. Bonifacino, J.S., C o s s o n , P., S h a h . N . and Klauser, R . D . (1991) E M B O J. 10, 2783-2793 99. Zvaritch, E., Vellani, F„ Guerini, D . and Carafoli, E. (1995) J. Biol. C h e m . 270, 2679-2688 100. T o l c h i n s k y , S., Y u k , M . H . , A y a l o n , M „ L o d i s h , H . F . and L e d e r k r e m e r , G . Z . ( 1 9 9 6 ) J . B i o l . Chem. 271,14496-14503 101. Stafford, F.J. and Bonifacino, J.S. (1991) J . Cell Biol. 115, 1225-1236 102. Vertel, B . M . , Velasco, A., LaFrance, S., Walters, L. and K a c z m a n - D a n i e l , K. (1989) J. Cell Biol. 109, 1827-1836 103. L i p p i n c o t t - S c h w a r t z , J . , B o n i f a c i n o , J . S . , Yuan, L . C . and K l a u s e n e r , R . D . (1988) Cell 54, 209-220 104. Wikstrom, L. and L o d i s h , H.F. (1992) J . Biol. C h e m . 2 6 7 , 5 - 8 105. 106. 107. 108. 109.

Y o u n g , J., Kane, L.P., E x l e y , M . and Wileman, T. (1993) J. Biol. C h e m . 268, 19810-19818 A l o n s o , M., H i d a l g o , J., Hendricks, L. and Velasco, A . (1996) Biochem. J . 3 1 6 , 4 8 7 - 4 9 5 Cotner, T. (1992) J. Immunol. 1 4 7 , 2 1 6 3 - 2 1 6 8 Milgram, S.L. and Mains, R.E. (1994) J. Cell Sci. 107, 737-745 H r e s k o , R . C . , M u r a t a , H . , M a r s h a l l , B . A . and Mueckler, M . (1994) J . Biol. C h e m . 269, 32110-32119

209

R . Q a n b a r and C . E . M a c h a m e r

210

110. Persson, R. and Pettersson, R.F. (1991) J . Cell Biol. 1 1 2 , 2 5 7 - 2 6 6 111. Balch, W.E., M c C a f f e r v , J . M . , Plutner, H . a n d F a r q u h a r , M . G . (1994) Cell 76, 8 4 1 - 8 5 2 112. M i z u n o , M . and Singer, S.J. (1993) Proc. N a t l . A c a d . Sci. U . S . A . 90, 5 7 3 2 - 5 7 3 6 113. L u z i o , J.P. and Banting, G . (1993) Trends B i o c h e m . Sci. 18, 3 9 5 - 3 9 8 114. N i l s s o n , T. and Warren, G . (1994) Curr. O p i n . Cell Biol. 6, 517-521 115. Sonnichsen, B., Fullekrug, J., Van N g u y e n , P., D i e k m a n n , W., R o b i n s o n , D . G . and M i e s k e s , G . (1994) J. Cell Sci. 1 0 7 , 2 7 0 5 - 2 7 1 7 116. Pelham, H . R . (1995) Curr. O p i n . Cell Biol. 7 , 5 3 0 - 5 3 5 117. B o l e , D . G . , D o w i n , R., D o r i a u x , M . and J a m i e s o n , J . D . ( 1 9 8 9 ) J . H i s t o c h e m . C y t o c h e m . 37, 1818-1823 118. M i e s e n b o c k , G . and R o t h m a n , J . E . (1995) J. Cell Biol. 129, 3 0 9 - 3 1 9 119. M u n r o , S. and Pelham, H . R . (1987) Cell 48, 8 9 9 - 9 0 7 120. H a u g e j o r d e n , S.M., Srinivasan, M . and G r e e n , M . (1991) J . Biol. C h e m . 2 6 6 , 6 0 1 5 - 6 0 1 8 121. N a p i e r , R . M . , F o w k e , L . C . , H a w e s , C . , L e w i s , M . and P e l h a m , H . R . ( 1 9 9 2 ) J . C e l l Sci. 102, 261-271 122. D e n e c k e , J., D e Rycke, R. and B o t t e r m a n . J . (1992) E M B O J . 1 1 , 2 3 4 5 - 2 3 5 5 123. Vennema, H . , Heijnen, L „ Rottier, P.J., H o r z i n e k , M . C . and Spaan, W.J. (1993) Adv. E x p . Med. Biol. 3 4 2 , 2 0 9 - 2 1 4 124. Zhen, L „ Rusiniak, M . E . and S w a n k , R.T. (1995) J . Biol. C h e m . 270, 11912-11920 125. L e w i s , M.J. and Pelham, H . R . (1990) N a t u r e ( L o n d o n ) 3 4 8 , 1 6 2 - 1 6 3 126. L e w i s , M.J. and Pelham, H . R . (1992) Cell 68, 353-364 127. Semenza, J . C . , H a r d w i c k , K . G . , Dean, N . and Pelham, H . R . (1990) Cell 6 1 , 1 3 4 9 - 1 3 5 7 128. Semenza, J . C . and Pelham, H . R . (1992) J. M o l . Biol. 2 2 4 , 1 - 5 129. H a r d w i c k , K . G . , B o o t h r o y d , J . C . , R u d n e r , A . D . a n d P e l h a m , H . R . ( 1 9 9 2 ) E M B O J . 11, 4187—4195 130. 131. 132. 133. 134.

Beh, C.T. and R o s e , M . D . (1995) Proc. N a t l . A c a d . Sci. U . S . A . 9 2 , 9 8 2 0 - 9 8 2 3 Townsley, F.M., Frigerio, G . and Pelham, H . R . (1994) J . Cell Biol. 1 2 7 , 2 1 - 2 8 Sweet, D . J . and Pelham, H . R . (1992) E M B O J. 11, 4 2 3 - 1 3 2 Peter, F., Van N g u y e n , P. and Soling, H . D . (1992) J . Biol. C h e m . 2 6 7 , 1 0 6 3 1 - 1 0 6 3 7 J a c k s o n , M . R . , N i l s s o n , T. and Peterson, P. A . (1990) E M B O J. 9, 3 1 5 3 - 3 1 6 2

135. Schutze, M.P., Peterson, P.A. and J a c k s o n , M . R . (1994) E M B O J. 1 3 , 1 6 9 6 - 1 7 0 5 136. Schindler, R „ Itin, C „ Zerial, M., Lottspeich, F. and H a u r i , H.P. (1993) Eur. J. Cell Biol. 6 1 , 1 - 9 137. S c h r o d e r , S., S c h i m m o l l e r , F., S i n g e r - K r u g e r , B . and R i e z m a n , H . ( 1 9 9 5 ) J . C e l l B i o l . 131, 895-912 138. C o s s o n , P. and Letourneur, F. (1994) Science 263, 1629-1631 139. L e t o u r n e u r , F., G a v n o r , E . C . , H e n n e c k e , S., D e m o l l i e r e , C . , E m r , S . D . , R i e z m a n , H . and C o s s o n , P. (1994) Cell 7 9 , 1 1 9 9 - 1 2 0 7 140. Kappeler, F., Itin, C . , Schindler, R. and H a u r i , H.P. (1994) J. Biol. C h e m . 269, 6 2 7 9 - 6 2 8 1 141. Itin, C „ Schindler, R. and H a u r i , H.P. (1995) J. Cell Biol. 1 3 1 , 5 7 - 6 7 142. N a r a y f e j e s t o t h , A . and Fejestoth, G . (1996) J . Biol. C h e m . 2 7 1 , 1 5 4 3 6 - 1 5 4 4 2 143. K u r o k i , K „ R u s s n a k , R. and G a n e m , D . (1989) M o l . Cell. Biol. 9, 4 4 5 9 - M 6 6 144. Ahn, K., S z s z e s n a - S k o r u p a , E . and Kemper, B. (1993) J . Biol. C h e m . 2 6 8 , 1 8 7 2 6 - 1 8 7 3 3 145. Finley, E . M . and K o r n f e l d , S. (1995) J . Biol. C h e m . 2 6 9 , 3 1 2 5 9 - 3 1 2 6 6 146. S z s z e s m a - S k o r u p a , E., A h n , K., C h e n , C . D . , D o r a y , B . and K e m p e r , B. (1995) J . Biol. C h e m . 270,24327-24333 147. M u r a k a m i , K., Mihara, K . and O m u r a , T. (1994) J . B i o c h e m . ( T o k y o ) 116, 164-175 148. Futterer, A., K r u p p a , G „ K r a m e r , B . , L e m k e , H . a n d K r o n k e , M . ( 1 9 9 5 ) M o l . B i o l . Cell 6, 161-170 149. Schiller, M . R . , Mains, R . E . and Eipper, B . A . (1995) J. Biol. C h e m . 270, 2 6 1 2 6 - 2 6 1 3 8 150. K e l l o k u m p u , S., S o r m u n e n , R., H e i k k i n e n , J . a n d M y l l y l a , R. ( 1 9 9 4 ) J . B i o l . C h e m . 2 6 9 , 30524-30529 151. Mallabiabarrena, A . J i m e n e z , M.A., Rico, M . and Alarcon, B. (1995) E M B O J . 1 4 , 2 2 5 7 - 2 2 6 8 152. C h e n , S.Y., M a t s u o k a , Y. and C o m p a n s , R.W. (1991) Virology 183, 3 5 1 - 3 6 5 153. Letourneur, F., Hennecke, S „ Demolliere, C . and C o s s o n , P. (1995) J . Cell Biol. 1 2 9 , 9 7 1 - 9 7 8 154. M e l i n , L., P e r s s o n , R . , A n d e r s s o n , A . , B e r g s t o r m , A . , R o n n h o l m , R. and P e t t e r s s o n , R.F. (1995) Virus Res. 3 6 , 4 9 - 6 6 155. B u p p , K., Stillmock, K. and G o n z a l e z s c a r a n o , F. (1996) Virology 220, 4 8 5 - 4 9 0

Localization t o endoplasmic reticulum and Golgi

156. Bernascone, E , Fasel, N . and Wittek, R. (1996) J. Cell Sci. 109, 1195-1201 157. Machamcr, C . E . (1993) Curr. O p i n . Cell Biol. 5 , 6 0 6 - 6 1 2 158. Gleeson, P.A., Teasdale, R . D . and Burke, J. (1994) G l y c o c o n j . J . 1 1 , 3 8 1 - 3 9 4 159. H e n d r i c k s , L . C . , G a b e l , C . A . , Suh, K. and F a r q u h a r , M . G . ( 1 9 9 1 ) J . Biol. C h e m . 266, 17559-17565 160. Machamer, C . E . and Rose, J . K . (1987) J . Cell Biol. 105, 1205-1214 161. Swift, A . M . and Machamer, C . E . (1991) J. Cell Biol. 115, 19-30 162. Tang, B.L., Wong, S.H., L o w , S.H. and H o n g , W. (1995) J . Biol. C h e m . 267 , 10122-10126 163. N i l s s o n , T., L u c o c q , J . M . , Mackav, D . and Warren, G . (1991) E M B O J. 10, 3 5 6 7 - 3 5 7 5 164. R u s s o , R . N . , Shaper, N . L . , Taatjes, D . J . and Shaper, J . H . (1992) J. Biol. C h e m . 267 , 9241-9247 165. B a n f i e l d , D . K . , L e w i s , M . J . , R a b o u i l l e , C . , Warren, G . and Pelham, H . R . ( 1 9 9 4 ) J . Cell Biol. 127,357-371 166. 167. 168. 169.

Bretscher, M.S. and M u n r o , S. (1993) Science 261 , 1280-1281 M u n r o , S. (1995) Biochem. Soc. Trans. 2 3 , 5 2 7 - 5 3 0 M u n r o , S. (1995) E M B O J. 1 4 , 4 6 9 5 - 4 7 0 4 N i l s s o n , T „ R a b o u l l i e , C „ H u i , N „ W a t s o n , R. and Warren, G . ( 1 9 9 6 ) J. Cell Sci. 109, 1975-1989

170. Bankaitis, V.A., Malehorn, D . E . , Emr, S.D. and Greene, R. (1989) J. Cell Biol. 108, 1271-1281 171. B a n k a i t i s , V.A., A i t k e n , J . R . , C l e v e s , A . E . and D o w h a n , W. (1990) N a t u r e ( L o n d o n ) 347, 561-562 172. M c G e e , T.P., Skinner, H . B . , Whitters, E . A . , H e n r y , S.A. and Bankaitis, V.A. ( 1 9 9 4 ) ] . Cell Biol. 124,273-287 173. Terui, T., Kahn, R . A . and R a n d a z z o , P.A. (1994) J . Biol. C h e m . 2 6 9 , 2 8 1 3 0 - 2 8 1 3 5 174. K t i s t a k i s , N . T . , B r o w n , H . A . , Sternweis, P.C. a n d R o t h , M . G . (1995) P r o c . N a t l . A c a d . Sci. U.S.A. 9 2 , 4 9 5 2 ^ 9 5 6 175. D e Camilli, P., Emr, S . D , McPherson, P.S. and N o v i c k , P. (1996) Science 271, 1533-1539 176. N i l s s o n , T., H o e , M . H . , S u s a r e w i c z , P , R a b o u i l l e , C . , Watson, R „ H u n t e , F., Watzele, G . , Berger, E . G . and Warren, G . (1994) E M B O J. 13, 562-574 177. N i l s s o n , T „ Susarewicz, P., H o e , M . H . and Warren, G . (1993) F E B S Lett. 330, l ^ t 178. M a c h a m e r , C . E . , G r i m , M . G . , E s q u e l a , A . , C h u n g , S.W., Rolls, M. and R y a n , K . (1993) M o l . Biol. Cell 4 , 6 9 5 - 7 0 4 179. Weisz, O . A , Swift, A . M . and Machamer, C . E . (1993) J. Cell Biol. 1 2 2 , 1 1 8 5 - 1 1 9 6 179a. Sevier, C.S. and Machamer, C . E . (1998) Biochemistry 37, 185-192 180. C o l e , N . B . , Smith, C . L . , Sciaky, N , Terasaki, M , Edidin, M. and Lippincott-Schwatz, J. (1996) Science 273, 797-801 181. G o n a t a s , J . O . , Mezitis, S . G , Stieber, A., Fleischer, B. and G o n a t a s , N . K . (1989) J . Biol. C h e m . 264,646-653 182. J o h n s t o n , P.S., Stieber, A . and G o n a t a s , N . K . (1994) J. Cell Sci. 107, 529-537 183. Harris, S.L. and Waters, G . M . ( 1996) J. Cell Biol. 1 3 2 , 9 8 5 - 9 9 8 184. M o l l o y , S . S , T h o m a s , L . , VanSlyke, J . K , Stenberg, P.E. and T h o m a s , G . (1994) E M B O J . 13, 18-33' 185. 186. 187. 188. 189.

Redding, K , H o l c o m b , C . and Fuller, R.S. (1991) J. Cell Biol. 113, 527-538 Wilcox, C . A , Redding, K „ Wright, R. and Fuller, R.S. (1992) Mol. Biol. Cell 3, 1353-1371 H u m p h r e y , J . S , Peters, P . J , Yuan, L . C . and B o m f a c i n o , J.S. (1993) J . Cell Biol. 120, 1123-1135 Seeger, M . and Payne, G.S. (1992) J. Cell Biol. 1 1 8 , 5 3 1 - 5 4 0 Schafer, W , Stroh, A , B e r g h o f e r , S „ Seller, J., Vey, M , K r u s e , M . L , Kern, H . F , K l e n k , H . D . and G a r t e n , W. (1995) E M B O J . 1 4 , 2 4 2 4 - 2 4 3 5

190. Scherer, P . E , L e d c r k r e m e r , G . Z , Williams, S , F o g l i a n o , M , Baldinin, G . and L o d i s h , H . F . (1996) J. Cell Biol. 1 3 3 , 2 5 7 - 2 6 8

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12 Import and export of proteins at the nucleus Naoko ImamotolYoichi Miyamoto and Yoshihiro Yoneda*

Department of Anatomy and Cell Biology, Osaka University Medical School, 2-2 Yamada-oka, Suita, Osaka 565-0871 ,Japan

Introduction Transport across the nuclear envelope, to a region where genes are sequestered in the nuclear compartment, is dissimilar to protein translocation across other types of biological membranes, such as mitochondria, endoplasmic reticulum and peroxisomes, in that transport occurs in both directions. Bidirectional transport provides an important avenue of communication between cytoplasm and nucleus. To enter and exit the nucleus, molecules translocate through a large proteinaceous structure called the nuclear pore complex (NPC), which spans the double lipid bilayer of the nuclear envelope (reviewed in [1-4]). This structure, of approx. 125 MDa, contains aqueous channels of 9 nm diameter that permit the passive diffusion of small molecules, such as ions, low-molecular-mass metabolites and proteins smaller than 20-40 kDa. Nearly all macromolecules are transported through the gated channels of the NPC by active and signal-mediated mechanisms. This review focuses on aspects of the mechanism for the bidirectional transport of proteins with emphasis on the properties of signals that direct molecules into and out of the nucleus, along with transport factors involved in these processes.

Nuclear import of proteins Nuclear uptake of proteins is highly selective, and considerable information regarding the permeability of NPCs and the selectivity of proteins was provided by early studies using living cells (reviewed in [5,6]). The discovery of the nuclear localization signal (NLS) and the development of in vitro systems [7,8] that reliably mimic in vivo transport have provided powerful tools for the investigation of the mechanism of nuclear protein import, and have led to the identification of the transport factors listed in Table 1. Experimentally, both in living cells and in the in vitro system, transport can be essentially divided into two steps: an NLS-dependent

*7o whom correspondence should be addressed.

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Table 1

Nuclear import factors

Vertebrate proteins

_____

Drosophila homologues

Yeast homologues

Pendulin/0H031

SRPIp/KAP65

hsp70, hsc70 Importina Karyopherin α NLS receptor PTAC58 hSRPI/NPI I Rchl/hSRPI-a Importin β

KAP95

Karyopherin β

PTAC97 p97 Gsp lp, Gsp2p, Cnrl p,

Ran/T C4 Cnr2p ρ 10/NTF2

B2 but energy-independent NPC binding step (first step), and an energy-dependent NPC translocation step (second step) [9,10],

NLSs The selective import of nuclear proteins into the nucleus is directed by a sequence of amino acids, referred to as the NLS, which is present within the primary structure of the nuclear protein. The concept of the NLS arose from studies by Dingwall et al. [11], who provided clear evidence that the nuclear import of nucleoplasmin, a 165 kDa pentameric nuclear protein in Xenopus , is dependent on the C-terminal 'tail' portion that does not bind to the interior structure of the nucleus. The presence of the NLS was established from a series of precise studies on a short amino acid sequence that directs simian virus 40 (SV40) T-antigen to the nucleus. These studies, which originated from the identification of a naturally occurring mutant virus defective in the nuclcar localization of T-antigen, showed that the minimum sequence PKKKRKV was both necessary and sufficient for directing proteins into the nucleus [12-17]. Numerous NLS structures have now been identified in a variety of nuclear proteins. These structures show complexity and divergence in structure, with a weak consensus, except that in most cases basic residues play a crucial role in determining activity (reviewed in [18-20]). Robbins et al. [21] first discovered the bipartite-type NLS from nucleoplasmin. This type of NLS contains two short basic

Import and export of proteins at the nucleus

amino acid clusters separated by about 10 spacer amino acids. Manipulation of the spacer region and the basic amino acid clusters indicated that the two basic domains are in close proximity in the tertiary structure of the NLS and function as one unit. More recently, the NLS of NGFI-A, a transcription factor encoded by an immediate-early gene, was shown to have a much more complex structure, involving 105 amino acids that constitute three highly basic C 2 H 2 zinc fingers [22], Substitution of cysteine residues, which disrupt the zinc-finger structure, led to the disruption of nuclear localization activity, thus providing evidence that the overall zinc-finger structure acts as an NLS. Several nuclear proteins have been found to contain two or more NLSs, which can function interdependently or independently. For example, the glucocor­ ticoid receptor has a hormone-inducible NLS and a second signal which becomes constitutively active only after the hormone-binding domain is deleted [23]. The muscle-specific transcription factor MyoD has two adjacent NLSs which appear to function independently of each other [24], The c-abl proto-oncogene product, whose cytostatic function is absolutely dependent on its nuclear localization, was shown to possess three structurally different NLSs which can function indepen­ dently [25]. On the other hand, there are many nuclear-localized proteins whose NLSs have not yet been identified. The structural analysis of NLSs (primary, secondary and tertiary), together with the regulation of activity of NLSs under cellular conditions in animal tissues, promise to become important topics in the next decade, with respect to the divergency of NLS receptor proteins described in a later section.

Identification of NLS-recognizing cytoplasmic factors Goldfarb et al. [14] provided early evidence, based on saturation kinetics, that nuclear import is a receptor-mediated process. The issue of whether NLS receptor proteins are present in the cytoplasm or the NPC was debated for some time, until several lines of evidence indicated that NLS receptor proteins are cytosolic. Breeuwer and Goldfarb [26] demonstrated the presence of a saturable NLSbinding protein in the cytoplasm of tissue-cultured mammalian cells by examining the nuclear accumulation of cytoplasmically injected small nuclear proteins, such as histone HI, and small reporter proteins conjugated to an SV40 T-antigen NLS peptide. In spite of the fact that they were sufficiently small to diffuse passively through the NPC, these proteins did not diffuse into the nucleus when injected into the cytoplasm. The authors proposed that small karyophilic proteins are complexed by an NLS receptor that is present in the cytoplasm. Newmeyer and Forbes [27], using Xenopus egg extracts and reconstituted nuclei, showed that the N-ethylmaleimide (NEM)-sensitive activity of a cytosolic fraction, called NIFl, was capable of restoring the binding step of the import process. This provided the first biochemical evidence that the receptor proteins are probably soluble/ cytosolic components.

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A breakthrough in the identification of NLS receptor proteins, as well as other transport factors described in later sections, was achieved as a result of the development of an in vitro transport system using digitonin-permeabilized semiintact cells [8], As in living cells, transport in this system depends on the NLS and energy, and is inhibited by wheat germ agglutinin [28,29], which recognizes the unique O-Iinked GlcNAc of nucleoporins. Adam and Gerace [30] purified the 54/56 kDa NLS-binding protein(s) [31] and showed, using a digitonin-permeabilized cell-free system, that this protein functionally supports NLS-mediated import. The 54/56 kDa protein, which they termed the NLS receptor, supported import in the presence of other cytosolic factors, one of which was shown to be a 97 kDa NEM-sensitive factor (referred to as p97) [32]. This protein co-operates with the 54/56 kDa NLS receptor in the binding step of nuclear import. Moore and Blobel [33] demonstrated that one of two complementary fractions obtained from a Xenopus egg extract, termed fraction A, supported binding to the NPC of an SV40 T-antigen-NLS conjugate in the permeabilized cell-free assay. This fraction contained an NEM-sensitive active component that elutes at approx. 200 kDa in a sizing column. In later studies, this fraction was shown to contain a 97 kDa protein (called karyopherin β), which interacts with several nucleoporins [34,35], and an NLS receptor (karyopherin a) [36], Gorlich et al. [37] purified and cloned a 60 kDa protein from Xenopus egg extracts, termed importin (now called importin a), which mediated the NPC binding of nucleoplasmin in the permeabilized cell-free assay. The primary structure of this protein revealed that it is a Xenopus homologue of a previously identified yeast (Saccbaromyces eerevisiae) protein called SRPlp [38]. In a subsequent study, it was observed that a 90 kDa protein (now called importin β), which co-precipitates with the 60 kDa protein, stimulates the binding step of the import process in conjunction with the 60 kDa protein [39]. On the other hand, using the digitonin-permeabilized cell-free transport assay and fractionated cytosol obtained from mouse Ehrlich ascites tumour cells, our studies found that a karyophilic protein containing an SV40 T-antigen NLS forms a stable complex with cytosolic components, and this complex shows NPCtargeting activity [40]. The complex, termed the nuclear-pore-targeting complex (PTAC), was sufficiently stable to permit one-step affimtv purification from the crude fraction. PTAC contained four cytosolic proteins of 54, 56,66 and 90 kDa. In vitro reconstitution of the complex revealed that the NPC-targeting activity of PTAC was dependent on the presence of both the 54 and 90 kDa components. cDNA cloning of the two essential components of PTAC revealed that the smaller protein is a mouse homologue of Xenopus importin a and yeast SRPlp, having a calculated molecular mass of 58 kDa [41]. The larger protein was found to be a novel protein of calculated molecular mass 97 kDa [42], which was found to be homologous with importin β [39], p97 [43] and karyopherin β [35] that were cloned independently at the same time.

Import and export of proteins at the nucleus

Cytoplasmically injected affinity-purified antibodies to a 58 kDa component of the PTAC (PTAC58) inhibited nuclear import and co-precipitated a karyophile in the cytoplasm. This provided in vivo evidence that this protein is involved in nuclear import through association with an NLS in the cytoplasm before the import substrate binds to nuclear pores in NLS-mediated nuclear import [41]. Using recombinant proteins, it was shown in vitro that the three components of the PTAC target nuclear pores as a single entity [39,42-45].

The SRP1 family A number of SRPlp-related proteins were identified using various biological screening techniques, in addition to the nuclear import assay. SRPl was originally identified as a suppressor of certain temperature-sensitive mutations of RNA polymerase I in S. cerevisiae [38]. The gene product, SRPlp, was shown by indirect immunofluorescence to localize to the nuclear rim. Belanger et al. [46] found that this protein shows genetic and physical interactions with the yeast nucleoporins Nuplp and Nup2p. SRPl is an essential gene, and its mutation leads to pleiotropic phenotypes, such as defects in transcription, RNA processing, nuclear division and segregation, as well as fragmentation of the nucleolus [38,47]. A Drosophila homologue of yeast SRPlp, known as pendulin or 0H031, was identified as a tumour suppressor in haematopoietic cells [48,49]. Human homologues of yeast SRPlp, called Rchl [50] and hSRPl/NPIl [51,52], were identified in yeast twohybrid screens by their interaction with the recombination-activating protein RAG-I and influenza nucleoprotein. Weis et al. [53] identified human Rchl (described as hSRPl-α) in yeast two-hybrid screens by its interaction with mRNA cap-binding protein 80 (CBP80). Analysis of protein sequence databases revealed a number of proteins that are closely related to SRPlp (amino acid sequence identity between 40 and 99%) present in yeast, animal, plant and mammalian cells. PTAC58 shows, respectively, 94, 65, 52 and 46% amino acid identity with human Rchl, Xenopus importin a, Drosophila pendulin/0H031 and yeast SRPlp. However, it shows only 44% amino acid identity with hSRP1 /NPI1. Rch1 and hSRP 1ρ were both obtained from a HeLa cell cDNA library, showing that a single cell contains at least two or more SRPlp-related proteins. The Xenopus cDNA library yielded six closely related clones of importin, which differed by between one and 22 amino acids [37], The 56 and 66 kDa proteins that were incorporated in the PTAC obtained from mouse Ehrlich ascites tumour cells [40] show specific NLS-binding activity and cross-react weakly with antibodies raised against PTAC58, suggesting that these two proteins may also be members of the SRPlp family (N. Imamoto and Y. Yoneda, unpublished work). Such divergency in the SRPlp family raises new questions. Unlike yeast SRP1, the Drosophila gene encoding pendulin/0H031 is not an essential gene [48,49], strongly suggesting that substituting factor(s) exist. However, the tissuespecific tumour suppressor activity of pendulin/0H031 suggests that certain

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members of the family possess specific functions in specific tissues. In this respect, it is noteworthy that the developmentally regulated and tissue-specific expression of certain members of the SRPlp family has been reported [48,49,54], Whether divergency in the SRPlp family reflects the divergency of NLSs, or is present for another purpose, remains to be determined. Indirect immunofluorescence studies using affinity-purified antibodies against PTAC58 located this protein in the cytoplasm, nuclear rim and nuclear interior, while nucleolar staining was not observed during the interphase of mammalian cultured cells [41]. A portion of cytoplasmically injected antibodies migrated rapidly into the nucleus in a temperature-dependent manner, providing in vivo evidence for the dynamic movement of this protein across the nuclear envelope [41]. The digitonin-permeabilized cell-free import assay revealed that this protein accumulates in the nucleus, presumably with an NLS substrate, during active nuclcar import [44,45], The nuclcar accumulation of this protein was shown to be directed by the conserved N-terminal 41 amino acids, which confer binding to its 97 kDa import partner [55,56]. At the transition between the G2 and M phases, Drosophila pendulin/ 0H031 was shown to accumulate massively in the nucleus and become concen­ trated around the chromosomes during mitosis [48,49], This was also observed for PTAC58 in cultured mammalian cells (N. Imamoto, M. Matsubae and Y. Yoneda, unpublished work) and for Xenopus importin α in cultured Xenopus A6 cells (M. Ochi, N. Imamoto and Y. Yoneda, unpublished work). The properties of the cellcycle-dependent nuclear localization of this protein family and its physiological significance remain to be resolved. Many members of the SRPlp family of proteins contain eight repeating motifs of 42 or 43 hydrophobic amino acids, known as armadillo or arm repeats [57]. This repeating motif was originally identified in the Drosophila segmentpolarity gene product armadillo, and has been found in several proteins that have diverse cellular functions, such as armadillo's vertebrate homologues (β-catenin, plakoglobin and ρ 120), the tumour suppressor adenomatous polyposis coli, and an exchange factor for Ras-related small GTP-binding proteins (smgGDS). The arm repeats are thought to mediate protein-protein interactions. For the SRPlp family, at least one of the functions of the arm repeats is specific NLS binding (Figure 1).

The 97 kDa component A 97 kDa component of PTAC (PTAC97, importin β, karyopherin β, p97) is, thus far, a unique protein. However, database searches indicate the presence of related proteins. In some cell extracts, e.g. Xenopus eggs, two additional protein bands that are slightly larger than PTAC97 can be detected specifically by affinity-purified antibodies against this protein (M. Ochi, N. Imamoto and Y. Yoneda, unpublished work), although their function is unknown. A product of the gene L8300 has been shown to be a S. cerevisiae homologue of the mammalian 97 kDa component (termed KAP95 [58]). This protein forms a complex with yeast SRPlp (termed

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Figure 1

The armadillo structure of PTAC58 has binding activity for the NLS of SV40Tantigen A) 444_529(aa)

Full

GST

N-Arm GST

Arm

GST

•

= arm repeat sequence = non repeat sequence

Βϋ(-) •Wild-T •Rev-T

S ©

¢/5 ω

Full

N-Arm

Arm

(A) Schematic structures of full-length and two deletion mutants of PTAC58. PTAC58 contains eight arm repeats within amino acids (aaj 99—443. N-Arm is a mutant protein in which the C-terminal sequence (aa 444—529) ofPTAC58 is deleted.Arm is a mutant protein in which both the N-terminal (aa 1-99) and C-terminal (aa 444—529) sequences ofPTAC58 are deleted.All three recombinant proteins were expressed as glutathione S-transferase (CST) fusion proteins. (B)An

125I-Iabelled

SV40T-antigen NLS

conjugate [synthetic peptide CYGGPKKKRKVEDP conjugated with biotinylated BSA (T-bBSA)] was incubated with full-length PTAC58 or one of the two deletion mutant proteins (fused to GST) in the absence (-) or presence of non-labelled wild-type SV40T-antigen NLS conjugate (WiId-T) or of a transport-inactive reverse T-antigen NLS (CYGGPDEKVKRKKKP) conjugate (Rev-T).After incubation, the GST fusion proteins were trapped in glutathione—Sepharose, and the amount of radioactivity associated with each GST fusion protein was measured using a y-radiation counter.

KAP60) and targets the SV40 T-antigen NLS substrate to the nuclear rim of digitonin-permeabilized mammalian cells, indicating that NLS-recognizing cytoplasmic components are conserved between yeast and higher eukaryotes. Three biochemical interactions involving this 97 kDa protein have been identified. First, it binds directly to a heterogeneous SRPlp family member

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[39,42,44]. Combination of this protein with the SRPlp-related protein in a 1:1 molar ratio completely restores the nuclear rim binding of NLS substrate in digitomn-permeabilized cells [32,39,42]. In in vitro experiments, PTAC58 and PTAC97 associate in a 1:1 molar ratio [42]. The second interaction is its ability to associate with the NPC. Direct interaction of this protein with several mammalian or yeast nucleoporins containing FXFG repeats (and one containing a GLFG repeat) has been demonstrated using ligand-overlay assays [34,59,60]. The third interaction is the ability of this component to bind the small GTPase Ran/TC4 ([61]; S. Rose, N. Imanioto, T. Tachibana and Y. Yoneda, unpublished work). These three interactions serve to connect the fundamental transport machineries involved in NLS-mediated nuclear import.

Identification of soluble factors mediating the second step of nuclear import The identification of soluble factors involved in the translocation step was achieved using the digitomn-permeabilized cell-free transport assay. Moore and Blobel [33] showed that one of two fractions obtained from Xenopus egg extracts (fraction B) mediated the translocation step of nuclear import. By analysing the active components in this fraction, they identified two cytosolic components: Ran/TC4 [62] and a dimeric protein called plO [63]. Independently, Melchior et al. [64] found that a non-hvdrolvsable GTP analogue inhibited import in the digitoninpermeabilized cell-free assay. By analysing the factor responsible for this inhibition, they identified Ran/TC4 as a component involved in nuclear transport. Paschal et al. [65] found that the transport activity of unfractionated cytosol can be depleted by incubation with immobilized mammalian nucleoporin p62. The adsorbed protein responsible for the transport activity was identified and found to be identical to protein pi C (referred to as NTF2).

Small GTPase Ran/TC4 Ran/TC4 is the only known GTPase of the Ras superfamily that is present in the mammalian cell nucleus. As with other members of the Ras superfamilv, the GTPase cycle of Ran is regulated by the guanine-nucleotide-exchange factor (GEF), which facilitates the exchange of GTPase-bound GDP with GTP, and bv a GTPase-activating protein (GAP) [66]. The only known GEF for Ran is RCCl, which is a chromatin-associated nuclear protein containing an NLS (reviewed in [67]). GAPs for Ran have been identified both in the cytoplasm [68,69] and in the nucleus [7C], Three additional GTP-Ran binding proteins have been identified: the 24 kD a soluble protein RanBPl [71,72], a 358 kDa nuclear pore protein, RanBP2 Nup35S [73,74], and PTAC97. Association of GTP-Ran with RanBPl stimulates the GAP-activated GTP hydrolysis of Ran [72], while association with PTAC97 inhibits this activity ([75]; T. Tachibana and Y. Yoneda, unpublished work). The biological significance of modulation of the GTPase cvele of Ran through such interactions remains to be elaborated.

Import and export of proteins at the nucleus

Ran is highly conserved from yeast to higher eukaryotes. In addition, GEF and GAP proteins for Ran have been identified in yeast. Defects in Ran and its regulatory proteins lead to pleiotropic phenotypes, suggesting that Ran is involved in a wide variety of cellular processes, such as cell cycle checkpoint control, RNA processing and export, maintenance of nuclear structure, and nuclear protein import (reviewed in [67,76]). Among these diverse cellular functions, a direct role for Ran has been established in nuclear protein import using a digitoninpermeabilized cell-free transport assay. The role of the RCCl-Ran/TC4 system in nuclear import was examined in living mammalian cells using temperature-sensitive mutant cells (tsBN2) [77]. tsBN2 cells are derived from a BHK21 cell line, and possess a point mutation in RCCl which leads to the rapid inactivation and degradation of this protein at nonpermissive temperatures [78]. Nuclear accumulation of a cytoplasmically injected SV40 T-antigen NLS conjugate was significantly decreased in tsBN2 cells cultured at a non-permissive temperature. However, the defect in nuclear import was incomplete. If the requirement for Ran is absolute, as indicated in the permeabilized cell-free system, this implies the presence of another GEF activity for Ran in addition to the activity for which RCC1, or other small GTPases, can partially substitute. The most unexpected finding in this study was the dominant-negative phenotype of tsBN2 cells observed in the heterokaryon of tsBN2 and wild-type BHK21 cells. In addition, substrate did not accumulate in the nuclei of digitoninpermeabilized non-permissive tsBN2 cells even in the presence of normal cytosol. The results show that loss of RCCl leads to pleiotropic defects in nuclear import, such as a decline in import competence of the nucleus and the accumulation of a factor in the cytoplasm that suppresses nuclear import. Therefore the GTPase cycle of Ran may be involved in a number of steps in nuclear import, such as co­ ordination and regulation of transport, in addition to the role that has been established in the in vitro transport assay. ρ 10/NTF2 plO/NTF2 supports the translocation step of nuclear import in conjunction with Ran/TC4. This protein binds to mammalian nucleoporin p62 [65] and to GDP-Ran [79]. A report by Nehrbass and Blobel [79] may shed light on the role of this protein in nuclear import. Using recombinant proteins of yeast pi 0/NTF2, KAP60 and KAP95, and a truncation product of yeast nucleoporin Nup36 containing FXFG repeats but lacking the Ran-binding domain, they showed that these four components form a transient pentameric complex with GDP-Ran. plO/NTF2 appears to play a key role in this complex-formation. Addition of GTP to this pentameric complex leads to its dissociation, which could be caused by the generation of GTP-Ran from GDP-Ran (see below). If this were the case, plO could be involved in the GEF activity present in the NPC that is required for NLSmediated nuclear import.

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Translocation model and questions Two cytosolic components of PTAC (importin α and β; karyopherin α and β) were shown to dissociate during the translocation step of NLS-mediated nuclear import by using the digitonin-permeabilized cell-free transport assay. Using yeast recombinant proteins, Rexach and Blobel [61] have provided biochemical data for a translocation model. They showed that (i) incubation of the KAP60-KAP95 complex with the FXFG repeat region of nucleoporins facilitates the dissociation of NLS substrate from the complex; and (ii) GTP-Ran causes dissociation of the NLS substrate from the KAP60-KAP95 complex, and even disassembly of the complex, by binding directly to KAP95. The reaction was shown to occur specifically with the GTP-bound form of Ran, but in the absence of GTP hydrolysis. The authors proposed that the translocation step of nuclear import involves repeated docking of NLS-recognizing cargo to FXFG-repeat-containing nucleoporins, and a Randependent undocking process in which vectorial movement of NLS substrate through the NPC is achieved through the lower-affinity docking site of NPC towards the higher-affinity docking site. The types of interactions among transport factors and NPC components, and how these relate to the vectorial movement of molecules through the NPC, however, remain open questions. Another as yet unresolved issue deals with the energy-dependency of the second step of nuclear import, which has been demonstrated repeatedly in many experimental systems. A requirement for GTP hydrolysis was proposed on the basis of in vitro evidence that Ran, when charged with a non-hydrolysable GTP analogue, potently inhibits nuclear import [64], The GTP hydrolysis of Ran was shown to occur at its NPC-binding site with, in all probability, RanBP2 [80]. This hydrolysis has been suggested to commit the translocation step of nuclear import. However, using the digitonin-permeabilized cell-free transport assay, there is, thus far, no direct evidence for a requirement for Ran GAP in the translocation step of import, although a requirement for this protein in nuclear import was shown by using a yeast mutant [81]. A requirement for ATP is much more vague. Early studies suggested that ATP hydrolysis may be required for expansion of the gated channels of the NPC for translocation of substrates [82], or for the movement of substrates along filaments postulated to traverse the NPC aided by an ATPase/motor molecule [83]. Thus far, however, there is little experimental evidence to support these proposals.

Heat-shock proteins of 70 kDa The involvement of a 70 kDa heat-shock protein (Hsp70 and Hsc70) in nuclear import was demonstrated both in living cells [84] and with the digitoninpermeabilized cell-free system [85-87], In living cells, the cytoplasmic injection of affinity-purified anti-Hsc70 antibodies strongly inhibited NLS-mediated nuclear import, but not the diffusive nuclear entry of small non-karyophilic proteins. The inhibitory effect of the antibody was not the result of cell death or depletion of ATP, since RNA synthesis continued normally, and the effect was reversible. In the

Import and export of proteins at the nucleus

digitonin-permeabilized cell-free transport assay, a requirement for Hsc/Hsp70 was demonstrated by depletion of this protein from the unfractionated cytosol, a procedure that led to a decline in transport activity. In addition, transport activity was restored by addition of purified Hsc/Hsp70 to the depleted cytosol. Interestingly, Yang and Defranco [87] showed that depletion of Hsc70 caused a decline in the transport of SV40 T-antigen, but not in the hormone-regulated nuclear import of the glucocorticoid receptor. If the low-affinity NLS-binding ability of Hsc70 [84,88,89] indeed has a role in nuclear import, the chaperone protein may function to present NLS to the high-affinity NLS receptor (SRPlp family), as proposed by Dingwall and Laskey [90]. Hormone binding to the glucocorticoid receptor may trigger presentation of the NLS to the receptor protein as well, and thus may not require chaperone activity. However, we must also consider an alternative role for Hsc70 in nuclear protein import. In spite of the potent inhibitory effect of anti-Hsc70 antibodies in living cells, and the decline in transport activity in Hsc/Hsp70-depleted unfrac­ tionated cytosol, almost no stimulation of transport activity was observed upon addition of purified Hsc70 to a reconstituted transport system comprising recombinant proteins of two cytosolic components of the PTAC, Ran and plO/NTF2 (N. Imamoto and Y. Yoneda, unpublished work). In crude cytosol, the two cytosolic components of the PTAC appear to associate independently with several soluble proteins in addition to their import partners (under investigation in our group; also indicated in [43,56]). The assembly of the PTAC, as well as the recycling of its components, may involve the disassembly (and assembly) of these associating proteins, which may require chaperone activity for co-ordination. Further studies are required to assess these possibilities.

Future research Significant progress has been made over the past two or three years in terms of understanding NLS-mediated nuclear import. The divergence of NLS receptors and their developmentally regulated, tissue-specific expression may provide an important avenue for tissue-specific gene regulation and cell growth. The direct involvement of Ran, which is also one of the key components of cell-cycle checkpoint control, suggests that nuclear import is closely related to cell-cycle progression. It is conceivable that.presently unidentified factors exist that modulate transport factors and NPC components. Moreover, it should be noted that transport factors described here were identified and confirmed using limited substrates, such as SV40 T-antigen NLS and nucleoplasmin. Thus the mechanisms of different import pathways, including the import of small nuclear ribonucleoproteins [91-93] and conditional transport, remain to be elucidated.

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Nuclear export of proteins In contrast with the nuclear import of proteins as described above, the nuclear export of proteins remains poorly understood [4,94,95]. The biological significance of nuclear export was first demonstrated by evidence for the existence of nuclear proteins that shuttle into and out of the nucleus [96-98]. One of the most fundamental cellular functions requiring protein shuttling is ribosome biogenesis [99], Ribosomal proteins are synthesized in the cytoplasm and transported into the nucleus to be assembled into pre-ribosomal subunits in the nucleolus. These subunits are then transported to the cytoplasm to mature. The nucleocytoplasmic shuttling of proteins was first demonstrated experimentally in heterokarvons by using nucleolar proteins as a probe. In these experiments, Borer et al. [100] demonstrated that chicken nucleolar proteins, such as nucleolin, migrate from chick nuclei to mouse nuclei in chick-mouse heterokarvons. Moreover, some proteins, e.g. the progesterone receptor [101], have been found to shuttle in a manner similar to nucleolin. Since then, various experimental approaches have been used to investigate the molecular mechanisms of the nuclear export of proteins. Schmidt-Zachmann et al. [102], using three classes of proteins (nucleolin, one of the first shuttling proteins identified, artificial nuclear import substrate containing the NLS, and lamins, proteins having the ability to be incorporated into the nuclear lamina), demonstrated that the export activity of proteins depends on their nuclear retention ability, i.e. their intranuclear interactions. The in vivo results also suggested that positively acting export signals may not be required for protein export from the nucleus. In contrast, Guiochon-Mantel et al. [103] showed, by means of heterokarvon formation and microinjection experiments, that the same NLSs play an important role in the bidirectional movement (i.e. import and export) of proteins through the nuclear envelope. They also showed that the nuclear export of proteins does not require energy, and suggested that nucleocytoplasmic shuttling may be a general biological phenomenon for nuclear proteins. Moreover, Moroianu and Blobel [104] demonstrated, using digitonin-permeabilized cells, that the nuclear export of NLS-containing transport substrates requires Ran GTPasc and GTP hydrolysis, but not ATP hydrolysis. Thus, in contrast with the mechanism for the nuclear import of proteins, it appears that the nuclear export of proteins does not require a specific signal, nor does it require ATP hydrolysis. However, these results do not exclude the possibility that some proteins are exported in a signal-mediated, energy-dependent manner.

Nuclear export signal (NES) During studies on the movement of the catalytic (C)-subunit of cAMP-dependent protein kinase, interesting information was obtained about the nuclear export of proteins themselves, in addition to that of RNA-protein complexes. Fantozzi et al. [105] and Wen et al. [106] showed that a thermostable inhibitor of cAMP-

Import and export of proteins at the nucleus

dependent protein kinase (PKI) enhances the export of the C-subunit of the kinase from the nucleus and carries a nuclear export signal. The C-subunit was found to be transported rapidly from the nucleus as a complex with PKI, and its export was dependent on temperature and energy. More recently, Wen et al. [107] actually identified the NES residing on PKI required for rapid, active export of the Csubunit-PKI complex from the nucleus. Residues 37^6 of PKI (LALKLAGLDI, as shown in Figure 2), when fused to heterologous proteins, were found to be sufficient for rapid export and to function as a minimal functional sequence. When hydrophobic residues such as the leucines or the isoleucine were replaced with alanines, a significant loss of export activity was observed, indicating that these hydrophobic residues are critical for activity. These authors also showed that Rev, an RNA-binding protein of HIV-1, has a similar sequence to act as a NES. Independently, in studies on the role of the Rev protein in HIV infection, especially in the nuclear export mechanism of unspliced and partially spliced HIV mRNAs encoding the viral proteins, Fischer et al. [108] identified the NES of HIV1 Rev. Later in HIV infection, the incompletely spliced and unspliced viral mRNAs are required to be transported to the cytoplasm. This nuclear export of viral mRNAs is mediated by the interaction of the Rev activation domain in the Rev protein with its binding site, the Rev response element, in the viral mRNAs. It was found that BSA conjugated with peptides containing the activation domain of Rev inhibited Rev-mediated export of RNAs containing the Rev response element, and that the BSA-peptide conjugates were exported from the nucleus in an active, saturable manner. These results indicate that the Rev activation domain (LPPLERLTL) acts as a NES required for the nuclear export of the Rev protein and of viral RNAs containing the Rev response element. Thus the NESs of both PKI and Rev consists of leucine-rich sequences of approx. 10 amino acids. On the other hand, one of the most abundant heterogeneous nuclear ribonucleoproteins, Al, which shuttles continuously between the nucleus and the cytoplasm, was found to have a NES with no sequence similarity to the NESs of PKI and Rev. Michael et al. [109] showed that a 38-amino-acid sequence within Al (termed M9; see Figure 2), which is required for the nuclear localization of A1, also acts as a NES, by placing M9 on the nucleoplasmin core domain. That is, although Figure 2

Nuclear export signals

PKI

LALKLAGLDI

Rev

LPPLERLTL

hnRNPA1

NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY

Abbreviation: hnRNP, heterogeneous nuclear ribonucleoprotein.

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the nuclcoplasmin corc domain, even when fused with classical NLSs, is not exported from the nucleus, it can be exported in a temperature-dependent manner when fused with M9. These results suggest that several different pathways may exist for the nuclear export of proteins.

Nuclear export machinery Since the NESs were discovered only very recently compared with the NLSs, studies on the mechanism of the nuclear export of proteins have just started. By using a yeast two-hybrid system, a protein that interacts with the Rev activation domain was identified in both humans (termed Rab/hRIP) [110,111] and yeast (termed Riplp) [112]. Although it has some repeats similar to those in nucleoporins and, at least, yeast Riplp was found to be localized in nuclear pores, the subcellular localization of human Rab does not indicate that Rab is a nucleoporin. However, Rab may interact with nucleoporins to target both Rev and Rev-binding RNA to the NPC by acting like an NES receptor in a manner similar to that in which nucleoporins interact with each other. Alternatively, Rev may interact with Rab and sequentially with nucleoporins to export its binding RNA. In yeast, it has been suggested that Rev may function as a carrier for Rev-binding RNA, taking it to Riplp located in the NPC.

Future research The NPC directs the bidirectional translocation of proteins. The mechanisms by which the nuclear import and export of proteins are regulated systematically via the NPC in cells present an intriguing question. At the present time, considerable fundamental information is available concerning protein and RNA export from the nucleus. In the near future, it will be determined whether or not several factors that have already been demonstrated to be involved in the nuclear import of proteins are also required for nuclear export. Thus, the more information we can obtain about the mechanism and machinery for the nuclear import and export of proteins, the nearer we will be to a complete understanding of the bidirectional exchange of cellular information through the NPC. We are grateful to members of our laboratory (Takuya Sbimamoto, Taro Tacbibana, Sbingo Kose, Tosbihiro Sekimoto, Masami Matsubae, Mariko Ocbi, Fumihiko Yokoyd', Mikt Hieda, Yosuke Matsuoka, and Sbunsuke Yuba), and to our collabo­ rators, Tosbifumi Takao and Yasutsugu Sbimonisbi from the Institute for Protein Research, Osaka University, Japan, for their persistent efforts throughout the course of our project, and for helpful discussions and comments.

References 1. 2. 3. 4.

Davis, L. ¢1995) Annu. Rev. Biochem. 64, 865-896 Fabre, E. and Hurt, E.C. (1994) Curr. Opin. Cell Biol. 6,335-342 Rout, MP. and Wente 1 S.R. (1994) Trends Cell Biol. 4, 357-365 Pante, N. and Aebi, U. (1995) Int. Rev. Cvtol. 162B, 225-255

Import and export of proteins at the nucleus

5. 6.

D i n g w a l l , C . and Laskev, R . A . (1986) A n n u . Rev. Cell Biol. 2, 3 6 7 - 3 9 0 Feldherr, C . (1992) in N u c l e a r Trafficking (Feldherr, C , ed.), p p . 15-28, A c a d e m i c Press, N e w York

7.

N e w m e y e r , D . D . , Finlay, D . R . and F o r b e s , D . J . (1986) J . Cell Biol. 103, 2 0 9 1 - 2 1 0 2

8. 9.

A d a m , S . A , Sterne-Marr, R. and Gerace. L. (1990) J . Cell Biol. 111, 8 0 7 - 8 1 6 R i c h a r d s o n , W . D , Mills, A . D , D i l w o r t h , R . A . , L a s k e v , R. and D i n g w a l l , C . ( 1 9 8 8 ) C e l l 52, 655-664

10. 11. 12.

N e w m e y e r , D . D . and F o r b e s , D . J . (1990) Cell 5 2 , 6 4 1 - 6 5 3 Dingwall, C , Sharnick, S.V. and Laskev, R . A . (1982) Cell 30, 4 4 9 - 4 5 8 L a n d f o r d , R . E . and Butel, J.S. (1984) Cell 37, 8 0 1 - 8 1 3

13.

K a l d e r o n , D . , R i c h a r d s o n , W.D., M a r k h a m , A.F. and Smith, A . E . (1984) N a t u r e ( L o n d o n ) 311, 33-38 G o l d f a r b , D . S . , G r a i e p v , J , S c h o o l n i k , G . and K o r n b e r g , R . D . ( 1 9 8 6 ) N a t u r e ( L o n d o n ) 332, 641-644

14. 15. 16. 17.

L a n d f o r d , R . E , K a n d a , P. and Kennedy, R . C . (1986) Cell 46, 5 7 5 - 5 8 2 Y o n e d a , Y , A r i o k a , T , I m a m o t o - S o n o b e , N . , S u g a w a , H , S h o m o n i s h i , Y. a n d U c h i d a , T. (1987) E x p . Cell Res. 170, 4 3 9 - 4 5 2 Chelsley, D , R a l p h , R. and J o n a k , G . (1989) M o l . Cell. Biol. 9, 2 4 8 7 - 2 4 9 2

18. 19.

Dingwall, C . and Laskey, R . A . (1991) Trends B i o c h e m . Sci. ! 6 , 4 7 8 - 4 8 1 G a r c i a - B u s t o s , J , H e i t m a n , J . and Hall, M . N . (1991) Biochim. B i o p h y s . Acta 1071, 83-101

20. 21.

L a C a s s e , E . C . and L e f e b r e , Y.A. (1995) N u c l e i c A c i d s Res. 23, 1647-1656 R o b b i n s , J , D i l w o r t h , S . M , Laskey, R . A . and D i n g w a l l , C . (1991) Cell 64, 15-23

22. 23. 24.

Matheny, C „ Day, M . L . and Milbrandt, J . C . (1994) J . Biol. C h e m . 269, 8176-8181 Picard, D . and Y a m a m o t o , K . R . (1987) E M B O J. 6, 3 3 3 3 - 3 3 4 0 V a n d r o m m e , M , C a v a d o r e , J . - C , Bonnie, A , Froeshle, A , L a m b , A . and F e r n a n d e z , A . (1995) Proc. N a t l . A c a d . Sci. U . S . A . 92, 4 6 4 6 - 4 6 5 0

25. 26. 27.

Wen, S . - T , J a c k s o n , P.K. and Etten, R.A.V. (1996) E M B O J . 1 5 , 1 5 8 3 - 1 5 9 5 Breeuwer, M . and G o l d f a r b , D . S . (1990) Cell 60, 9 9 9 - 1 0 0 8 N e w m e y e r , D . D . and F o r b e s , D . J . (1990) J . Cell Biol. 1 1 0 , 5 4 7 - 5 5 7

28. 29.

Finlay, D . R , N e w m e y e r , D . D , Price, T.M. and F o r b e s , D . J . (1987) J . Cell Biol. 104, 189-200 Y o n e d a , Y , I m a m o t o - S o n o b e , N „ Y a m a i z u m i , M . and U c h i d a , T. ( 1 9 8 7 ) E x p . Cell R e s . 173, 586-595

30. 31. 32.

A d a m , S . A . and G e r a c e , L . (1991) Cell 66, 8 3 7 - 8 4 7 A d a m , S . A , L o b l , T . J , Mitchell, M . A . and G e r a c e , L . (1989) N a t u r e ( L o n d o n ) 3 3 7 , 2 7 6 - 2 7 9 A d a m , E . J . H . and A d a m , S.A. (1994) J. Cell Biol. 1 2 5 , 5 4 7 - 5 5 5

33. 34. 35. 36. 37.

M o o r e , M . and Blobel, G . (1992) Cell 69, 9 3 9 - 9 5 0 R a d u , A , M o o r e , M.S. and Blobel, G . (1995) Cell 8 1 , 2 1 5 - 2 2 2 R a d u , A , B l o b e l , G . and M o o r e . M.S. (1995) Proc. N a t l . A c a d . Sci. U . S . A . 92, 1769-1773 M o r o i a n u , J , Blobel, G . and R a d u , A. (1995) Proc. N a t l . Acad. Sci. U . S . A . 9 2 , 2 0 0 8 - 2 0 1 1 G o r l i c h , D , Prehn, S , Laskey, R . A . and H a r t m a n n , E. (1994) Cell 79, 767-778

38.

Y a n o , R , O a k e s , M , Y a m a g i s h i , M „ D o d d , J . A . and N o m u r a , M . (1992) M o l . C e l l . B i o l . 12, 5640-5651 G o r l i c h , D , K o s t k a , S , K r a f t , R , D i n g w a l l , C , L a s k e y , R . A , H a r t m a n n , E . and P r e h n , S. (1995) Curr. Biol. 5, 3 8 3 - 3 9 2

39. 40. 41.

I m a m o t o , N , T a c h i b a n a , T , M a t s u b a e , M . and Y o n e d a , Y. ( 1 9 9 5 ) J . B i o l . C h e m . 2 7 0 , 8559-8565 I m a m o t o , N , Shimamoto, T , Takao, T , Tachibana, T , Kose, S , Matsubae, M , Sekimoto, T , Shimonishi, Y. and Y o n e d a , Y. (1995) E M B O J . 1 4 , 3 6 1 7 - 3 6 2 6

42.

I m a m o t o , N , Shimamoto, T , Kose, S , Takao, T , Tachibana, T , Matsubae, M , Sekimoto, T , Shimonishi, Y. and Yoneda, Y. (1995) F E B S Lett. 368, 4 1 5 - 1 1 9

43. 44.

C h i , N . C , A d a m , E . J . H . and A d a m , S.A. (1995) J . Cell Biol. 1 3 0 , 2 6 5 - 2 7 4 M o r o i a n u , J , H i j i k a t a , M , B l o b e l , G . and R a d u , A . ( 1 9 9 5 ) P r o c . N a t l . A c a d . Sci. U . S . A . 92, 6532-6536

45.

G o r l i c h , D , Vogel, F , Mills, A . D . , H a r t m a n n , E . and L a s k e y , R . A . ( 1 9 9 5 ) N a t u r e ( L o n d o n ) 377, 2 4 6 - 2 4 8

46.

Belanger, K . D , K e n n a , M . A , Wei, S. and Davis, L.I. (1994) J. Cell Biol. 126, 6 1 9 - 6 3 0

227

228

N. I m a m o t o e t a l .

47.

Y a n o , R . , O a k e s , M., T a b b , M . M . a n d N o m u r a , M . ( 1 9 9 4 ) P r o c . N a t l . A c a d . Sci. U . S . A . 91, 6880-6884

48. 49.

Kiissel, P. and Frasch, M . (1995) J . Cell Biol. 1 2 9 , 1 4 9 1 - 1 5 0 7 T o r o k , I., Strand, D . , Schmitt, R . , T i c k , G . , T o r e k , T., K i s s , I. and Mechler, B . M . (1995) J. Cell Biol. 129, 1473-1489

50.

C u o m o , C . A . , Kirch, S.A., G v u r i s , J . , Brent, R . and Oettinger, M . A . ( 1 9 9 4 ) P r o c . N a t l . A c a d . Sci. U . S . A . 9 1 , 6 1 5 6 - 6 1 6 0

51. 52.

C o r t e s , P., Ye, Z.S. and Baltimor, D . (1994) Proc. N a t l Acad. Sci. U . S . A . 91, 7 6 3 3 - 7 6 3 7 O ' N e i l l , R . E . a n d P a l e s e , P . (1995) V i r o l o g y 206, 116-125

53. 54.

Weis, K., Mattaj, I. and L a m o n d , A. (1995) Science 2 6 8 , 1 0 4 9 - 1 0 5 1 Prieve, M . G . , G u t t r i d g e , K . L . , M u n g n i a , J . E . a n d W a r e r m a n , M . L . (1996) J . Biol. C h e m . 271, 7654-7658

55. 56. 57.

G o r l i c h , D , Henklein, P., Laskey, R . A . and H a r t m a n n , E . (1996) E M B O J . 1 5 , 1 8 1 0 - 1 8 1 7 Weis, K., Ryder, U . and L a m o n d , A.I. (1996) E M B O J . 15, 1818-1825 Peifer, M „ Berg, S. and R e y n o l d s , A . B . (1994) Cell 76, 789-791

58. 59.

Enenkel, C . , Blobel, G . and Rexach, M . (1995) J. Biol. C h e m . 2 7 0 , 1 6 4 9 9 - 1 6 5 0 2 Kraemer, D . M . , Strambio-de-Castillia, C . , Blobel, G . and R o u t , M.P. (1995) J. Biol. C h e m . 270, 19017-19021

60. 61. 62. 63. 64. 65. 66. 67.

Iovine, M . K . , W a t k i n s . J . L . a n d W e n t e , S . R . (1995) J . Cell Biol. 1 3 1 , 1 6 9 9 - 1 7 1 3 Rexach, M . and Blobel, G . (1995) Cell 8 3 , 6 8 3 - 6 9 2 M o o r e , M.S. and Blobel, G . (1993) N a t u r e ( L o n d o n ) 365, 6 6 1 - 6 6 3 M o o r e , M.S. and Blobel, G . (1994) Proc. N a t l . A c a d . Sci. U . S . A . 9 1 , 1 0 2 1 2 - 1 0 2 1 6 Melchior, F.B., Paschal, J., Evans, J. and G e r a c e , L . (1993) J . Cell Biol. 123, 1649-1659 Paschal, B . M . and Gerace, L . (1995) J. Cell Biol. 1 2 9 , 9 2 5 - 9 3 7 Sazer, S. (1996) Trends Cell Biol. 6, 8 1 - 8 5 D a s s o , M . (1993) Trends B i o c h e m . Sci. 18, 96-101

68. 69.

Tragila, H . N . , A t k i n s o , N . S . and H o p p e r , A . K . (1989) M o l . Cell. Biol. 9 , 2 9 8 9 - 2 9 9 9 B i s c h o f f , F.R., Kreber, H „ K e m p f , T „ H e r m e s , I. and Ponstingl, H . C . (1995) Proc. N a t l . Acad. Sci. U . S . A . 92, 1749-1753 H a t t o r i , M . , T s u k a m o t o , N . , N u r - E - K a m e l , M . S . A . , R u b i n f e l d , B . , Iwai, K . , K u b o t a , H . , Maruta, H . and M i n a t o , N . (1994) M o l . Cell. Biol. 15, 5 5 2 - 5 6 0

70. 71.

C o u t a v a s , E., R e n , M., O p p e n h e i m , J . D . , D ' E u s t a c h i o , P. a n d R u s h , M . G . ( 1 9 9 3 ) N a t u r e ( L o n d o n ) 366, 5 8 5 - 5 8 7

72.

B i s c h o f f , F.R., K r e b b e r , H . , S m i r n o v a , E . , D o n g , W. a n d P o n s t i n g l , H . ( 1 9 9 5 ) E M B O J. 14, 705-715

73.

Y o k o y a m a , N . , H a y a s h i , N . , Seki, T., Pante, N . , O h b a , T., Nishii, K., K u m a , K., H a y a s h i d a , T., Mivata, T., Aebi, U . , et al. (1995) N a t u r e ( L o n d o n ) 376, 184-188 Wu, J . , M a t u n i s , M . J . , K r a e m e r , D . , B l o b e l , G . a n d C o u t a v a s , E . ( 1 9 9 5 ) J . B i o l . C h e m . 270, 14209-14213

74. 75. 76. 77.

Floer, M . and Blobel, G . (1996) J. Biol. C h e m . 271, 5 3 1 3 - 5 3 1 6 Melchior, F. and Gerace, L. (1995) Curr. O p i n . Cell Biol. 7 , 3 1 0 - 3 1 8 Tachibana, T., I m a m o t o , N . , Seino, H . , N i s h i m o t o , T. and Yoneda, Y. (1994) J. Biol. C h e m . 269, 24542-24545

78. 79. 80.

N i s h i m o t o , T., Eilen, E . and Basilico, C . (1978) Cell 1 5 , 4 5 7 - » 8 3 N e h r b a s s , U . and Blobel, G . ( 1996) Science 272, 120-122 Melchior, F., G u a n , T., Y o k o y a m a , N . , N i s h i m o t o , T. and G e r a c e , L . (1995) J . Cell Biol. 131, 571-581

81.

C o r b e t t / A . H . , K o e p p , D . M . , S c h l e n s t e d t , G . , L e e , M . S . , H o p p e r , A . H . , K o e p p , D . M . and Silver, P . A . ( 1995) J. Cell Biol. 130, 1017-1026 A k e y , C . W . and G o l d f a r b , D.S. (1989) J. Cell Biol. 1 0 9 , 9 7 1 - 9 8 2 Akey, C.W. and Radermacher, M. (1993) J. Cell Biol. 122, 1 - 1 9 I m a m o t o , N . , M a t s u o k a , Y., K u r i h a r a , T., K o h n o , K . , M i y a g i , M . , S a k i v a m a , F., O k a d a , Y., Tsunasawa, S. and Yoneda, Y. (1992) J. Cell Biol. 119, 1047-1061 Shi, Y. and T h o m a s , J . O . (1992) M o l . Cell. Biol. 12, 2 1 8 6 - 2 1 9 2 O k u n o , Y„ I m a m o t o , N . and Yoneda, Y. (1993) Exp. Cell Res. 206, 134-142 Yang, J. and D e f r a n c o , D . B . (1994) M o l . Cell. Biol. 14, 5088-5098

82. 83. 84. 85. 86. 87.

Import and export of proteins at the nucleus

88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 110. 111. 112.

Y o n e d a , Y , I m a m o t o , N , M a t s u o k a , Y , I w a m o t o , R , K i h o , Y. and U c h i d a , T. ( 1 9 8 8 ) Science 242,275-278 I m a m o t o , N , M a t s u o k a , Y , S e m b a , T „ O k a k d a , Y., U c h i d a , T. and Y o n e d a , Y. ( 1 9 9 0 ) J . Biol. C h e m . 265, 16504-16508 Dingwall, C . and L a s k e y , R. (1992) Science 258, 9 4 2 - 9 4 7 M i c h a u d , N . and G o l d f a r b , D.S. (1991) J. Cell Biol. 1 1 2 , 2 1 5 - 2 2 3 Fischer, U , D a r z v n k i e w i c z , E , T a h a r a , S . M , D a t h a n , N . A , L u r m a n n , R . and M a t t a j , I.W. (1991) J. Cell Biol. 1 1 3 , 7 0 5 - 7 1 4 M i c h a u d , N . and G o l d f a r b , D . S . (1992) J. Cell Biol. 1 1 6 , 8 5 1 - 8 6 2 Gerace, L . (1995) Cell 82, 341-344 Yoneda, Y. (1996) Trends Microbiol. 4, 1 - 2 Pante, N . and A e b i , U . (1996) Curr. O p i n . Cell Biol. 8, 3 9 7 - 4 0 6 W o o d c o c k , C . L . , L a F o n d , R . E . , W o o d c o c k , H , B a l d w i n , L . A . and B h o r j e e , J . S . ( 1 9 8 4 ) E x p . Cell Res. 1 5 4 , 1 5 5 - 1 7 0 M a d s e n , P., Nielsen, S. and Celis, J . E . (1986) J . Cell Biol. 1 0 3 , 2 0 8 3 - 2 0 8 9 Sommerville, L. (1986) Trends B i o c h e m . Sci. 1 1 , 4 3 8 - 4 4 2 Borer, R . A , Lehner, C . F , Eppenberger, H . M . and N i g g , E . A . (1989) Cell 56, 3 7 9 - 3 9 0 G u i o c h o n - M a n t e l , A , L e s c o p , P , C h r i s t i n - M a i t r e , S , L o o s f e l t , H , P e r r o t - A p p l a n a t , M . and M i l g r o m , E. (1991) E M B O J . 10, 3 8 5 1 - 3 8 5 9 S c h m i d t - Z a c h m a n n , M.S., D a r g e m o n t , C „ K u h n , L . C . and N i g g , E . A . (1993) Cell 7 4 , 4 9 3 - 5 0 4 G u i o c h o n - M a n t e l , A , D e l a b r e , K , L e s c o p , P. and M i l g r o m , E . ( 1 9 9 4 ) P r o c . N a t l . A c a d . Sci. U.S.A. 91,7179-7183 M o r o i a n u , J . and Blobel, G . (1995) Proc. N a t l . A c a d . Sci. U . S . A . 92, 4 3 1 8 - 4 3 2 2 F a n t o z z i , D . A , H a r o o t u n i a n , A . T , Wen, W , T a y l o r , S.S., F e r a m i s c o , J . R , T s i e n , R.Y. and M e i n k o t h , J . L . (1994) J . Biol. C h e m . 2 6 9 , 2 6 7 6 - 2 6 8 6 Wen, W , H a r o o t u n i a n , A . T , A d a m s , S . R , F e r a m i s c o , J , Tsien, R.Y'., M e i n k o t h , J . L . and Taylor, S.S. (1994) J . Biol. C h e m . 269, 3 2 2 1 4 - 3 2 2 2 0 Wen, W , M e i n k o t h , J . L , Tsien, R.Y. and Taylor, S.S. (1995) Cell 82, 4 6 3 - 4 7 3 Fischer, U , H u b e r , J , Boelens, W.C.. Mattaj, I.W. and L u h r m a n n , R. (1995) Cell 8 2 , 4 7 5 - 4 8 3 Michael, W . M , C h o i , M . and D r e v f u s s , G . (1995) Cell 8 3 , 4 1 5 - 4 2 2 B o g e r d , H . P , Fridell, R . A , M a d o r e , S. and C u l l e n , B . R . (1995) Cel l 8 2 , 4 8 5 - 4 9 4 Fritz, C . C , Z a p p , M . L . and G r e e n , M . R . (1995) N a t u r e ( L o n d o n ) 376, 530-533 Stutz, F„ Neville, M . and R o s b a s h , M . (1995) Cell 8 2 , 4 9 5 - 5 0 6

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13 Mitochondrial targeting and import Ruud Hovius Laboratory of Physical Chemistry of Polymers and Membranes, Swiss Federal Institute of Technology, I Ol 5 Lausanne1SwitzerIand

Introduction Mitochondria contain hundreds of different proteins, of which only a handful are encoded by the mitochondrial genome. Most of these mitochondrially encoded proteins are key components of the multisubunit complexes involved in oxidative phosphorylation. The majority of the nuclear-encoded proteins are synthesized in the cytosol on free polysomes and contain signals coding for their destination. They are complexed by cytosolic factors (chaperonins) and guided towards the mitochondria, where an intricate machinery takes care of import and correct sorting. The import of proteins into mitochondria is complex, since these organelles contain two membranes, enclosing between them the intermembrane space, with the matrix delimited by the inner membrane. Although an important part of our knowledge on mitochondrial protein import has been obtained from studies with yeast and Neurospora crassa, the general features have been confirmed in mammals. An overview of the different requirements for a protein to be imported into mitochondria and the components involved in import and maturation will be presented. The import paths followed by different proteins are discussed briefly and have been reviewed recently [1-5]. The recently proposed nomenclature is followed [6], whereby proteins of the outer and inner membranes involved in protein translo­ cation are called Tom and Tim respectively, for Translocation across Outer/Inner Membrane, followed by a number indicating their molecular mass (in kDa).

Mitochondrial protein import: co- or post-translational? No evidence is available to prove that protein import into mitochondria can occur solely via a co-translational route. However, such a route has been demonstrated. Some mitochondrial precursor proteins could start import before translation had been completed [7], and cytosolic ribosomes attached to the outer membrane [8-12] might extrude nascent proteins into mitochondria.

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On the other hand, post-translational import of many precursor proteins has been demonstrated in vitro (e.g. [13]), with or without the help of additional proteins (chaperonins) that keep the nascent protein in a translocation-competent form. Post-translational import has also been shown in vivo. Yeast cells accumulated some mitochondrial precursor proteins upon dissipation of the proton gradient with the uncoupler carbonyl cyanide w-chlorophenylhvdrazone [14,15], Thus, taking all the points together, mitochondrial protein import seems to be mainly post-translational.

Targeting sequences and destination It has been demonstrated that several mitochondrial precursor proteins are synthesized on free polysomes as larger precursors with an N-terminal extension [13] or targeting sequence. It was shown that the presequence alone [16] could compete for import of other precursor proteins. Moreover, presequences allowed the import into the mitochondrial matrix not only of passenger proteins but even of attached single-stranded and double-stranded DNA [17]. No consensus for mitochondrial targeting sequences has been discovered. A high proportion of randomly generated sequences could function as import signals [18], as long as they could adopt basic, amphipathic structures (Chapter 2). These artificial targeting sequences were less efficient than the naturally occurring signals [19]. In fact, isolated mitochondrial preseqences are unstructured in solution, but in the presence of lipids they form amphipathic structures [20], which are often, but not exclusively, helical [21]. In primitive eukarvotes such as Trichomonas (which contains organelles called hvdrogenosomes which resemble mitochondria) or Trypanosoma, proteins destined for the mitochondria contain short (5-12 amino acid) N-terminal extensions that are rich in hydrophobic and hvdroxylated amino acids and poor in acidic residues [22]. Arginine is frequently encountered at position -2 relative to the matrix processing peptidase (MPP) cleavage site. These short sequences were also able to target passenger proteins into yeast mitochondria, although they were not very efficient, and removal of the presequence by the MPP was not always observed. The import machinery thus seems to have been relatively well conserved throughout eukarvotic evolution. In higher eukarvotes the best characterized sorting tags are the matrix targeting signals, which guide a passenger protein towards the matrix, where the signal is removed by the MPP. In general, they are composed of 20-35 amino acids, but can be as long as 70 residues [23]. They are rich in basic and hvdroxylated amino acids, whereas acidic amino acids are virtually absent, as in the case of the primitive eukarvotes mentioned above. Often arginine, and sometimes lysine, is found at position -2 relative to the MPP cleavage site, while at position +2 there is a slight preference for serine and alanine [24].

Mitochondrial targeting and import

To arrest a protein in a mitochondrial membrane, a hydrophobic stoptransfer signal is added after the matrix targeting signal, and together they form a bipartite signal. Upon entry into the translocation channel, this sequence blocks further translocation and gains access to the membrane itself. Outer-membrane proteins often have a targeting sequence starting with about 10 residues rich in basic and hydroxylated amino acids, directly followed by a hydrophobic transmembrane domain of about 20 residues, as in the case of Tom70 [25,26]. The relative position of the stop-transfer sequence determines the final destination of the imported protein. For example, when it is placed directly behind the matrix targeting signal, the protein remains in the outer membrane, whereas if it is more distant, the protein ends up in the inner membrane [27], This suggests that, in the latter case, the distance between the stop-transfer and matrix targeting signals is sufficient for the matrix targeting signal to reach over the intermembrane space towards the innermembrane import receptor Tim23. Proteins destined for the intermembrane space that contain an N-terminal targeting sequence, such as cytochrome b 2 , are first imported into the inner membrane using a matrix stop-transfer signal; then, after the matrix targeting signal and the stop-transfer signal are removed proteolytically, the mature protein is released into the intermembrane space [28]. Sorting of imported proteins is not always absolute, as some proteins are sorted to two different locations. One example is NADH-cytochrome b i reductase, which is directed towards both the outer membrane and the intermem­ brane space [29], Furthermore, many proteins have been found to have a non-cleavable N-terminal targeting signal. For outer-membrane proteins, proteolytic removal of the targeting sequence is not observed, indicating that the targeting information is contained within the mature protein itself. Moreover, the targeting signal is not always in the N-terminal part of the sequence. For the outer-membrane proteins Bcl2 [30] and porin [31], targeting information seems to be encoded in their C terminal part. Also, for the ATP/ADP carrier (an inner-membrane protein), the targeting information seems to present in the C-terminal two-thirds of the protein [32,33].

Cytosolic chaperonins Flow d o the precursors end up at the mitochondria without prior (mis)folding or aggregation? Improperly folded proteins are degraded rapidly by proteolysis [34], but a properly folded precursor cannot be imported into mitochondria [35,36]. Molecular chaperones have been shown to interact with mitochondrially destined precursors and to keep them in a translocation-competent form. Several chaperonins have been shown to be specific for mitochondrial precursors, while

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234

others are non-specific (Figure 1). The relative importance of each chaperonin is determined by its affinity for the individual precursor [37]. The heterodimeric protein MSF (mitochondrial import stimulating factor) catalyses the binding to and import into mitochondria of precursors. MSF recognizes the matrix targeting sequence [38], and is therefore specialized for mitochondrial precursor recognition. MSF can actively reverse precursor aggregation and folding in an ATP-dependent manner [39]. In fact, the ATPase activity of MSF is stimulated by import-incompetent precursors [39]. The MSF-precursor complex is able to bind to a receptor on the outer membrane [40] formed by Tom37 and Tom70, after which MSF is released into the cytosol in an ATP-dependent and 7V-ethylmaleimide-sensitive manner [41].

Figure 1

Generalized

representation

of protein import into

mitochondria

(MSF)

(MSf

(Hsp70j

I^l5140/PSC £

ATR

Outer membrane ΔΨ Mature

F

17

3

FMGC

ATP (mHsp70l||l||

Inner membrane

(D) {(30

ATP / ΔΨ

ATP Cpr3

mHsp70j ATP

Mature

A precursor protein containing a basic targeting signal is bound by chaperonins (MSF and Hsp70). In the outer membrane, the precursor is handed over to the Tom machinery.Via the outer-membrane channel, the precursor can either enter the outer membrane (step A) or be handed over to the Tim machinery (step B).There the precursor can enter the inner membrane direaly (step Cj, be imported into the matrix (step D) or be imported into the matrix and re-exported to the inner membrane (step EJ. Striped lozenges indicate presequences. Abbreviations used: ΛΨ, membrane potential; PSC, peptide-sensitive channel; MCC, multiple conductance channel; (m)Hsp, (mitochondrial) heat-shock protein; MPP, matrix processing pep dase; IMP, inner-membrane peptidase; ·,acidic domain.The Tom and Tim proteins are each identified by a number denoting the molecular mass.

Mitochondrial targeting and import

The 70 kDa heat-shock protein (Hsp70) binds to exposed hydrophobic domains of non-folded proteins in general, and keeps the proteins in a translocation-competent state. Hsp70 binds to precursor proteins selectively: it did not bind to the cytosolic isoform of aspartate aminotransferase, whereas it did bind to and prevented folding of the mitochondrial precursor of this protein [42], Also, as for MSF, the intrinsic ATPase activity of Hsp70 is activated by the interaction with the precursor protein. Upon delivery of the bound precursor to the Tom20-Tom22 receptor in the outer membrane, Hsp70 is released in an ATP-independent manner [37], without itself binding to the receptor complex [43]. Two additional proteins have been reported to play a role in the import of proteins into mitochondria. The so-called presequence binding factor was purified from reticulocyte lysate and shown to maintain the precursor of ornithine transcarbamylase in a translocation-competent state, independent of ATP [44], Presequence binding factor seems to bind to the targeting sequence of the precursor, as different synthetic presequences competed for this binding [44]. Also, a 28 kDa protein, termed targeting factor, was purified from reticulocyte lysate by presequenceaffinity chromatography, and antisera raised against this protein blocked the binding of precursors to mitochondria [45].

Binding to the outer membrane It was realized quite early on that the import of proteins into mitochondria is mediated by a proteinaceous factor on the mitochondrial surface. Treatment of mitochondria with proteases abolished, or at least slowed down, the import of precursor proteins [46-48]. Two receptor complexes are found on the outer membrane which interact reversiblv with each other via the so-called tetratrico domains [49]. Each receptor interacts with a subset of precursors. The first receptor is composed of Tom20 and Tom22, and interacts with the positively charged, amphipathic N-terminal mitochondrial targeting sequence [50] via 'acid bristles', i.e. clusters of acidic amino acids in the cytosolic domains of Tom 20 and Tom22. Mutations in these acid bristles diminished the binding of precursors to yeast mitochondria [51 ], and deletion of this domain decreased the binding of different precursor proteins to the recombinant cytosolic domain of human Tom20 [52]. However, it was reported that deletion of the acid-bristle domain of rat Tom20, expressed in Atom20 yeast, had no effect on the respirationdependent growth of yeast [53], Cross-linking studies indicated that Tom20 and Tom22 are intimately arranged [54] and that both interact with precursors [55,56]. Hsp70 delivers the precursors to this receptor in an ATP-independent way [37], The second receptor is a 1:1 dimer of Tom37 and Tom70 [57]. This receptor deals with authentic mitochondrial precursor proteins [58] that are bound by MSF [40]. Tom70 was initially identified as the receptor for the precursor of the

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ATP/ADP carrier [59]. Tom70 has a large cytosolic domain containing seven tetratrico peptides to facilitate interactions with other Tom proteins. The two receptor pathways are not mutually exclusive, since deletion of Tom20 [60], Tom37 [57] or Tom70 [60,61] did not abolish import. Only Tom22 was found to be essential [56,62], which might be due to the involvement of the intermembrane-space domain of Tom22 in the translocation of precursor proteins over the outer membrane (see below). Experiments with the purified recombinant cytosolic domains of yeast Tom20, Tom22 and Tom70 indicated that these receptors display a sliding scale of selectivity: while Tom22 was specific for precursors carrying targeting sequences and Tom70 preferentially bound precursors with internal signals, Tom20 recognized both groups of precursors [63]. The two receptors interact with each other via Tom20 and Tom70. Precursor proteins that have been delivered by MSF to the Tom37-Tom70 complex are transferred to the Tom20-Tom22 complex before further insertion into the membrane [49,64].

Translocation over the outer membrane via a dynamic Tom machinery Precursors bound by the import receptor are transferred to the general insertion pore, a proteinaceous channel in the outer membrane that seems to be formed by Tom40 [65]. The interaction between the receptor and the pore Tom40 is regulated by small outer-membrane proteins. Tom6 facilitates the association of the Tom20-Tom22 receptor with Tom40, and seems to establish co-operativity between the receptor and the pore and promote the release of the precursor from the receptor [66]. Tom5, which could be cross-linked to translocation intermediates [67], has recently been proposed to transfer precursor proteins from Tom20-Tom22 to the general insertion pore Tom40 using its acidic N-terminal cytosolic domain [68], Finally, Tom7 tends to dissociate Tom20-Tom22 from Tom40, thereby possibly enabling proteins destined for the outer membrane, such as porin, to leave the channel and to move towards the outer membrane itself [69]. Deletion of Tom7 inhibited the import of porin, whereas the import of precursors destined for the mitochondrial interior was hardly affcctcd [69]. Also, the released and emptied Tom20-Tom22 is set free to scavenge the outer membrane for newly arrived precursor proteins. A reversible, dynamic interaction between the Tom proteins is essential for correct protein import and sorting. However, is Tom40 the actual pore? It has been proposed that the peptidesensitive channel is the pore responsible for the translocation of proteins across the outer membrane [70], The peptide-sensitive channel is a cation channel present in the outer membrane which has a large conductance upon reconstitution in vesicles (about 330 pS at -40 mV) and which was blocked by the peptide dynorphin B and the presequence of cytochrome c oxidase subunit 4. The peptide-sensitive channel is associated with Tom40, as monoclonal antibodies against Tom40 depleted the

Mitochondrial targeting and import

channel from detergent-solubilized mitochondria. Also, dynorphin could function as a targeting sequence for the import of a cytosolic protein into mitochondria [71], Once the targeting sequence of the precursor has traversed the outer membrane, it seems to be recognized and bound on the inside of the outer membrane. The intermembrane-space domain of Tom22, which is rich in acidic amino acids, has been implicated, since its ir«»s-domain has been shown to bind specifically to mitochondrial targeting peptides with submicromolar affinity [72,73]. However, the role of this domain is not clear. Deletion of the trans-domain of Tom22 has been seen to cause the import of certain matrix and inner-membrane proteins to be retarded [72,74], but another study showed hardly any effect [75]. Import of proteins into the outer membrane was unaffected [74]. The reversible binding of a targeting sequence to the intermembrane-space domain of Tom22 might catalyse its transfer towards the inner-membrane translocation machinery. It would be of interest to investigate a possible interaction between Tom22 and the inner-membrane receptor Tim23. The only precursor known not to use the Tom import machinery described above is apocytochrome c, which lacks a cleavable signal sequence. This precursor seems to interact reversibly with and insert into the lipids of the outer membrane, as was suggested by experiments with model membranes [76-78], However, the mitochondrial protein Cyc2 was found to increase the efficiency of apocytochrome c import [79]. The outer-membrane translocation machinery can act in the absence of an intermembrane space or inner-membrane components. Outer-membrane vesicles have been shown to bind and import several outer-membrane proteins and to translocate certain intermembrane-space proteins [80,81]. However, precursors destined to be translocated into the inner membrane or matrix were only bound to the outer-membrane vesicles [80]. Protein insertion into the outer membrane is thus independent of the innermembrane potential and of matrix components (ATP, peptidases, chaperonins).

Intermembrane-space components Few proteins of the intermembrane space have been identified as being essential for or involved in protein import into mitochondria. So far, only the role of cytochrome c haem lyase has been clearly established for the import of apocytochrome c, a protein following an extraordinary import pathway. The driving force for apocytochrome c import seems to be its interaction with the intermembrane-space enzyme cytochrome c haem lyase, and the attachment of the haem group to the apo-enzyme resulting in a tight folding of the protein around this prosthetic group [82,83]. Two other proteins, MRS5 and MRSl 1, have been identified by suppression of a respiration-dcficicnt phenotype in yeast. These proteins have 35%

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sequence identity and similar functions, with both being essential for the viability of yeast cells [84]. Depletion of these proteins caused the accumulation of the precursor of mitochondrial Hsp60, defective sorting of cytochrome b j reductase and an inability to form cytochromes [84—86].

Translocation across the inner membrane Once the presequence has traversed the outer membrane and arrived in the intermembrane space, it is pulled over the inner membrane in a membranepotential-dependent manner, without the need for ATP [87,88]. Six proteins have been identified as being involved in this translocation across the inner membrane. Tim23 has an N-terminal domain that protrudes into the intermembrane space, followed by four transmembrane domains. Tim23 can, stimulated by the inner-membrane potential, dimerize via the leucine repeats in its intermembranespace domain [89], As a dimer, Tim23 is believed to expose acidic domains, attracting positively charged matrix targeting signals. The binding of a matrix targeting signal induces monomer formation [89]. Tim23 can then form a complex with Timl 7, a protein similar to Tim23 but lacking the intermembrane-space domain [90,91], This complex might be part the inner-membrane channel that allows passage of the precursors. The multiple conductance channel is a highly conducting (up to 1 nS) cation-selective channel that can be blocked transiently by isolated matrix targeting sequences, but not by presequence peptides of outer-membrane proteins [92]. This block was dependent upon the presence of Tim23 [93]. It is tempting to speculate that the receptor for precursor proteins, i.e. Tim23 (alone or in complex with Tim 17), hands over the precursor to the multiple conductance channel for translo­ cation across the inner membrane. This would parallel the situation in the outer membrane, where Tom20-Tim22 passes on the precursor to Tom40 and the peptide-sensitive channel. Upon protruding into the matrix, the presequence is bound by mitochondrial Hsp70 (mHsp70). Since mHsp70 binds only with low affinity to the presequence, further translocation into the matrix is initiallv dependent on the membrane potential to prevent back-slip [94], This action of mHsp70 determines the unidirectionality of protein import, since in the absence of mHsp70 precursors diffused out of the mitochondria [95], Then, in an ATP-consuming evele, mHsp70 pulls the precursor into the matrix until translocation has been completed [96,97], Co-operation between Tim44, anchored in the inner membrane via a Cterminal hydrophobic sequence, and mHsp70 was essential only for the import of folded precursors [98], and not for unfolded ones [99], This suggests that, under situations of high-power demand, mHsp70 uses Tim44 as a scaffold to pull the precursor over the inner membrane.

Mitochondrial targeting and import

The binding and dissociation of mHsp70 from Tim44 and the precursor is modulated by the co-chaperonin or nucleotide-exchange factor MGEl [97], ADPcontaining mHsp70 is preferentially Tim44-bound [100]. It was shown recently that mHsp70 can also interact directly with Tim23-Timl7 [101], and that Tim23 also interacts with Tim44 [101]. A second, independent import pathway is presented by a further innermembrane protein, Tim22, which is specifically implicated in the import of innermembrane proteins lacking an N-terminal matrix target signal, such as the ADP/ATP carrier [102], This protein was not associated with the Tim23-Timl7 system, and the import of precursors with a targeting signal was independent of Tim22. Nevertheless, Tim22 shows sequence similarity with Tim23 and Timl 7. Finally, Timll has been cross-linked to a preprotein destined for the innermembrane space [103]; however, its function remains unclear.

Dynamic contact sites between the outer and inner membranes Originally observed 30 years ago by electron microscopy, contacts between the mitochondrial outer and inner membranes were suggested to play a role in the transport of proteins into mitochondria [104]. Later, contact sites were also implicated in the energy metabolism of mitochondria [105] and in the import of phospholipids [106], However, these latter two processes seem to involve a different type of contact site than that involved in protein import. Direct proof for the involvement of contact sites in protein import came from the demonstration that translocation intermediates spanned both the outer and inner membranes at the contact sites [107], The N-terminal targeting sequence was removed by the MPP, while the C-terminus was still accessible to antibodies or proteases. A stretch of about 50 amino acids was sufficient to span the contact sites, suggesting that precursors are translocated in an extended, unfolded confor­ mation [108]. The dynamic character of contact sites was nicely demonstrated by a progressive 'zippering' of the outer and inner membranes upon accumulation of translocation intermediates [109]. Quantification of the intermediates yielded estimates of 100-1000 contact sites per yeast mitochondrion [110]. The Tom machinery seemed to be randomly distributed over the surface of the outer membrane [111] and to be in excess over the Tim machinery [112], This might suggest that the rate-limiting step in protein import is the first: an encounter between the precursor and one of the outer-membrane receptors. Once bound, the precursor is imported swiftly. No direct interaction between the Tim and Tom machineries has been observed. Upon completion of translocation over the outer membrane, the

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contact sites disappeared, indicating that the two membranes are connected through the precursor.

Sorting towards the inner membrane Two major pathways have been described for protein import into the inner membrane; these involve either re-export from the matrix or direct insertion. The first pathway involves the 'conservative sorting' of precursor proteins that carry relatively long N-terminal targeting signals, consisting of a matrix targeting signal and a stop-transfer domain (bipartite signal). The precursors are imported initially into the mitochondrial matrix, where the MPP removes the matrix targeting signal. The stop-transfer signal is thought to 'persuade' Hsp60 to keep the intermediate form of the precursor in a translocation-competent state [113] and to trigger re-export of the protein towards the inner membrane, where the stoptransfer signal finally acts as membrane anchor, as in the case of the sorting of subunit 9 of Neurospora crassa F 0 -ATPase [114]. In this case, export was shown to be dependent on matrix ATP and a proton gradient over the inner membrane [115]. Also, the polytopic Oxal protein is first imported into the matrix and then re­ inserted into the inner membrane in a membrane-potential-dependent manner, ending up in an N o u t /C i n orientation [116]. It is supposed that proteins that are imported via conservative sorting are derived from an endosymbiotic, prokaryotic ancestor. After import into mitochondria, these proteins are the exported via prokaryotic pathways [114], For the second pathway, proteins are directed via a matrix stop-transfer signal (Chapter 4) to the Tim machinery in the inner membrane. Complete translo­ cation into the matrix is prevented by the hydrophobic stop-transfer sequence, which is thought to provoke opening of the translocation channel, yielding access to the inner membrane. Reconstitution of the inner-membrane insertion machinery in proteoliposomes has demonstrated that a membrane potential is required for insertion, but that Tim44, the ATP-dependent translocation motor mHsp70 and Hsp60 are not needed [117,118]. Proteins without cleavable signal sequences, such as the ATP/ADP carrier [114,119] and other exchange proteins, are imported via this pathway. It is suggested that these proteins do not have an endosymbiotic origin and therefore follow a specialized pathway differing from conservative sorting. This might also be corroborated by the use of the specialized import receptor Tim22 [102].

Towards the intermembrane space Several pathways have been described for protein import into the intermembrane space. Apocytochrome c takes the most direct route. This precursor interacts

Mitochondrial targeting and import

reversibly with and inserts into the lipids of the outer membrane, as was suggested by experiments with model membranes [76-78], and the formation of the holoprotein gives the directionality to its translocation into the intermembrane space [82,83]. The second pathway is via the Tom machinery directly into the intermembrane space, independent of an inner-membrane potential and matrix ATP. An example is cytochrome c haem lyase, which has a non-cleavable signal sequence [120]. In a third pathway, yeast cytochrome b 1 is first anchored in the inner membrane by an N-terminal stop-transfer signal, which arrests translocation through the Tim machinery, until the protein has completely traversed the outer membrane [103]. Then the MPP removes the matrix targeting signal. The mature protein is released into the intermembrane space by the inner-membrane protease, located at the outer face of the inner membrane [121,122], However, in Neurospora crassa, cytochrome b 2 is transported via the matrix; after removal of the matrix targeting signal by MPP, it is re-translocated back across the inner membrane (conservative sorting) v f/ in an Nin /C out orientation. Hsp60 and matrix ATP were required to keep the protein in a translocationcompetent form [113]. Another precursor that is imported via conservative sorting is a fusion protein between the presequence of cytochrome c oxidase subunit 2 and mouse dihydrofolate reductase: an intermediate form could be accumulated in the matrix and then be exported to and released into the intermembrane space [123].

Maturation by peptidases In the matrix, the targeting sequence is cleaved off from precursor proteins by a metal-ion-dependent MPP [124] consisting of two subunits of about 50 kDa (aMPP and β-ΜΡΡ). Fluorescence studies with MPP purified from N. crassa suggested that one α-subunit interacts with two β-subunits [125], The catalytic domain seems to reside on β-ΜΡΡ [126], but both subunits are necessary for activity [127]. Both the location of and the interaction between the two subunits is species-dependent. Whereas in N. crassa both subunits are found in the matrix as individual proteins [127], in the rat MPP is present in the matrix as a heterodimer, and in plants MPP is, as one protein, an integral part of the bc { complex of the respiratory chain [128], Sequence identity indicates that MPP belongs to a large family that also includes prokaryotic proteases [129]. MPP does not seem to recognize a defined amino acid sequence in the precursor. An arginine residue, often found at position -2 or -3 with respect to the cleavage site, had been proposed as the recognition site of MPP [130], but some processed precursors do not contain such a residue, and some that do are not cleaved by MPP. Furthermore, it was shown that removal of only four amino acids at the N-terminus of a precursor protein abolished its processing by a soluble MPP

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[131]. In addition to the arginine just before the cleavage site, the arrangement of the presequence in a helix-linker-helix motif seems to be necessary to present the cleavage site in a correct way to MPP [128,132], The presequence of aldehyde dehydrogenase comprises two α-helices connected by a flexible linker; upon deletion of the linker, the precursor was properly imported but was no longer processed [133]. The helix-linker-helix model is further supported by the observation that the presequences of two mitochondrial precursor proteins that are not processed bv MPP form long extended helices [134]. A second metalloprotease is found in the matrix: the mitochondrial intermediate peptidase [135]. Several proteins, such as ornithine transcarbamylase, are, upon arrival in the matrix, first processed by MPP, but then the mitochondrial intermediate peptidase removes a further eight amino acids with the consensus sequence (F/L/I)XX(T/S/G)XXXX [136,137], Finally, a third metalloprotease, the inner-membrane protease, displays its activity on the outer face of the inner membrane. This enzyme is most probably heteromeric, and has certain features in common with Escherichia coli leader peptidase and the signal peptidase in the endoplasmic reticulum [138], This enzyme liberates intermembrane-space proteins that have initially been anchored in the inner membrane by a stop-transfer sequence.

Protein folding in the matrix To be imported into the matrix, proteins must be in an unfolded, extended state; after import these proteins must fold to obtain their mature structure. They do this either independently or aided by different proteins. Soluble mHsp70 stabilizes unfolded proteins against aggregation and rapid degradation in an ATP-dependent manner, thus giving the precursor time to fold properly. mHsp70 is modulated by mMdj1, which influences the tightness and reversibility of precursor binding [139], and by MGE1, which acts as a nucleotideexchange factor [140]. The proline isomerase Cpr3 speeds up folding bv cis-trans isomerization of peptide bonds of proline-containing proteins [141]. Co-operativitv between mHsp70 and Cpr3 has been shown in the folding of Su9-DHFR, a fusion of a matrix targeting signal with mouse dihydrofolate reductase [142]. Proteins needing more help are handled by Hsp60, which catalyses folding in an ATP-dependent fashion [143], under the regulation of HsplO [144]. The three-dimensional structures of Hsp60 and HsplO (which are homologues of GroEL and GroES respectively from E. coli) have been solved [145]. Inside a cavity formed by two heptameric rings of Hsp60 and heptameric lids of Hsp 10, the unfolded precursor is kneaded until it folds into its native structure, after which it is released [4]. Rhodanese imported into the matrix required Hsp60 for folding, and did not interact with soluble mHsp70 [142].

Mitochondrial targeting and import

Are lipids involved in protein import? Porin binds to isolated mitochondria both with a nanomolar affinity to a saturable site and with low affinity to a site with a much larger binding capacity [46,146]. The high-affinity binding was abolished by protease treatment of the mitochondria before binding [46], and was inhibited by antibodies against Tom20 [46,147]. Lipids were proposed to comprise the low-affinity binding site, since the affinities of porin for shaved mitochondria and for lipid vesicles were similar [46]. Negatively charged lipids, especially cardiolipin, have been suggested to play an important role at several stages during the import of proteins into mitochondria. Specific targeting of precursor proteins to the mitochondria could be due to cardiolipin, a lipid that is synthesized solely [148] and found exclusively [149] in mitochondria, and which has been shown to be present on the outer surface of these organelles [150]. Lipid-directed organelle-specific precursor protein sorting was suggested by specific interactions between (1) the presequence of cytochrome c oxidase subunit IV and vesicles made from a mitochondrial outer-membrane lipid extract, and (2) the precursor of the chloroplast protein ferredoxin and vesicles formed from a chloroplast outer-envelope membrane lipid extract [151]. The binding of precursors and presequences to lipid vesicles is highly dependent on the presence of negatively charged lipids, especially cardiolipin [77,152]. Lipid vesicles containing the negatively charged lipid phosphatidylserine could compete successfully with mitochondria for the binding of apocytochrome c, whereas vesicles comprising zwitterionic phosphatidylcholine were ineffective [78]. The unfolding of (partially) folded proteins by the negatively charged lipids on the surface of mitochondria has been implicated in the stimulation of protein import [153,154], Translocation of isolated presequences [155] and of apocytochrome c [77] across lipid vesicles containing negatively charged lipids has been described. Moreover, the translocation of presequences into lipid vesicles was membrane potential dependent [155], as is precursor protein import into mitochondria. Finally, presequences were able to induce, in a cardiolipin-dependent way, contacts between membranes [156]. The ability of several presequences to induce interbilayer contacts paralleled their efficiency of import into mitochondria [156], This suggests that the formation of dynamic contact sites during mitochondrial protein import might be a presequence-directed process. Although lipids alone seem to be able to perform or mimic many of the properties of mitochondrial protein import, they are rather inefficient in doing so. A minimal role for the proteinaceous components of the import machinery may be merely to make the whole process more efficient [157], perhaps by keeping the precursor in a translocation-competent form [158].

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An outlook Many proteins involved in mitochondrial protein import have now been identified, their function or phenotvpe has been described, and in many cases the sequence in which they act during protein import has been established. However, our molecular, mechanistic understanding is still limited. Also, the interaction between the outer- and inner-membrane translocation complexes is poorly understood. More and more purified proteins are becoming available, either from native tissues or from overexpression. This will enable both structural studies of single proteins and reconstitution of the import machinery from purified components. In vitro experiments combining genetic, biochemical and biophysical methods will continue to yield a wealth of clues as to the complex interactions implicated in protein import. In parallel, new methods for the study of mitochondrial protein import in vivo might allow the verification of results obtained in the svstems mentioned above, and facilitate the rapid identification of important factors under physiological conditions. Examples are the transient expression of mitochondrial precursor proteins [159] and visualization of the import of a presequence fused to the Green Fluorescent Protein [160].

References 1. 2. 3.

Neupert, W. (1997) Annu. Rev. Biochem. 66, 863-917 Ptanner, N. and Mcijcr, M. (1997) Curr. Biol. 7, 100-103 Haucke, V. and Lithgow, T. (1997) J. Bioenerg. Biomembr. 29, 11-17

4. 5.

Martin, J. (1997) J. Bioenerg. Biomembr. 29, 35—43 Ryan, M.T., Naylor, D.J., Hoj, RB., Clark, M.S. and Hoogenraad, N.J. (1997) Int. Rev. Cytol. 174, 127-193 Pfanner, N., Douglas, M.G., Endo, T., Hoogenraad, N.J., Jensen, R .E., Meijer, M., Neupert, W., Schatz, G., Schmitz, U.K. and Shore, G.C. (1996) Trends Biochem. Sci. 21, 51-52

6. 7. 8. 9.

Verner, K. (1993) Trends Biochem. Sci. 18,366-371 Kellems, R.E., Allison, V.F. and Butow, R.A. (1975) J. Cell Biol. 65, 1-14 Kellems, R.E. and Butow, R.A. (1972) J. Biol. Chem. 247, 8043-805C

10. 11.

Kellems, R.E., Allison, V.F. and Butow, R.A. (1974) J. Biol. Chem. 249,3297-3303 Kellems, R.E. and Butow, R.A. (1974) J. Biol. Chem. 249, 3304-331C

12.

Pon, L., Moll, T,, Vestweber, D., Marshallsay, B. and Schatz, G. (1989) J. Cell Biol. 109, 2603-2616 Maccecchim, M.L., Rudin, Y., Blobel, G. and Schatz, G. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 343-347 Reid, G.A. and Schatz, G. (1982) J. Biol. Chem. 257, 13056-13061 Reid, G.A. and Schatz, G. (1982)J. Biol. Chem. 257, 13062-13067

13. 14. 15. 16. 17. 18. 19.

Ito, A., Ogishima, T., Ou, W., Omura, T., Aoyagi, H., Lee, S., Mihara, H. and Izumiva, N. (1985) J. Biochem. (Tokyo) 98, 1571-1582 Vestweber, D. and Schatz, G. (1989) Nature (London) 338, 170-172 Baker, A. and Schatz, G. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 3117-3121 Vassarotti, A., Stroud, R. and Douglas, M. (1987) EMBO J. 6, 705-711

20.

Roise, D., Horvath, S.J., Tomich, J.M., Richards, J.H. and Schatz, G. (1986) EMBO J. 5, 1327-1334

21.

Roise, D.,Theiler, E, Horvath, S.J., Tomich, J.M., Richards, J.H., Allison, D.S. and Schatz, G. (1988) EMBO J. 7, 649-653 Hausler, T., Stierhof, YD., Blattner,J. and Clayton, C. (1997) Eur. J. Cell Biol. 73, 240-251 Roise, D. and Schatz, G. (1988) J. Biol. Chem 263, 4509—1511

22. 23.

Mitochondrial targeting and import

24.

Gavel, Y. and v o n Heijne, G . (1990) Protein E n g . 4, 3 3 - 3 7

25. 26. 27. 28.

H a s e , T „ Muller, U . , R i e z m a n , H . and Schatz, G . (1984) E M B O J. 3, 3 1 5 7 - 3 1 6 4 M c B r i d e , H . M . , Millar, D . G . , L i , J . M . a n d Shore, G . C . (1992) j . Cell Biol. 119, 1451-1457 N g u y e n , M . , Bell, A.W. and Shore, G . C . (1988) J. Cell Biol. 1 0 6 , 1 4 9 9 - 1 5 0 5 T o k a t l i d i s , K . , J u n n e , T., M o e s , S., S c h a t z , G . , G l i c k , B . S . and K r o n i d o u , N . ( 1 9 9 6 ) N a t u r e ( L o n d o n ) 384, 5 8 5 - 5 8 8 H a h n e , K., H a u c k e , V., R a m a g e , L. and Schatz, G . (1994) Cell 79, 8 2 9 - 8 3 9 N g u y e n , M . , Millar, D . G . , Y o n g , V.W., K o r s m e y e r , S.J. and Shore, G . C . (1993) J . Biol. C h e m . 268,25265-25268 C o u r t , D . A . , Kleene, R., N e u p e r t , W. and Lill, R. (1996) F E B S Lett. 390, 7 3 - 7 7 Pfanner, N . , H o e b e n , P., T r o p s c h u g , M . and N e u p e r t , W. ( 1 9 8 7 ) J . B i o l . C h e m . 2 6 2 , 14851-14854 Smagula, C . S . and D o u g l a s , M . G . (1988) J . Cell. B i o c h e m . 36, 3 2 3 - 3 2 7 Jentsch, S. (1992) A n n u . Rev. Genet. 26, 179-207 R a s s o w , J . , G u i a r d , B „ W i e n h u e s , U . , H e r z o g , V., H a r t l , F . - U . and N e u p e r t , W. (1989) J . Cell Biol. 109, 1421-1428 Wienhues, U., Becker, K., Schlever, M., G u i a r d , B „ T r o p s c h u g , M., H o r w i c h , A . L . , Pfanner, N . and N e u p e r t , W. (1991) J . Cell Biol. 1 1 5 , 1 6 0 1 - 1 6 0 9 K o m i y a , T „ Sakaguchi, M . and Mihara, K. (1996) E M B O J. 1 5 , 3 9 9 - 1 0 7 K o m i y a , T., H a c h i y a , N . , S a k a g u c h i , M., O m u r a , T. and Mihara, K . (1994) J . Biol. C h e m . 269, 30893-30897 H a c h i y a , N . , A l a m , R . , S a k a s e g a w a , Y „ S a k a g u c h i , M „ M i h a r a , K . and O m u r a , T. ( 1 9 9 3 ) Biochem. M o l . Biol. Int. 2 9 , 4 2 9 - 4 3 4 H a c h i y a , N . , K o m i y a , T., A l a m , R., I w a h a s h i , J . , S a k a g u c h i , M . , O m u r a , T. and M i h a r a , K .

29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62.

(1994) E M B O J . 13, 5146-5154 H a c h i y a , N . , M i h a r a , K . , S u d a , K., H o r s t , M . , S c h a t z , G . and L i t h g o w , T. ( 1 9 9 5 ) N a t u r e (London) 376,705-709 L a i n , B., Iriate, A . , M a t t i n g l y , J . R . , M o r e n o , J.1. a n d M a r t i n e z - C a r r i o n , M . ( 1 9 9 5 ) J . Biol. Chem. 270,24732-24739 K o m i y a , T., R o s p e r t , S., Schatz, G . and Mihara, K. (1997) E M B O J . 16, 4 2 6 7 - J 2 7 5 M u r a k a m i , K . and M o r i , M . (1990) E M B O J. 9 , 3 2 0 1 - 3 2 0 8 O n o , H . and T u b o i , S. (1990) Arch. B i o c h e m . B i o p h y s . 2 8 0 , 2 9 9 - 3 0 4 Pfaller, R. and N e u p e r t , W. (1987) E M B O J. 6, 2 6 3 5 - 2 6 4 2 Zwizinski, C . , Schleyer, M . and N e u p e r t , W. (1984) J . Biol. C h e m . 259, 7 8 5 0 - 7 8 5 6 Pfaller, R., Pfanner, N . and N e u p e r t , W. (1989) J. Biol. C h e m . 264, 3 4 - 3 9 H a u c k e , V., H o r s t , M „ Schatz, G . and L i t h g o w , T. (1996) E M B O J. 1 5 , 1 2 3 1 - 1 2 3 7 H a u c k e , V., L i t h g o w , T., R o s p e r t , S., H a h n e , K . and S c h a t z , G . ( 1 9 9 5 ) J . B i o l . C h e m . 270, 5565-5570 Bolliger, L „ J u n n e , T., Schatz, G . and Lithgow, T. (1995) E M B O J. 14, 6318-6326 Schleiff, E., Shore, G . C . and G o p i n g , I.S. (1997) J. Biol. C h e m . 272, 17784-17789 I w a h a s h i , J ., Y a m a z a k i , S., K o m i y a , T., N o m u r a , N . , N i s h i k a w a , S., E n d o , T. and M i h a r a , K . (1997) J . Biol. C h e m . 2 7 2 , 1 8 4 6 7 - 1 8 4 7 2 Mayer, A., N a r g a n g , F.E., N e u p e r t , W. and Lill, R. (1995) E M B O J . 14, 4204-1211 H a u c k e , v., L i t h g o w , T „ R o s p e r t , S „ H a h n e , K . and S c h a t z , G . ( 1 9 9 5 ) J. Biol. C h e m . 2 7 0 , 5565-5570 Honlinger, A., Kubrich, M., M o c z k o , M., Gartner, F., Mallet, L., Bussereau, F , E c k e r s k o r n , C., Lottspeich, F., Dietmeicr, K „ Jacquet, M „ et al. (1995) Mol. Cell. Biol. 15, 3 3 8 2 - 3 3 8 9 G r a t z e r , S., L i t h g o w , T., Bauer, R . E . , L a m p i n g , E., P a l t a u f , F., K o h l w e i n , S . D . , H a u c k e , V., J u n n e , T „ Schatz, G . and H o r s t , M. (1995) J. Cell Biol. 129, 25-34 H i n e s , V. and Schatz, G . (1993) J . Biol. C h e m . 268, 449-154 Sollner, T „ Pfaller, R., Griffiths, G „ Pfanner, N . and N e u p e r t , W. (1990) Cell 62, 107-115 G a r t n e r , F., V o o s , W., Q u e r o l , A., Miller, B . R . , C r a i g , E . A . , C u m s k v , M . G . and Pfanner, N . (1995) J . Biol. C h e m . 2 7 0 , 3 7 8 8 - 3 7 9 5 H i n e s , V., B r a n d t , A., G r i f f i t h s , G „ H o r s t m a n n , H „ Brutsch, H . and Schatz, G . (1990) F . M B O J. 9,3191-3200 N a r g a n g , F.E., Kunkele, K.P., Mayer, A., Ritzel, R . G . , N e u p e r t , W. and Lill, R. (1995) E M B O J . 14, 1099-1108

245

246

R.Hovius

63. 64.

Brix, J., Dietmeier, K. and Pfanner, N . (1997) J. Biol. C h e m . 2 7 2 , 2 0 7 3 0 - 2 0 7 3 5 K o m i y a , T. and Mihara, K . (1996) J . Biol. C h e m . 2 7 1 , 2 2 1 0 5 - 2 2 1 1 0

65.

Vestweber, D , Brunner, J , Baker, A. and Schatz, G . (1989) N a t u r e ( L o n d o n ) 3 4 1 , 2 0 5 - 2 0 9

66.

A l c o n a d a , A , K u b r i c h , M., M o c z k o , M , H o n l i n g e r , A . and Pfanner, N . (1995) M o l . Cell. Biol. 15,6196-6205 Sollner, T , R a s s o w , J , W i e d m a n n , M , S c h l o s s m a n n , J . , K e d , P , N e u p e r t , W. and Pfanner, N .

67. 68. 69.

(1992) N a t u r e ( L o n d o n ) 355, 8 4 - 8 7 Dietmeier, K , Honlinger, A , B o m e r , U , D e k k e r , P . J , E c k e r s k o r n , C , Lottspeich, E , Kubrich, M . and Pfanner, N . (1997) N a t u r e ( L o n d o n ) 388, 195-200 H o n l i n g e r , A , B o m e r , U , A l c o n a d a , A , E c k e r s k o r n , C , L o t t s p e i c h , F , D i e t m e i e r , K . and Pfanner, N . (1996) E M B O J . 15, 2 1 2 5 - 2 1 3 7

70. 71. 72.

Henrv, J . P , Juin, P., Vallette, F. and Thieffry, M . (1996) J . Bioenerg. B i o m e m b r . 28, 101-108 Juin, P , Thieffry, M , H e n r y , J.P. and Vallette, F.M. (1997) J. Biol. C h e m . 2 7 2 , 6 0 4 4 - 6 0 5 0 Bolliger, L , J u n n e . T , Schatz, G . and Lithgow, T. (1995) E M B O J . 14, 6 3 1 8 - 6 3 2 6

73. 74.

Mayer, A , N e u p e r t , W. and Lill, R. (1995) Cell 80, 127-137 C o u r t , D . A . , N a r g a n g , F.E., Steiner, H , H o d g e s , R . S , N e u p e r t , W. and Lill, R. (1996) M o l . Cell. Biol. 1 6 , 4 0 3 5 - 4 0 4 2

75. 76.

N a k a i , M „ Kinoshita, K. and E n d o , T . (1995) J. Biol. C h e m . 2 7 0 , 3 0 5 7 1 - 3 0 5 7 5 Rietveld, A , Ponjee, G . A . , Schiffers, P , J o r d i , W., van de C o o l w i j k , P., D e m e l , R . A , M a r s h , D . and de Kruijff, B. (1985) Biochim. B i o p h y s . A c t a 8 1 8 , 3 9 8 - 4 0 9 Rietveld, A , J o r d i , W. and de Kruijff, B. (1986) J . Biol. C h e m . 2 6 1 , 3 8 4 6 - 3 8 5 6

77. 78. 79. 80.

J o r d i , W , H e r g e r s b e r g , C . and d e Kruijff, B. (1992) Eur. J . B i o c h e m . 204, 841-846 D u m o n t , M . E , Schlichter, J . B , C a r d i l l o , T . S , H a y e s , M . K . , B e t h l e n d y , G . and S h e r m a n , F. (1993) M o l . Cell. Biol. 1 3 , 6 4 4 2 - 6 4 5 1 Mayer, A , Lill, R . and N e u p e r t , W. (1993) J. Cell Biol. 1 2 1 , 1 2 3 3 - 1 2 4 3

81. 82.

Iwahashi, J , Takaichi, S „ Mihara, K. and O m u r a , T. (1994) J. B i o c h e m . ( T o k y o ) 1 1 6 , 1 5 6 - 1 6 3 N i c h o l s o n , D . W , H e r g e r s b e r g , C . and N e u p e r t , W. (1988) J . Biol. C h e m . 2 6 3 , 1 9 0 3 4 - 1 9 0 4 2

83. 84. 85.

N i c h o l s o n , D . W , Kohler, H . and N e u p e r t , W. (1987) Eur. J. B i o c h e m . 164, 147-157 J a r o s c h , E , R o d e l , G . and Schweyen, R.J. (1997) M o l . G e n . Genet. 2 5 5 , 1 5 7 - 1 6 5 Koehler, C . M , J a r o s c h , E „ Schweyen, R.J. and Shatz, G . S . (1996) M o l . Biol. Cell 7, 800

86. 87.

J a r o s c h , E , Tuller, G , D a u m , G , Waldherr, M „ V o s k o v a , A . and Schweyen, R.J. (1996) J. Biol. C h e m . 271, 17219-17225 Pfanner, N . and N e u p e r t , W. (1985) E M B O J . 4 , 2 8 1 9 - 2 8 2 5

88. 89.

Martin, J , Mahlke, K . and Pfanner, N . (1991) Eur. J . B i o c h e m . 2 0 0 , 4 6 3 - 4 6 9 Bauer, M . F , Sirrenberg, C , N e u p e r t , W. and Brunner, M . (1996) Cell 87, 33-41

90. 91.

B l o m , J , D e k k e r , P.J. and Meijer, M . (1995) Eur. J . B i o c h e m . 232, 309-314 B e r t h o l d , J . , Bauer, M . F , Schneider, H . C , K l a u s , C , Dietmeier, K., N e u p e r t , W. and Brunner, M . (1995) Cell 8 1 , 1 0 8 5 - 1 0 9 3

92. 93. 94. 95. 96. 97.

Lohret, T.A. and Kinnally, K.W. (1995) J . Biol. C h e m . 2 7 0 , 1 5 9 5 0 - 1 5 9 5 3 Lohret, T . A , Jensen, R . E . and Kinnally, K.W. (1997) J. Cell Biol. 137, 3 7 7 - 3 8 6 U n g e r m a n n , C „ G u i a r d , B „ N e u p e r t , W. and Cyr, D . M . (1996) E M B O J . 15, 735-744 U n g e r m a n n , C , N e u p e r t , W. and Cyr, D . M . (1994) Science 266, 1250-1253 I Iorst, M „ Oppliger, W , Feifel, B , Schatz, G . and Glick, B.S. (1996) Protein Sci. 5, 7 5 9 - 7 6 7 Schneider, H . C , Westermann, B , N e u p e r t , W. and Brunner, M . (1996) E M B O J. 15, 5796-5803

98. 99.

Glick, B . S , Wachter, C , Reid, G . A . and Schatz, G . (1993) Protein Sci. 2, 1901-1917 Voos, W „ von, A h s e n , O , Muller, H , G u i a r d , B „ R a s s o w , J . and Pfanner, N . (1996) E M B O J. 15,2668-2677

100. H o r s t , M „ O p p l i g e r , W , R o s p e r t , S „ S c h o n f e l d , H . J , Schatz, G . and A z e m , A . (1997) E M B O J. 1 6 , 1 8 4 2 - 1 8 4 9 101. B o m e r , U , Meijer, M , M a a r s e , A . C , H o n l i n g e r , A , D e k k e r , P . J , Pfanner, N . and R a s s o w , J. (1997) E M B O J. 16, 2 2 0 5 - 2 2 1 6 102. Sirrenberg, C , 384,582-585 103. T o k a t l i d i s , K , ( L o n d o n ) 384, 104. H a c k e n b r o c k ,

Bauer, M . F , G u i a r d , B , N e u p e r t , W. and Brunner, M . (1996) N a t u r e ( L o n d o n ) J u n n e , T „ M o e s , S „ S c h a t z , G „ G l i c k , B . S . and K r o n i d o u , N . ( 1 9 9 6 ) N a t u r e 585-588 C . R . J . (1968) J . Cell Biol. 37, 3 4 5 - 3 6 9

Mitochondrial targeting and import

105. N i c o l a y , K., R o j o , M., Wallimann, T., D e m e l , R. and H o v i u s , R. (199C) Biochim. B i o p h y s . Acta 1018, 2 2 9 - 2 3 3 106. H o v i u s , R . , F a b e r , B., B r i g o t , B., N i c o l a y , K . a n d d e K r u i j f f , B . ( 1 9 9 2 ) J . B i o l . C h e m . 2 6 7 , 16790-16795 107. Schleyer, M . and N e u p e r t , W. (1985) Cell 4 3 , 3 3 9 - 3 5 0 108. R a s s o w , J . , H a r t l , F . U . , G u i a r d , B., Pfanner, N . and N e u p e r t , W. ( 1 9 9 0 ) F E B S L e t t . 275, 190-194 109. Schulke, N . , Sepuri, N . B . and Pain, D . (1997) Proc. N a t l . Acad. Sci. U . S . A . 94, 7 3 1 4 - 7 3 1 9 110. V e s t w e b e r . D . and Schatz, G . (1988) J . Cell Biol. 1 0 7 , 2 0 3 7 - 2 0 4 3 111. van der Klei, I.J., Veenhuis, M . and N e u p e r t , W. (1994) Microsc. Res. Tech. 27, 2 8 4 - 2 9 3 112. D e k k e r , P.J.T., M a r t i n , F., M a a r s e , A . C . , G u i a r d , B . , Meijer, M . , R a s s o w , J . and Pfanner, N . (1997) E M B O J. 16, 5408-5419 113. K o l l , H . , G u i a r d , B., R a s s o w , J . , O s t e r m a n n , J . , H o r w i c h , A . L . , N e u p e r t , W. and H a r t l , F . - U . (1992) Cell 68, 1163-1175 114. Mahlke, K., Pfanner, N . , Martin, J., H o r w i c h , A . L . , Hartl, F.-U. and N e u p e r t , W. (1990) J . Biol. Chem. 265,16541-16547 115. 116. 117. 118.

R o j o , E . E . , Stuart, R . A . and N e u p e r t , W. (1995) E M B O J . 14, 3445-3451 H e r r m a n n , J . M . , N e u p e r t , W. and Stuart, R . A . (1997) E M B O J. 1 6 , 2 2 1 7 - 2 2 2 6 H a u c k e , V. and Schatz, G . (1997) E M B O J . 16, 4 5 6 0 - 4 5 6 7 Mahlke, K., Pfanner, N . , Martin, J., H o r w i c h , A . L . , Hartl, F.-U. and N e u p e r t , W. (1990) Eur. J. B i o c h e m . 192, 5 5 1 - 5 5 5

119. Pfanner, N . and N e u p e r t , W. (1985) E M B O J. 4 , 2 8 1 9 - 2 8 2 5 120. Lill, R., S t u a r t , R . A . , D r y g a s , M . E . , N a r g a n g , F.E. and N e u p e r t , W. ( 1 9 9 2 ) E M B O J . 11, 449-156 121. Reid, G . A . , Yonetani, T. and Schatz, G . (1982) J . Biol. C h e m . 257, 13068-13074 122. D a u m , G . , G a s s e r , S . M . a n d Schatz, G . (1982) ]. Biol. C h e m . 2 5 7 , 1 3 0 7 5 - 1 3 0 8 0 123. H e r r m a n n , J . M . , Koll, H „ C o o k , R.A., N e u p e r t , W. and Stuart, R . A . (1995) J. Biol. C h e m . 270, 27079-27086 124. Bohni, P.C., D a u m , G . and Schatz, G . (1983) J. Biol. C h e m . 258, 4 9 3 7 - J 9 4 3 125. Boteva, R., Fillippi, B. and Salvato, B. (1996) Arch. Biochem. B i o p h y s . 332, 329-334 126. Luciano, P. and G e l i , V . (1996) Experientia 52, 1077-1082 127. H a w l i t s c h e k , G . , S c h n e i d e r , H . , S c h m i d t , B., T r o p s c h u g , M . , H a r t l , F . - U . a n d N e u p e r t , W. (1988) Cell 53, 7 9 5 - 8 0 6 128. Glaser, E., S j o l i n g , S., S z i g y a r t o , C . and E r i k s s o n , A . C . (1996) B i o c h i m . B i o p h y s . A c t a 1275, 33-37 129. Braun, H.P. and Schmitz, U . K . (1995) Trends B i o c h e m . Sci. 20, 171-175 130. Heijne, G.V., Steppuhn, J . and H e r r m a n n , P.G. (1989) Eur. J. Biochem. 180, 5 3 5 - 5 4 5 131. H u r t , E . C . , Allison, D.S., Muller, U . and Schatz, G . (1987) J. Biol. C h e m . 262, 1420-1424 132. S j o l i n g , S., Waltner, M . , K a l o u s e k , F., G l a s e r , E. and Weiner, H . (1996) Eur. J . B i o c h e m . 242, 114-121 133. T h o r n t o n , K . , W a n g , Y., Weiner, H . a n d G o r e n s t e i n , D . G . (1993) J. Biol. C h e m . 268, 19906-19914 134. H a m m e n , P.K., Gorenstein, D . G . and Weiner, H . (1994) Biochemistry 33, 8610-8617 135. K a l o u s e k , F., H e n d r i c k , J.P. and R o s e n b e r g , L . E . ( 1 9 8 8 ) P r o c . N a t l . A c a d . Sci. U . S . A . 85, 7536-7540 136. Isaya, G , M i k l o s , D . and Rollins, R.A. (1994) Mol. Cell. Biol. 1 4 , 5 6 0 3 - 5 6 1 6 137. Peralta, D „ L i t h g o w , T „ H o o g e n r a a d , N . J . and H o j , P.B. (1993) Eur. J . B i o c h e m . 2 1 1 , 8 8 1 - 8 8 9 138. Schneider, A., Behrens, M . , Scherer, P., Pratje, E., Michaelis, G . and Schatz, G . (1991) E M B O J. 10, 2 4 7 - 2 5 4 139. P r i p - B u u s , C . , W e s t e r m a n , B., Schmitt, M . , L a n g e r , T., N e u p e r t , W. and S c h w a r z , E. (1996) F E B S Lett. 3 8 0 , 1 4 2 - 1 4 6 140. L a l o r a y a , S., D e k k e r , P.J., V o o s , W., C r a i g , E . A . and Pfanner, N . (1995) J. M e m b r . Biol. 148, 169-176 141. M a t o u s c h e k , A . , R o s p e r t , S „ Schmid, K., G l i c k , B.S. and Schatz, G . (1995) Proc. N a t l . A c a d . Sci. U . S . A . 9 2 , 6 3 1 9 - 6 3 2 3 142. R o s p e r t , S., L o o s e r , R . , D u b a q u i e , Y., M a t o u s c h e k , A., G l i c k , B.S. and S c h a t z , G . (1996) E M B O J. 1 5 , 7 6 4 - 7 7 4

247

248

R.Hovius

143. O s t e r m a n n , J , H o r w i c h , A . L , N e u p e r t , W. a n d H a r t l , F . - U . ( 1 9 8 9 ) N a t u r e ( L o n d o n ) 341, 125-130 144. H o h f e l d , J. and Hartl, F.-U. (1994) J. Cell Biol. 126, 3 0 5 - 3 1 5 145. X u , Z., H o r w i c h , A . L . and Sigler, P.B. (1997) N a t u r e ( L o n d o n ) 388, 7 4 1 - 7 5 0 146. Pfaller, R , F r e i t a g , H , H a r m e y , M . A . , B e n z , R . and N e u p e r t , W. ( 1 9 8 6 ) J . B i o l . C h e m . 260, 8188-8193 147. Sollner, T , G r i f f i t h s , G , Pfaller, R , Pfanner, N . and N e u p e r t , W. (1989) Cell 59, 1061-1070 148. Hostetler, K.Y. and B o s c h , H . (1972) Biochim. B i o p h y s . A c t a 260(3), 3 8 0 - 3 8 6 149. Marinetti, G . V , E r b l a n d , J. and Stotz, E. (1985) J. Biol. C h e m . 233, 5 6 2 - 5 6 5 150. H o v i u s , R , T h i j s s e n , J , van d e r L i n d e n , P , N i c o l a y , K . and de K r u i j f f , B . ( 1 9 9 3 ) F E B S Lett. 330, 7 1 - 7 6 151. van't H o f , R , van K l o m p e n b u r g , W , Pilon, M , K o z u b e k , A , de K o r t e - K o o l , G , D e m e l , R . A , Weisbeek, P.J. and de Kruijff, B. (1993) J . Biol. C h e m . 2 6 8 , 4 0 3 7 - 4 0 4 2 152. O u , W . J , I t o , A , U m e d a , M , I n o u e , K . a n d O m u r a , T. (1988) J . B i o c h e m . ( T o k y o ) 103, 589-595 153. E n d o , T , Eilers, M . and Schatz, G . (1989) J . Biol. C h e m . 2 6 4 , 2 9 5 1 - 2 9 5 6 154. Eilers, M „ E n d o , T. and Schatz, G . (1989) J . Biol. C h e m . 2 6 4 , 2 9 4 5 - 2 9 5 0 155. M a d u k e , M . and R o i s e , D . (1993) Science 2 6 0 , 3 6 4 - 3 6 7 156. L e e n h o u t s , J . M . , T o r o k , Z , D e m e l , R . A . , d e G i e r , J . a n d d e K r u i j f f , B . ( 1 9 9 6 ) M o l . M e m b r . Biol. 1 1 , 1 5 9 - 1 6 4 157. L e e n h o u t s , J . M , T o r o k , Z , M a n d i e a u , V , G o o r m a g h t i g h , E. and de K r u i j f f , B . ( 1 9 9 6 ) F E B S Lett. 388, 3 4 - 3 8 158. Roise, D . (1997) J . Bioenerg. B i o m e m b r . 2 9 , 1 9 - 2 7 159. H u c k r i e d e , A , H e i k e m a , A . , W i l s c h u t , J . a n d A g s t e r i b b e , E. ( 1 9 9 6 ) E u r . J . B i o c h e m . 237, 288-294 160. Yano, M , K a n a z a w a , M . , Terada, K , N a m c h a i , C , Y a m a i z u m i , M , H a n s o n , B , H o o g e n r a a d , N . and M o r i , M.V. (1997) J . Biol. C h e m . 272, 8 4 5 9 - 8 4 6 5

14 Translocation of proteins into and across the thylakoid membrane Colin Robinson*, Alexandra Mant and Susanne Brink Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, U.K.

Introduction Chloroplast biogenesis is a complex process, requiring protein trafficking on a massive scale. This organelle is one of the most complex known in structural terms, comprising three distinct membranes (outer and inner envelopes and the thylakoid membrane) that enclose, in turn, three distinct soluble phases (intermembrane space, stroma and thylakoid lumen). The chloroplast contains its own genetic system and synthesizes about 10-20% of the organellar proteins; the remainder are imported after synthesis in the cytosol. In total, several hundred different proteins are imported from the cytosol and distributed to all six chloroplastic subcompartments; hence the processes of protein import and intra-organellar protein sorting are of central importance in plant cell biology. The biogenesis of the photosynthetic apparatus in the thylakoid membrane is of particular interest in this respect. Most of

the photosynthetic proteins arc organized into large

protein-pigment complexes that contain a mixture of integral membrane proteins and hydrophilic, loosely bound proteins. Because most of the proteins are synthesized in the cytosol, these proteins must be transported across the two envelope membranes and the soluble stromal phase to reach their target membrane. Each of these complexes also contains chloroplast-encoded polypeptides, so the plant cell is faced with the considerable problem of co-ordinating the expression of the two genetic systems during the biogenesis of the photosynthetic apparatus. The complexity of the protein targeting/assembly requirements is exemplified by Figure 1, which shows a model for the structure of the photosystem II (PSII) complex. PSII contains numerous integral membrane proteins with varying numbers of transmembrane spans; some of these are chloroplast-encoded, whereas others are imported from the cytosol. The complex also contains hydrophilic proteins that are loosely bound to the lumenal face of the membrane (thepsbO,psbP andpsbQ gene products, often referred to as 33K, 23K and 16K, and the psbTgene product), and the import of these nuclear-encoded lumenal proteins has attracted considerable attention because these proteins must "To whom correspondence should, be addressed.

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Figure 1

Structure of the PSII complex in higher plants

Stroma

LHCIJ

Lumen

PsbQ (16K)

PsbP (23K)

PsbO (33K)

The diagram illustrates a madel far the PSII camplex. in which chlaroplast-encoded subunits are all shaded. The diagram also shows the pathway of electron flow from water to plastoquinone. via tyrosine residues (ZTyr). a manganese cluster. the reaction ffiffffi. mm




+ + + +

(b) α -Helix formation

(c) Insertion

^TPP (d) Processing

(e)

The model is based on similarities between these thylakoid proteins and M13 procoat, and is entirely untested, (a) Interaction of the positively charged precursor with the negatively charged surface of the thylakoid membrane, (b) The two Η-domains (one in the thylakoid transfer signal, the other in the mature protein) form α-helices, either before or after binding to the membrane, (c) The two Η-domains insert into the membrane, and the intervening acidic region flips across, (d) Cleavage by thylakoidal processing peptidase (TPP) releases the presequence and leaves the mature protein in a transmembrane form (e).

numbers of negative charges. Nevertheless, the basic insertion mechanisms may turn out to be similar in important respects, and initial impressions suggest that this may be a preferred method for integrating those thylakoid proteins that contain a single membrane span and a small N-terminal section in the lumen. Studies in bacteria (Chapters 4 and 5) have shown that the translocation of larger hydrophilic regions across the membrane tends to require the Sec apparatus (in particular SecA), and the same may well apply in thvlakoids.

Integration of more complex thylakoid membrane proteins Many imported thylakoid membrane proteins contain multiple membranespanning regions, but very few of these proteins have been studied in terms of integration mechanism. The majority are synthesized only with envelope transit

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signals, and the lack of a cleavable thylakoid transfer signal means that the required integration signals must reside in the mature proteins. This has been confirmed experimentally in studies on the major thylakoid membrane protein, the lightharvesting chlorophyll-binding protein of PSII (LHC II), which contains three membrane-spanning regions. Replacement of the LHC II presequence with that of an imported stromal protein has no effect on integration efficiency, and the mature LHC II protein is, furthermore, able to insert into isolated thylakoids [37,38]. To date, LHC II is the only complex membrane protein whose integration has been analysed rigorously. In vitro assays for insertion into isolated thylakoids have shown that this process requires NTP hydrolysis, with GTP being the preferred energy source [21,39], The thylakoidal ΔρΗ is also required for efficient integration, and the process requires, in addition, the presence of a stromal protein factor which probably serves to maintain the LHC II in a soluble, integrationcompetent form [40], More recent studies [41] have identified this factor (or a part thereof) as a homologue of the 54 kDa polypeptides of signal recognition particles (SRPs). SRPs are involved in guiding proteins to the translocation apparatus in the endoplasmic reticulum (Chapter 10) and the bacterial plasma membrane (Chapter 6) and, although eukaryotic SRPs contain six polypeptides and an RNA molecule, the bacterial counterpart appears to be a simpler entity that comprises a homologue of the eukaryotic 54 kDa subunit together with an RNA molecule. It is not yet clear whether the chloroplast stromal SRP also contains RNA, but the molecule appears more similar to bacterial than to eukaryotic SRPs, suggesting that this pathway, like the Sec pathway, was inherited from the cyanobacterial progenitor of the chloroplast. It appears likely that most multi-spanning membrane proteins utilize this pathway, although it remains a distinct possibility that some may associate with SecA, rather than SRP, in the stroma.

Conclusions Thylakoid protein targeting has proved to be a fascinating subject area in recent years, full of surprises. The various known targeting pathways are summarized in Figure 4, which illustrates the remarkable, and quite unexpected, complexity of the system. Many questions remain to be answered. The origins and mechanism of the ΛρΗ-driven system are of particular interest, mainly because this system has no known parallels in other protein-translocating membranes to date. In addition, we currently have very little information on the means by which the different import components recognize their cognate precursors, because all of the targeting signals have hydrophobic domains which appear superficially quite similar. Of course, it may be that there is some overlap in the system, with some proteins being targeted by more than one pathway, but because there is no evidence for such redundancy so far, we believe that the signals are recognized rather specifically by the appropriate import apparatus. The available import assays should go a long way towards

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Figure 4

Multiple pathways for thylakoid protein targeting

Cytosol

Pre -PsbW

Stroma

pre CF0II iPsbX iPsbW

'Spontaneous'

CF0II PsbW PsbX

The known substrate for the SRP-dependent pathway, LHC II, is synthesized with only a stroma targeting 'envelope transit' signal, and information within the mature protein specifies integration into the membrane by a GTP/ATP-dependent mechanism. Proteins on the other pathways are synthesized with bipartite presequences containing cleavable thylakoid transfer signals. Lumenal proteins are targeted either by the Sec- or ApH-dependent pathways, whereas CFJi, PsbW and PsbX integrate by a Sec/ ApH-independent, apparently spontaneous, mechanism.Most of these proteins are converted into intermediate forms in the stroma by stromal processing peptidase, although at least two proteins (CFJl and PSI-N) are targeted to thylakoids as the full precursor forms. PC, plastocyanin; the prefix V denotes an intermediate form of a protein.

answering these questions, although there is little doubt that complementary genetic and structural studies will be of enormous value in the final analysis.

References 1. 2. 3. 4. 5.

Thcg, S.M. and Scott, S.V. (1993) Trends Cell Biol. 3,186-190 Gray, J.C. and Row, P.E. (1995) Trends Cell Biol. 5,243-247 Schnell,D.J. and Blobel, G. (1993) J. Cell Biol. 120, 103-115 Olsen, L .J., Theg, S.M., Selman, B.R. and Keegstra, K. (1989) J. Biol. Chem. 264, 6724-6729 Theg, S.M., Bauerle, C.B., Olsen, L.J., Selman, B.R. and Keegstra, K. (1989) J. Biol. Chem. 264,

6. 7. 8.

6730-6736 Robinson, C. and Ellis, R.J. (1984) Eur. J. Biochem. 142, 337-342 Oblong, J.E. and Lamppa, G.K. (1992) EMBO J. 11,4401-4409 Hageman, J., Robinson, C., Smeekens, S. and Weisbeek, R (1986) Nature (London) 324,

9.

567-569 James, H.E., Bartling, D., Musgrove l J-E., Kirwin, P.M., Herrmann, R.G. and Robinson, C.

10.

(1991) J. Biol. Chem. 264, 19573-19576 Hageman, J., Baecke, C„ Ebskamp, M., Pilon, R., Smeekens, S. and Weisbeek, P. (1990) Plant Cell 2,479—194

258

C.Robinson etal.

11. 12.

K o , K . and C a s h m o r e , A . R . (1989) E M B O J . 8 , 3 1 8 7 - 3 1 9 4 N i e l s e n , V.S., Mant, A., K n o e t z e l , J . , Moller, B . L . and R o b i n s o n , C . (1994) J . Biol. C h e m . 269, 3762-3766

13.

K i r w i n , P.M., E l d e r f i e l d , P.E., W i l l i a m s , R . S . a n d R o b i n s o n , C . ( 1 9 8 8 ) ] . B i o l . C h e m . 2 6 3 , 18128-18132

14. 15.

von Heijne, G „ Steppuhn, J. and H e r r m a n n , R . G . (1989) Eur. J. Biochem. 180, 5 3 5 - 5 4 5 H a e h n e l , W., J a n s e n , T., G a u s e , K . , K l o s g e n , R . B . , Stahl, B., Michl, D . , H u v e r m a n n , B., K a r a s , M . and H e r r m a n n , R . G . (1994) E M B O J. 13, 1028-1038

16.

Seidler, A. and Michel, H . (1990) E M B O J. 9, 1743-1748

17. 18.

Briggs, L . M . , Peroraro, V.L. and M c i n t o s h , L. (1990) Plant M o l . Biol. 1 5 , 6 3 3 - 6 4 2 K u w a b a r a , T., R e d d y , K . J . a n d S h e r m a n , L . A . ( 1 9 8 7 ) P r o c . N a t l . A c a d . Sci. U . S . A . 84, 8230-8235 N a k a i , M . , Sugita, D . , O m a t a , T. and E n d o , T . ( 1 9 9 2 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 193, 228-234

19. 20. 21. 22. 23.

M o u l d , R . M . and R o b i n s o n , C . (1991) J. Biol. C h e m . 266, 12189-12193 Cline, K „ Ettinger, W. and Theg, S.M. (1992) J . Biol. C h e m . 267, 2 6 8 8 - 2 6 9 6 H u l f o r d , A., Hazell, L., M o u l d , R . M . and R o b i n s o n , C . (1994) J . Biol. C h e m . 269, 3251-3256 R o b i n s o n , C „ C a i , D . , H u l f o r d , A., B r o c k , I.W., Michl, D . , H a z e l l , L., Schmidt, I., H e r r m a n n , R . G . and K l o s g e n , R . B . (1994) E M B O J. 1 3 , 2 7 9 - 2 8 5

24.

N a k a i , M . , G o t o , A . , N o h a r a , T., S u g i t a , D . a n d E n d o , T. ( 1 9 9 4 ) J . B i o l . C h e m . 2 6 9 , 31338-31341

25. 26.

Yuan, J., Henry, R „ M c C a f f r e y , M . and Cline, K . (1994) Science 266, 7 9 6 - 7 9 8 Laidler, V., C h a d d o c k , A . M . , K n o t t , T . G . , Walker, D . and R o b i n s o n , C . (1995) J . Biol. C h e m . 270,17664-17667 M a n t , A., N i e l s e n , V.S., K n o t t , T . G . , Meller, B . L . and R o b i n s o n , C . (1994) J . Biol. C h e m . 269, 27303-27328

27. 28.

K a r n a u c h o v , I., C a i , D . , S c h m i d t , I., H e r r m a n n , R . G . and K l o s g e n , R . B . (1994) J . Biol. C h e m . 269,32871-32878

29.

M a n t , A., S c h m i d t , I., H e r r m a n n , R . G . , R o b i n s o n , C . and K l o s g e n , R . B . (1995) J . Biol. C h e m . 270,23275-23281

30.

C r e i g h t o n , A . M . , H u l f o r d , A., Mant, A., R o b i n s o n , D . and R o b i n s o n , C . (1995) J . Biol. C h e m . 270,1663-1669

30a.

Settles, M . A . , Y o n e t a n i , A . , B a r o n , A . , B u s h , D . R . , C l i n e , K . a n d M a r t i e n s s e n , R . ( 1 9 9 7 ) Science 278, 1467-1470 H e n r y , R . , K a p a z o g l o u , A . , M c C a f f e r y , M . and C l i n e , K . ( 1 9 9 4 ) J . B i o l . C h e m . 2 6 9 , 10189-10192

31. 32.

C h a d d o c k , A . M . , M a n t , A . , K a r n a u c h o v , I., B r i n k , S., H e r r m a n n , R . G . , K l o s g e n , R . B . a n d R o b i n s o n , C . (1995) E M B O J. 1 4 , 2 7 1 5 - 2 7 2 2

33.

L o r k o v i c , Z.J., Schroder, W.P., Pakrasi, H . B . , Irrgang, K . - D . , H e r r m a n n , R . G . and Oelmuller, R. (1995) Proc. N a t l . A c a d . Sci. U . S . A . 9 2 , 8 9 3 0 - 8 9 3 4 Michl, D . , R o b i n s o n , C . , Shackleton, J . B . , H e r r m a n n , R . G . and K l o s g e n , R . B . (1994) E M B O J . 13, 1310-1317

34.

-

35. 35a.

K i m , S.J., R o b i n s o n , D . and R o b i n s o n , C . (1996) F E B S Lett. 3 9 0 , 1 7 5 - 1 7 8 K i m , S.J., R o b i n s o n , C . and Mant, A . (1998) F E B S Lett. 424, 105-108

36. 37.

R o b i n s o n , D . , K a r n a u c h o v , I., H e r r m a n n , R . G . , K l o s g e n , R . B . and R o b i n s o n , C . (1996) Plant J. 10, 149-155 L a m p p a , G . K . (1988) J. Biol. C h e m . 263, 14996-14999

38. 39. 40.

Viitanen, P.V., D o r a n , E . R . and D u n s m u i r , P. (1988) J. Biol. C h e m . 263, 15000-15007 H o f f m a n , N . E . and Franklin, A. (1994) Plant Physiol. 1 0 5 , 2 9 5 - 3 0 4 F u l s o n , D . R . and Cline, K . (1988) Plant P h y s i o l . 8 8 , 1 1 4 6 - 1 1 5 3

41.

Li, X . , H e n r y , R „ Yuan, J . , C l i n e , K . and H o f f m a n , N . E . (1995) P r o c . N a t l . A c a d . Sci. U . S . A . 92,3789-3793

15 Principles of peroxisomal protein sorting and assembly J.A.K.W. Kiel, I.J. van der Klei and M.Veenhuis* Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Biological Centre, Kerklaan 30,9751NN Haren1The Netherlands

Introduction Microbodies (peroxisomes/glyoxysomes/glycosomes) are currently recognizcd as a class of important organelles, indispensable for the proper functioning of eukaryotic cells. Although their morphological structure is relatively simple, their physiological properties are remarkably complex. Initially, the organelles were thought to be involved in hydrogen peroxide generation and decomposition, Subsequently, various microbody functions have been described, including essential roles in photorespiration in plants, cholesterol metabolism and plasmalogen biosynthesis in mammals, penicillin biosynthesis in filamentous fungi, and C 1 and C, metabolism in fungi [1,2], Part of the problem in assigning a single function to the compartment is that the menu of peroxisomal proteins varies between organisms, cell types, the developmental stage of the cells/organism and the environment in which they occur. This functional flexibility is unique to peroxisomes, and is not observed for other essential organelles which have their own characteristic tasks (e.g. energy generation by mitochondria). Currently, our knowledge of the molecular mechanisms of microbody biogenesis and function is expanding rapidly, especially now that various peroxisome-deficient mutants have become available and the complementing genes isolated [3]. Topics of current research interest include organelle homoeostasis, sorting and assembly of matrix proteins and biosynthesis of the microbody membrane. In this contribution, we present an overview of recent achievements relating to the principles of peroxisomal protein import and assembly, and discuss how these findings fit into the current, generally accepted, concept of peroxisome biogenesis.

'To whom correspondence should be addressed.

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General properties of yeast microbodies Induction of microbodies In veasts, the induction and metabolic significance of microbodies (peroxisomes) is predominantly prescribed by environmental stimuli (e.g. growth conditions). In the methvlotrophic yeast Hansenulapolymorpha, maximal peroxisome induction is obtained during growth of cells on methanol; under these conditions, these organelles may take up 80% of the cytoplasmic volume [4] (Figure 1). Other substrates known to induce microbodies in fungi are fattv acids, C, compounds (e.g. ethanol), propionate, primary amines, D-amino acids and purines [1]. A charac­ teristic feature of these organelles is that they contain the key enzymes involved in the metabolism of the above carbon/nitrogen sources. Few exceptions have been encountered with respect to this general rule. For instance, in fully repressed yeast cells grown on glucose, only a single small organelle is present. The significance of this organelle in intermediary cellular metabolism is still unknown; however, it is not essential for the cell's viability, since peroxisome-deficient mutants, which lack intact microbodies, normally grow on glucose [5,6]. Present knowledge on the physiology and biochemistry of peroxisome induction now allows precise adjustment of both microbody proliferation and their protein composition bv manipulation of the growth conditions. Figure 1

Overall morphology of a wild-type H. polymorpha cell grown in a methanol-limited chemostat

The cell is crowded with peroxisomes. The scale bar represents I μ/η.

Intact peroxisomes are essential to support growth of methylotrophic yeasts on methanol as the sole carbon and energy source Peroxisome-deficient {pex) mutants of methylotrophic yeasts are unable to grow on methanol as the sole carbon source. In most of the pex mutants, the enzymes

Peroxisomal protein sorting and assembly

involved in methanol metabolism, including the peroxisomal enzymes alcohol oxidase (AO), catalase and dihydroxyacetone synthase, are normally present and active [7,8]. Although methanol is utilized by pex mutants, its metabolism is apparently hampered by the cytosolic location of these enzymes. This raises the intriguing question as to why compartmentalization of the key reactions of methanol metabolism is essential for growth. The use of methanol by Hansenula polymorpbapex mutants was studied in detail in glucose-limited chemostat cultures to which methanol was added as a second growth substrate. These experiments revealed that methanol is not used as a second carbon source by pex cells. However, it may serve as an additional energy source when added in relatively low amounts. At enhanced levels, methanol utilization resulted in severe metabolic disadvantages, reflected by a decrease in cell yield [7], This phenomenon^ appeared to be related to the accumulation of the first intermediates of methanol metabolism (hydrogen peroxide and formaldehyde) in the cytosol, which resulted in their further metabolism via energetically disadvantageous pathways, and suggested that the general major advantage of intact microbodies is that their presence permits the cell to adjust precisely the levels of different intermediates required for specific metabolic pathways. Another possibility, namely compartmentalization of the metabolism of toxic and/or reactive compounds such as glyoxylate and hydrogen peroxide, although essential under specific conditions (e.g. during growth on methanol), is less likely, since H. polymorpha pex strains grow normally on organic nitrogen compounds which require the activity of a hydrogen peroxide-generating oxidase (e.g. amine oxidase during growth of cells on methylamine [9]). Apparently, the cell can deal readily with considerable amounts of hydrogen peroxide, which have been shown to be decomposed by mitochondrial peroxidases [10], and it is of relevance to mention that catalase-negative mutants of Saccharomyces eerevisiae also grow well on oleic acid [11].

Peroxisomal protein import Only a subset of peroxisomes is capable of importing proteins in vivo A remarkable feature of methanol-hmited H. polymorpha cells is that they contain many large peroxisomes of comparable size together with a few smaller ones. Apparently, under these conditions, the organelles do not exceed a certain size. Similar observations were made in other organisms: the size (and number) of what we consider to represent 'mature' organelles is remarkably constant and apparently prescribed by the prevailing conditions [1]. Subsequent studies revealed that, in vivo, the small organelles in the peroxisomal population are capable of importing newly synthesized matrix proteins. Cytochemical studies demonstrated unequiv­ ocally that heterogeneity may exist among microbodies within one cell with respect

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to their capacity to incorporate newly synthesized proteins [12]. During the adaptation of Candida boidinii cells to a new growth environment, i.e. a shift of cclls from oleate to methanol, a functionally mixed population of microbodies was observed, of which the major part contained solely enzymes of the /3-oxidation pathway, while the other part contained newly synthesized key enzymes of methanol metabolism together with the same /3-oxidation enzymes [13]. In contrast, cells growing on oleate/methanol mixtures only contained one population of peroxisomes, which harboured the key enzymes associated with both growth substrates. This again stressed the functional complexity of the organelles, which may be involved simultaneously in the metabolism of two different carbon sources, and at the same time may also participate in the metabolism of unusual nitrogen sources such as D -amino acids or primary amines. This poses the question of how the cell manages to discriminate between mature and import-competent peroxisomes [14]. We have proposed that there must be functional protein complexes on the peroxisomal membrane that are involved in matrix protein import (see below). Upon maturation and subsequent fission of the growing organelle, these protein complexes may be donated to the newly developing organelle, thus resulting in a small protein import-competent organelle and a mature one which is metabolically active but has lost its developmental functions.

At least two separate matrix protein-sorting mechanisms exist In the initial concept of De Duve and co-workers, microbodies were thought to develop from the endoplasmic reticulum (ER) [15], This view was changed completely when Lazarow and Fujiki [16] demonstrated that precursors of peroxisomal matrix proteins are synthesized in the cytosol on free polysomes, indicating that import occurs post-translationally; in this respect it resembles import into other cell organelles such as mitochondria (Chapter 13) and chloroplasts (Chapter 14). Subsequent studies have identified the first peroxisomal targeting signals (PTSs), discrete amino acid sequences that are both necessary and sufficient to direct proteins to the peroxisomal matrix ([17]; Chapter 16). The majority of peroxisomal matrix proteins appear to contain a C-terminal tripeptide of the sequence Ser-Lys-Leu-COOH or a conservative variant (designated PTSl [17,18]). PTSl-containingpolypeptides appear not to be cleaved upon entry into the peroxisome. A second PTS, designated PTS2, was shown to consist of an Nterminal nonapeptide with the consensus sequence (R/K)(L/V/I)(X) S (H/Q)(L/A), which so far has been observed in only a limited number of peroxisomal proteins [17-19]. In mammals and plants, PTS2 appears to be processed upon import into the organelle. In contrast, in yeasts, PTS2-containing polypeptides are not cleaved, except for thiolase in Yarrowia lipolytica [20]. However, additional sorting mechanisms must exist [21,22], but have not yet been identified. Also, peroxisomal membrane proteins (Pmps) are not targeted to the peroxisome by PTSl or PTS2 sequences. Recently, Dyer et al. [23] showed that the targeting information of C. boidinii Pmp47 resides in a 20-amino-acid loop between two membrane spans. This

Peroxisomal protein sorting and assembly

loop appeared to contain a cluster of basic amino acids (KIKKR) that is conserved in other peroxisomal membrane proteins. PTSl and PTS2 are conserved from yeast to humans [24]. The use of both yeast and human systems selectively deficient in either PTSl- or PTS2-dependent protein import has allowed the identification of the PTSl and PTS2 receptors (Pex5p and Pex7p respectively; see Table 1). Surprisingly, the location of both receptors appears to be rather controversial. The PTSl receptor in the yeast Pichia pastoris was shown to be tightly associated with the peroxisomal membrane [25], In contrast, homologues in other yeasts, as well as in humans, exhibit different subcellular locations, ranging from completely cytosolic to completely intraperoxisomal [26-31]. Similar puzzling observations have been made for the only PTS2 receptor identified so far, S. cerevisiae Pex7p [32,33]. Rachubinski and Subramani [34] have suggested that the multiple subcellular localizations of these receptors either might reflect shuttling proteins with their location dependent on the physiological state of the cell, or might in part represent dead-end molecules that are no longer actively engaged in protein import. PTSl receptors belong to the tetratrico-peptide repeat family of proteins. These proteins share a loosely conserved 34-amino-acid repeat that has been shown to bind PTSl [25]. Similarly, S. cerevisiae Pex7p is a member of the so-called WD40 repeat family, and was shown to bind PTS2 [35,36], It has been reported that tetratrico-peptide repeat and WD40 proteins are functionally related. Recently, Rehling et al. [35] showed that the PTSl and PTS2 receptors indeed interact both in vivo (two-hybrid screening) and in vitro (affinity chromatography). This observation seems rather surprising in view of the fact that yeast mutants lacking the PTSl receptor normally import PTS2 proteins, and vice versa, suggesting that, despite the interaction between the receptors, the PTSl and PTS2 import pathways can apparently proceed independently. This appears not to be true for the human system. Wiemer et al. [29] showed that full complementation occurred when a cDNA encoding the human PTSl receptor was expressed in fibroblasts from certain patients suffering from the peroxisomal disorder Zellweger syndrome that were impaired in the import of both PTSl and PTS2 proteins. In this case, an interaction between components of the PTSl and PTS2 pathways seems to be essential for normal protein import. Clearly, additional evidence for the physio­ logical significance of the association between the receptors is required. An intriguing finding in organellar protein import research was that the peroxisomal import machinery could accomodate oligomeric proteins. Glover et al. [37] showed that PTS2-deficient thiolase molecules could be imported into peroxisomes provided that they were co-produced with wild-type thiolase. Similar observations have been made for the PTSl system [38] using the trimeric protein chloramphenicol acetyltransferase as a reporter protein. Furthermore, stabilization of the folded conformation of PTSl proteins did not affect their import into microbodies [39,40]. In fact, even 9 nm gold particles carrying PTSl can be a substrate for import into peroxisomes [39], Taken together, these data demonstrate

263

PEX5

I 12-127 kDa; belongs to the family of AAA ATPases; contains two AAA domains; has been localized to the peroxisomal

34-48 kDa; integral peroxisomal membrane protein; contains C3HC4 zinc-finger motif; suggested to be involved in peroxisome

PEXIO

43 kDa; peroxisomal membrane protein; C-terminal SH3 domain; binds the PTSI recognition factor; putative docking protein

PEX13

see [3,49].

38—40 kDa; peroxisomal membrane protein

Abbreviation.TPR, tetratrico-peptide repeat For references,

PEX14

31 kDa; integral peroxisomal membrane protein; contains degenerate C3HC4 zinc-finger motif

PEXI2

for peroxisomal protein import

27-32

PEXII

kDa; peroxisomal membrane protein; involved in peroxisome proliferation; deficiency results in giant peroxisomes

proliferation or lumen formation

42 kDa; integral peroxisomal membrane protein

aspects of the peroxisomal membrane

71-81 kDa; contains both a C-terminal PTSI and an N-terminal PTS2; has been localized to the peroxisomal matrix and inner

and the peroxisomal matrix

42 kDa; contains six WD40 motifs; PTS2 recognition factor; localized to the cytosol as well as to the peroxisomal membrane

membrane and the cytosol

PEX9

PEX8

PEX7

PEX6

64-69 kDa; contains at least six TPR motifs; PTSI recognition factor; localized to the cytosol as well as to the peroxisomal

PEX4

membrane and the peroxisomal matrix

51-52 kDa; integral peroxisomal membrane protein

21-24 kDa; ubiquitin-conjugating protein; associated with the peroxisomal membrane

PEX3

I 17-127 kDa; belongs to the family of AAA-type ATPases; contains two AAA domains; subcellular location not yet known

35-52 kDa; contains characteristic C3HC4 zinc-finger motif; integral peroxisomal membrane protein

PEXI

Gene

PEX2

Characteristics of PEX genes and their translation products

Peroxin characteristics

Table I

Peroxisomal protein sorting and assembly

that peroxisomal protein import is not just a variation on well known mechanisms (e.g. those in mitochondria; Chapter 13), but has several new features. Different models have been proposed to explain the ability of peroxisomes to incorporate these large, stable particles [38]. These range from the existence of either static or dynamic pores in the peroxisomal membrane to endocytosis events at the level of the peroxisomal membrane. However, convincing experimental evidence for either of these possibilities is lacking so far. As stated above, the sorting machinery that is essential for matrix protein import is apparently not required for the insertion of peroxisomal membrane proteins. This can be concluded from the finding that the majority of Apex strains, in which matrix protein import is impaired, contain peroxisomal membrane remnants ('ghosts') carrying peroxisomal membrane proteins [41]. This suggests that matrix protein import and membrane protein insertion proceed via different, independent, pathways.

Assembly of peroxisomal matrix proteins An intriguing aspect of peroxisome biogenesis involves the assembly and activation of matrix enzymes. In vivo, these processes are precisely regulated; this is indicated by the fact that, in wild-type yeast cells, the activity of peroxisomal enzymes is confined to the organellar matrix. Also, the cytosolic precursor pools are invariably undetectable or extremely low [42]. The observation that a dimeric peroxisomal protein such as thiolase can be translocated by the peroxisomal import machinery in a folded conformation suggests that this protein probably assembles in the cytosol [37], However, evidence is accumulating that specific peroxisomal factors are required for the assembly/activation of AO and probably also other oligomeric matrix proteins of methylotrophic yeasts.

AO assembles into an octameric flavoprotein inside peroxisomes The flavoprotein AO of methylotrophic yeasts (C. boidinii, H. polymorpha) has been used extensively as a model protein in peroxisomal protein assembly/ activation studies. In vivo, AO precursors are synthesized in the cytosol and sorted to their target organelle by a PTSl signal (-ARF.COOH), where the protein obtains its enzymically active conformation [42J. Besides oligomerization into a homooctamer, this also includes the non-covalent binding of the FAD cofactor. Numerous experiments designed with the ultimate goal of establishing the conditions needed to activate monomeric, inactive, AO into the active octamer in vitro have failed. A major obstacle in this approach is related to the intrinsic property of monomeric AO, which is that it easily forms aggregates upon removal of the denaturant. H. polymorpha AO is also not assembled and activated when synthesized in vitro in a reticulocyte lysate [43] or in heterologous hosts (e.g. Escherichia coll or S. cerevisiae [44,45]).

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When import is prevented in wild-type H. polymorpba cells, peroxisomal matrix proteins accumulate in the cytosol in an inactive monomeric form, where they often form aggregates. However, in pex mutants the matrix enzymes are correctly assembled and active in the cytosol [8,9]. Apparently, in the absence of peroxisomes, putative peroxisomal assembly factors are located in the cytosol, where they can perform their function normally [8,46].

FAD binding is essential in AO assembly The role of FAD binding in AO assembly was studied in riboflavin-auxotrophic mutants of H. polymorpba, in which the amount of available flavin could be manipulated by varying its concentration in the growth medium. These studies revealed that riboflavin limitation, and thus limitation of the cofactor FAD, resulted in the formation of both active and inactive AO proteins [47]. Two locations for inactive AO were observed, namely as small protein aggregates inside peroxisomes and soluble monomers in the cytosol. Octameric AO was exclusively peroxisomal, but had a lower FAD content and displayed reduced specific AO activity compared with AO protein isolated from wild-type cells. Subsequent studies showed that only FAD-containing monomers were able to assemble into the octameric, active protein [46], The AO aggregates in the peroxisomal matrix therefore were probably composed of FAD-deficient monomers, resulting from the reduced efficiency of AO oligomerization related to FAD limitation. The accumulation of cytosolic AO monomers may have resulted from saturation of the putative intra-peroxisomal assembly machinery, leading to a partial inhibition of AO import.

Biogenesis of peroxisomes Peroxisomal membrane biogenesis and protein import may be coupled processes Analysis of the available peroxisome-deficient yeast mutants has led to the identifi­ cation of several proteins involved in peroxisome biogenesis (Table 1). Interestingly, some of these seem to play a dual role, i.e. both in peroxisomal protein import and in the biogenesis of the peroxisomal membrane. In an H. polymorpba PEX3 disruption strain (Δpex3), the biogenesis of the peroxisomal membrane is fully disturbed, i.e. peroxisomal 'ghosts' or peroxisomal membrane remnants are completely absent [41,48]. This implies that Pex3p is essential for the formation of the peroxisomal membrane. This was also evident from experiments using a strain in which HpPEX3 had been placed under the control of a regulatable promoter. A sudden repression of PEX3 in a Pex3p-producing strain resulted in the gradual disintegration of the peroxisomal membrane, indicating a role for Pex3p in the maintenance of the membrane [48]. Other findings suggested a role for Pex3p in peroxisomal protein import. Overexpression of PEX3 in wild-type H. polymorpba cells resulted in a defect in peroxisome assembly [48a], reflected in the formation of

Peroxisomal protein sorting and assembly

numerous membrane vesicles that were characterized by the presence of Pex3p in conjunction with the cytosolic location of the major matrix proteins. Thus overex­ pression of PEX3 apparently results in enhanced peroxisomal membrane biosyn­ thesis, but simultaneously prevents peroxisomal matrix protein import. This indicates that these processes may be coupled. Similar effects have been observed on overexpression of HpPEX14 [49], which encodes a peroxisomal membrane protein implicated in peroxisomal matrix protein import. Most probably, the effects of PEX3 and PEX14 overexpression are not simply an artifact due to the overpro­ duction of a protein involved in peroxisome biogenesis (peroxin), because this is not generally the case for peroxins. Overproduction of HpPEXlO, which also encodes a peroxisomal membrane protein, resulted in the enhanced proliferation of peroxisomes; however, it did not result in a block in protein import [50].

Is the ER involved in peroxisome biogenesis? In yeasts, developing peroxisomes are invariably observed in close association with strands of ER. However, a distinct role for the ER in peroxisome formation is not yet substantiated. Only recently did targeting studies of Pex3p in H. polymorpba provide the first evidence for this possibility. In wild-type cells, on synthesizing a hybrid protein composed of the N-terminal 16 amino acids of Pex3p fused to a reporter protein, the majority of the reporter ended up in membranous layers that were continuous with the nuclear envelope and therefore most probably derived from the ER [48], However, when larger parts of the N-terminus of Pex3p (37 or 115 amino acids) were used, the reporter protein was predominantly located on the peroxisomal membrane. Based on these results, we speculate that H. polymorpha Pex3p is transported to peroxisomes via the ER. Apparently, the ER targeting information resides in the first 16 amino acids, whereas additional information for peroxisomal targeting is located in the next 21 amino acids. Interestingly, the putative targeting sequence QIKKR [23] is located at amino acids 53-57, indicating that HpPex3p may contain additional topogenic signals. In addition to HpPex3p, other Pmps may contain targeting information to direct the proteins to the ER/secretory pathway. For instance, overexpression of PASl (no PEX number assigned yet) in S. cerevisiae resulted in the mislocation of a portion of Pas21p at the ER [51]. Furthermore, Pas21p was found to be mislocalized to the plasma membrane when the C-terminal 55 amino acids of the protein were deleted. The most simple explanation for these phenomena is that Pex3p and Pas21ρ are first targeted to the ER and at a later stage are transported via vesicles to import-competent peroxisomes. Analogous to the formation of the vacuolar membrane or the plasma membrane, the peroxisomal membrane may thus also develop by fusion of vesicles derived from ER membranes. Such processes may also explain the significance of two peroxins belonging to the protein family of AAAtype ATPases (Pexlp and Pex6p; see Table 1) in peroxisome biogenesis. These proteins show similarity with cytosolic proteins involved in the fusion of

267

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membranes in eukaryotic cells (mammalian A/-ethylmaleimide-sensitive fusion protein and S. cerevisiae Secl8p) [52], Brcfeldin A has proved to be the tool of choice in studies on vesiclemediated transport pathways in eukaryotic cells, since it specifically inhibits the formation of coated vesicles from ER membranes [53]. In recent studies we showed that brefeldin A also interferes with peroxisomal protein sorting. Incubation of methanol-grown cells of H. polymorpba with brefeldin A resulted in the accumula­ tion of newly synthesized peroxisomal proteins at the ER (Figure 2; [53a]). It is not vet clear whether the brefeldin A-induced mistargeted peroxisomal matrix proteins were actually located in the ER lumen or bound to the cytosolic surface of the mem­ branes. So far, we have found no indications for protein modification (e.g. glycosylation), a process that is likely to occur when proteins enter the ER (Chapter 11). Figure 2

Effect of brefeldin A treatment on AO localization

I-® jr;-, "Λ v;·." ...

(A) Accumulation of AO on the ER (arrow) and nuclear membrane after incubation of methanol-grown H. polymorpha cells for I h in the presence of 20 μξ/ηπΙ brefeldin A. (B) Control cell (no brefeldin A added). AO protein is solely in the peroxisomal matrix. Abbreviations: N, nucleus; M, mitochondrion; P, peroxisome. The scale bar represents I μπ.

Hypothetical model for the role of vesicular trafficking in peroxisome biogenesis As shown in Figure 3, we can envisage a number of different pathways for the import of membrane and matrix proteins into peroxisomes which include a role for the ER and may explain the accumulation of peroxisomal membrane and matrix proteins at the ER in brefeldin Α-treated cells. The model proposes that specialized ER membranes are involved in the formation of (coated) vesicles, which are transported to pre-existing peroxisomes. Fusion of these vesicles with peroxisomes will result in growth of the organelle and incorporation of membrane components (proteins/lipids). Possibly, this process may also result in the incorporation of matrix proteins (Figure 3).

Peroxisomal protein sorting and assembly

Figure 3

269

Hypothetical model of peroxisome biogenesis in H. polymorpha

MP

AO Pex5p mRNA

iFAD ASF"

Nucleus mRNA (coated) vesicle

PMF

ER

PMB,

The development of peroxisomes in H. polymorpha is indicated on the left-hand side. Peroxisomes grow via the import of matrix proteins.This process is thought to be mediated by a functional protein complex which is donated to the newly formed bud upon fission of the organelle, resulting in a mature organelle which is metabolically active but has lost its developmental functions.These have been transferred to the new, small organelle.The synthesis and insertion/translocation of peroxisomal proteins is also presented. Membrane proteins involved in peroxisome biogenesis (PMBs;e.g. Pex3p) are synthesized in the cytosol and sorted to the ER, where they accumulate in small vesicles which are subsequently transported to the growing peroxisome. BrefeIdinA prevents the formation of these vesicles.The finding that matrix proteins (e.g. AO) also accumulate at the ER in the presence ofbrefeldinA can be explained by:fi) artificial binding to PMB complexes, which are prevented from migrating to their target organelle, or (ii) the possibility that this represents a real intermediate step in matrix protein import (route I). Possible implications of suggestion (i) are that the import of matrix proteins proceeds via already formed PMBcontoining vesicles (route II) or concurrent with docking of these vesicles by creation of a specific import site during fusion (route III). Membrane proteins involved in peroxisome function (PMFs; e.g. transporters) may be sorted directly from the cytosol to the peroxisome. Abbreviations:ASF, assembly factor; MP, matrix proteins; hsp, heat-shock protein; Pex5p, PTSI receptor.

We propose that the ER-derived vesicles contain peroxisomal membrane proteins involved in peroxisome biogenesis (PMBs). These membrane proteins tend to accumulate at the ER when vesicle formation is inhibited by brefeldin A. Likely candidates include H. polymorpha Pex3p and S. cerevisiae Pas2 Ip [48,51].

270

J .A.K.W. Kiel et al.

All possible import routes imply a direct or indirect temporal association of matrix proteins with components of the vesicle. In routes I and II (Figure 3), this inevitably results in the translocation of the matrix protein across the membrane, whereas in route III this may involve only binding to the outside of the vesicle. In any case, the model predicts that inhibition of vesicle formation by brefeldin A will result in ER accumulation of PMBs, including components with which matrix proteins associate [either during import into the ER (route I) or into the vesicle (route II), or when the matrix protein associates with the outside of the vesicle during the fusion process (route III)]. This may explain why both peroxisomal membrane and matrix proteins are mislocated at the ER in brefeldin Α-treated cells. Our current data do not allow us to predict which of the three possible pathways for matrix protein import (I, II or III) is actually used under physiological conditions. The major reason for this is that, so far, it is not known whether, in brefeldin Α-treated cells, the matrix proteins are located in the ER lumen or bound to the ER membrane at its cytosolic surface. We presume that not all peroxisomal membrane proteins are transported to peroxisomes via the ER. It can be envisaged that only those proteins involved in peroxisome biogenesis (PMBs) are sorted via the ER. Peroxisomal membrane proteins involved in microbody function (e.g. transporters) may be inserted directly from the cvtosol into the peroxisomal membrane. Likely candidates for this are Pmp47 from C. boidinn, which belongs to the family of mitochondrial solute transporters [54], the ABC-type transporter Pmp70 [55] and Pmp22 [56] from higher eukaryotes. The proposed model may also provide an elegant explanation for the surprising observation that the re-introduction of the HpPEX3 gene in a pex3 disruption strain results in peroxisome formation. As mentioned earlier, Apex3 strains do not contain any peroxisomal membrane remnants [48], In addition, it was demonstrated that peroxisomal membrane remnants may not be used as a target for matrix protein import [57], This raises the question of how the initial peroxisome is formed under complementing conditions. Our model predicts that re-introduction of the PEX3 gene in a Jpex3 strain results in the formation of vesicles from the ER that contain the major membrane proteins for peroxisome biogenesis. Subsequent binding of other proteins (peroxins) may give rise to a small pre-peroxisome, which will then grow and divide.

Conclusion One major outcome of recent studies on PEX genes is the notion that the basic principles of peroxisome biogenesis are conserved between yeast and humans. Moreover, analysis of the corresponding protein products (peroxins) has led to the view that the mechanisms of peroxisomal protein transport and delivery may not

Peroxisomal protein sorting and assembly

271

just r e p r e s e n t v a r i a t i o n s o n a well k n o w n t h e m e (e.g. e s t a b l i s h e d f o r

ER,

m i t o c h o n d r i a a n d c h l o r o p l a s t s ) , b u t have several n o v e l c h a r a c t e r i s t i c s

and

u n e x p e c t e d f e a t u r e s . A m a j o r c h a l l e n g e f o r the near f u t u r e is to u n d e r s t a n d t h e s e p r o c e s s e s at the m o l e c u l a r level. H o w e v e r , a m a j o r o b s t a c l e t o the d e t a i l e d functional analysis of presently k n o w n p e r o x i n s and those to b e identified in f u t u r e is that reliable in vitro assays for i m p o r t into yeast p e r o x i s o m e s are not yet available, d e s p i t e the e f f o r t s of several g r o u p s . T h e recent a c h i e v e m e n t s in v a r i o u s l a b o r a tories as an o u t c o m e of m o l e c u l a r s t u d i e s p r o v i d e c o m p e l l i n g a r g u m e n t s f o r the d e v e l o p m e n t of s u c h an in vitro s y s t e m . T h i s w o u l d enable dissection of the v a r i o u s s t e p s in p e r o x i s o m a l p r o t e i n s o r t i n g . S u c h s t u d i e s will also e l u c i d a t e w h e t h e r the E R is essential f o r p e r o x i s o m e b i o g e n e s i s and, related to this, give an a n s w e r t o the f u n d a m e n t a l q u e s t i o n of whether de novo p e r o x i s o m e biogenesis is possible. We thank

Richard

various parts of this

Baerends,

Florian Salomons

and Jan Zagersfor

their assistance

in

study.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

Veenhuis, M . a n d H a r d e r , W. ( 1 9 9 1 ) in T h e Y e a s t s , vol. 4 ( R o s e , A . , ed.), p p . 6 0 1 - 6 5 3 , A c a d e m i c Press van den B o s c h , H . , S c h u t g e n s , R . B . H . , W a n d e r s , R . J . H . and Tager, J . M . ( 1 9 9 2 ) A n n u . Rev. Biochem. 6 1 , 1 5 7 - 1 9 7 Distel, B., E r d m a n n , R . , G o u l d , S.J., Blobel, G . , C r a n e , D.I., C r e g g , J . M . , D o d t , G „ Fujiki, Y „ G o o d m a n , J . M . , J u s t , W.W., et al. (1996) J . Cell Biol. 135, 1-3 Veenhuis, M . and H a r d e r , W. (1989) Yeast 5, 517-524 E r d m a n n , R . , V e e n h u i s , M . , M e r t e n s , D . and K u n a u , W . - H . ( 1 9 8 9 ) P r o c . N a t l . A c a d . Sci. U . S . A . 86, 5 4 1 9 - 5 4 2 3 C r e g g , J . M . , Veenhuis, M „ van der Klei, I.J., Suiter, G.J. and Harder, W. (1990) Yeast 6 , 8 7 - 9 8 van der Klei, I.J., Harder, W. and Veenhuis, M . (1991) Arch. Microbiol. 156, 15-23 van der Klei, I.J., Harder, W. and Veenhuis, M . (1991) Yeast 7 , 1 9 5 - 2 0 9 Suiter, G.J., van der Klei, I.J., Harder, W. and Veenhuis, M . (1990) Yeast 6 , 5 0 1 - 5 0 9 Verduyn, C . , van Wijngaarden, C.J., Scheffers, W.A. and van Dijken, J.P. (1991) Yeast 7, 3 7 - J 6 van der K l e i , I.J., R y t k a , J . , K u n a u , W . H . and V e e n h u i s , M . ( 1 9 9 0 ) A r c h . M i c r o b i o l . 153, 313-517 V e e n h u i s , M „ v a n d e r Klei, I.J., Suiter, G . W . a n d H a r d e r , W. ( 1 9 8 9 ) A r c h . M i c r o b i o l . 151, 105-110 Waterham, H . R . , K e i z e r - G u n n i n k , L, G o o d m a n , J . M . , H a r d e r , W. and Veenhuis, M . (1992) J . Bacteriol. 1 7 4 , 4 0 5 7 - ^ 0 6 3 van der Klei, I.J. and Veenhuis, M . (1996) Ann. N . Y . Acad. Sci., 804, 47-59 D e D u v e , C . and B a u d h u i n , P. (1966) Physiol. Rev. 4 6 , 3 2 3 - 3 5 7 L a z a r o w , P.B. and Fujiki, Y. (1985) Annu. Rev. Cell Biol. 1 , 4 8 9 - 5 3 0 Subramani, S. (1993) A n n u . Rev. Cell Biol. 9, 4 4 5 - 1 7 8 de H o o p , M . J . and A b , G . (1992) Biochem. J. 286, 6 5 7 - 6 6 9 Swinkels, B.W., G o u l d , S.J., Bodnar, A . G . , Rachubinski, R . A . and Subramani, S. (1991) E M B O J. 1 0 , 3 2 5 5 - 3 2 6 2 Nuttlev, W.M., Brade, A . M . , F.itzen, G . A . , Veenhuis, M., Aitchison, J . D . , Szilard, R . K . , Glover, J . R . and Rachubinski, R . A . (1994) J. Biol. C h e m . 269, 556-566

21.

Kragler, F., Langeder, A., R a u p a c h o v a , J . A . , Binder, M . and Hartig, A. (1993) J. Cell Biol. 120,

22. 23.

665-673 Purdue, P.E. and L a z a r o w , P.B. (1994) J. Biol. C h e m . 269, 30065-30068 D y e r , J . M . , M c N e w , J . A . and G o o d m a n , J . M . (1996) J . Cell Biol. 1 3 3 , 2 6 9 - 2 8 0

272

J.A.K.W. Kiel e t a l .

24.

G o u l d , S.J., Keller, G . A . , Schneider, M . , H o w e l l , S . H . , G a r r a r d , L.J., G o o d m a n , J . M . , Distel, B., Tabak, H . and Subramani, S. (1990) E M B O J . 9, 8 5 - 9 0

25.

Terlecky, S.R., N u t t l e y , W.M., M c C o l l u m , D . , S o c k , E . and S u b r a m a n i , S. (1995) E M B O J . 14, 3627-3634

26.

van der Leij, I., F r a n s e , M . M . , E l g e r s m a , Y., Distel, B. and T a b a k . H . (1993) P r o c . N a t l . Acad. Sci. U . S . A . 9 0 , 1 1 7 8 2 - 1 1 7 8 6 van der Klei, I.J., H i l b r a n d s , R . E . , S w a v i n g , G . J . , W a t e r h a m , H . R . , Vrieling, E . G . , T i t o r e n k o , V.I., C r e g g , J . M . , Harder, W. and Veenhuis, M . (1995) J . Biol. C h e m . 270, 17229-17236

27. 28. 29.

D o d t , G . , Braverman, N . , W o n g , C , Moser, A., Moser, H.W., Watkins, P., Valle, D . and G o u l d , S.J. (1995) N a t u r e Genet. 9, 115-125 Wiemer, E . A . , Nuttley, W . M , Bertolaet, B . L . , Li, X . , Francke, U . , Wheelock, M . J , A n n e , U . K . , J o h n s o n , K . R . and Subramani, S. (1995) J. Cell Biol. 1 3 0 , 5 1 - 6 5

30.

S z i l a r d , R . K , T i t o r e n k o , V.I., V e e n h u i s , M . and R a c h u b i n s k i , R . A . ( 1 9 9 6 ) J. C e l l B i o l . 131 , 1453-1469

31.

F r a n s e n , M . , B r e e s , C , B a u m g a r t , E., V a n h o o r e n , J . C . T . , B a e s , M . , M a n n a e r t s , G.P. a n d Van Veldhoven, P.P. (1995) J. Biol. C h e m . 270, 7 7 3 1 - 7 7 3 6

32. 33. 34.

M a r z i o c h , M , E r d m a n n , R , Veenhuis, M . and K u n a u , W.-H. (1994) E M B O J. 1 3 , 4 9 0 8 - 4 9 1 8 Zhang, J.W. and L a z a r o w , P.B. (1995) J. Cell Biol. J 2 9 , 6 5 - 8 0 Rachubinski, R . A . and Subramani, S. (1995) Cell 83, 5 2 5 - 5 2 8

35.

R e h l i n g , P., M a r z i o c h , M . , N i e s e n , F., W i t t k e , E „ V e e n h u i s , M . and K u n a u , W . - H . (1996) E M B O J. 1 5 , 2 9 0 1 - 2 9 1 3 Zhang, J.W. and I.azarow, P.B. (1996) J. Cell Biol. 1 3 2 , 3 2 5 - 3 3 4 G l o v e r , J . R „ A n d r e w s , D.W. and R a c h u b i n s k i , R . A . ( 1 9 9 4 ) P r o c . N a t l . A c a d . Sci. U . S . A . 91, 10541-10545

36. 37. 38. 39 40. 41.

M c N e w . J . A . and G o o d m a n , J . M . (1994) J. Cell Biol. 127, 1245-1257 Walton, P . A , Hill, P.E. and Subramani, S. (1995) M o l . Biol. Cell 6 , 6 7 5 - 6 8 3 Hausier, T , Stierhof, Y . - D , Wirtz, E. and C l a y t o n , C . (1996) J. Cell Biol. 1 3 2 , 3 1 1 - 3 2 4 V e e n h u i s , M . , K o m o r i , M . , S a l o m o n s , F., H i l b r a n d s , R . E . , H u t , H . , B a e r e n d s , R . J . S . , Kiel, J . A . K . W . and van der Klei, I.J. (1996) F E B S Lett. 3 8 3 , 1 1 4 - 1 1 8

42

G o o d m a n , J . M . , S c o t t , C.W., D o n a h u e , P . N . and A t h e r t o n , J.P. (1984) J . Biol. C h e m . 259, 8485-8493 R o a , M . and Blobel, G . (1983) Proc. N a t l . A c a d . Sci. U . S . A . 80, 6 8 7 2 - 6 8 7 6 Distel, B , Veenhuis, M . and Tabak, H.F. (1987) E M B O J. 6, 3 1 1 1 - 3 1 1 6

43. 44. 45.

van der K l e i , I.J., Veenhuis, M . , van der Ley, I. and H a r d e r , W. (1989) F E M S M i c r o b i o l . Lett. 57,133-138

46. 47.

Evers, M . E , Harder, W. and Veenhuis, M . (1995) F E B S Lett. 3 6 8 , 2 9 3 - 2 9 6 Evers, M . E , T i t o r e n k o , V . I , van der Klei, I . J , H a r d e r , W. and Veenhuis, M . (1994) M o l . Biol. Cell 5, 8 2 9 - 8 3 7

48.

B a e r e n d s , R . J . S , R a s m u s s e n , S . W , H i l b r a n d s , R . E , van der H e i d e , M , Faber, K . N , R e u v e k a m p , P . T . W , Kiel, J . A . K . W , C r e g g , J . M , van der K l e i , I.J. and Veenhuis, M . (1996) J. Biol. C h e m . 271, 8887-8894

48a.

B a e r e n d s , R . J . S , S a l o m o n s , F . A , Faber, K . N , Keil, J . A . K . W , van der K l e i , I.J. and Veenhuis, M . (1997) Yeast 13, 1437-1448 K o m o r i , M , R a s m u s s e n , S . W , Kiel, J . A . K . W , B a e r e n d s , R . J . S , C r e g g , J . M , van der Klei, I.J. and Veenhuis, M . (1997) E M B O J. 1 6 , 4 4 - 5 3

49. 50. 51. 52. 53.

Tan, X , Waterham, H . R . , Veenhuis, M . and C r e g g , J . M . (1995) J. Cell Biol. 1 2 8 , 3 0 7 - 3 1 9 Elgersma, Y. (1995) Ph.D. Thesis, University of A m s t e r d a m R o t h m a n , J . E . (1996) Protein Sci. 5, 185-194

K l a u s n e r , R . D , D o n a l d s o n , J . G . and L i p p i n c o t t - S c h w a r t z , J. (1992) J . C e l l B i o l . 116, 1071-1080 53a. S a l o m o n s , F . A , van der Klei, I . J , K r a m , A . M . , Harder, W. and Veenhuis, M . (1997) F E B S Lett. 411,133-139 54. J a n k , B „ H a b e r m a n n , B , Sweyen, R.J. and Link, T . A . (1993) Trends Biochem. Sci. 1 8 , 4 2 7 - 4 2 8 55. K a m i j o , K , Taketani, S , Y o k o t a , S , O s u m i , T. and H a s h i m o t o , T. (1990) J. Biol. C h e m . 265, 4534-4540 56. Diestelkotter, P. and J u s t , W.W. (1993) J. Cell Biol. 123, 1717-1725 57. Waterham, H . R , T i t o r e n k o , V . I , Swaving, G . J , Harder, W. and Veenhuis, M . (1993) E M B O J. 12,4785—1794

16 Targeting of glyoxysomal proteins A. Baker* and B.Tugal Centre for Plant Biochemistry and Biotechnology, University of Leeds, Leeds LS2 9JT, U.K.

Introduction 'Peroxisomes' or 'microbodies' is a general term used to describe a class of organelles that are bounded by a single membrane bilayer, possess flavin-containing oxidases which generate hydrogen peroxide, and also contain catalase (EC 1.11.1.6), which degrades generated hydrogen peroxide ([1]; Chapter 15). Glyoxysomcs arc a specialized type of peroxisome that are defined by the presence of the glyoxylate pathway enzymes in the lumen [2], In addition, glyoxysomes (and other types of peroxisomes) contain enzymes which break down fatty acids to acetyl-CoA (βoxidation). Glyoxysomes are present in the cotyledons or endosperm tissue of oilstoring plant species, and in some fungi and algae when grown solely on fatty acids or acetate. In germinating seeds, and in fungi grown under limited nutritional conditions, the β-oxidation and glyoxylate pathways tie in to convert C, units into C 4 precursors for gluconeogenesis. Glyoxysomes are inducible organelles, the presence of which depends on the physiological requirements of the organism [3,4]; therefore the exact protein composition of glyoxysomes is dynamic, i.e. it changes during growth and development. Cells generally contain only one type of microbody at any given time. The one known exception to this is the case of Neurospora crassa, where the two unique enzymes of the glyoxylate pathway, namely isocitrate lyase (EC 4.1.3.1) and malate synthase (EC 4.1.3.2), induced during growth on acetate, are sequestered in an organelle which is distinct from that containing catalase and urate oxidase [5,6]. Currently our knowledge of peroxisomal/glyoxysomal biogenesis is not sufficient to explain this apparent anomaly. Mammals lack isocitrate lyase and malate synthase, and so cannot carry out gluconeogenesis from acetyl-CoA and hence do not possess glyoxysomes. However, mammalian peroxisomal β-oxidation plays a vital role in the metabolism of very-long-chain fatty acids, which cannot be handled by the mitochondrial βoxidation system. Morphologically, glyoxysomes are pleiomorphic organelles with a diameter of about 0.5-1.0 μιη, although, as with their composition, this may change during development. In plant cells they are often found juxtaposed to lipid bodies ••To whom correspondence should be addressed.

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and mitochondria. The basis of this phenomenon is not clear, but may be related to their biochemistry, which integrates pathways in these organelles; their close proximity may therefore facilitate the exchange of metabolites between them. The glyoxysomal matrix (and that of other types of peroxisome) is packed with proteins and appears in micrographs as an electron-dense mass. The dense nature of the matrix is assumed to be a result of the strong association between lumenal enzymes, leading to increased biochemical efficiency due to metabolite channelling, as proposed by Heupel et al. [7,8] for leaf peroxisomes, and perhaps also aiding the confinement of the toxic compounds that are produced and degraded in glyoxysomes and other types of peroxisome. In sucrose gradients, glyoxysomes sediment at a density of 1.23-1.26 g-cnr 3 , aiding their isolation for investigation [2], Much of the initial attention given to glyoxysomes and peroxisomes has focused on their morphology, composition and biochemistry. Such work has highlighted the plasticity of this organelle with respect to its function, stimulating interest in how this is achieved. It is now acknowledged that such functional plasticity is a direct result of the ability of this organelle to take up microbody-specific nuclear-coded proteins, as it does not contain any genetic information. This Chapterwill review the enzymic functions found in glyoxysomes and the information available concerning their targeting and import.

Metabolic functions of glyoxysomes Biochemical reactions within glyoxysomes are of central importance to the organism. In fungi, peroxisomes (glyoxysomes) are the sole cellular site of βoxidation and are therefore essential for growth on fatty acids as the sole carbon source. This has been exploited in genetic screens for mutants impaired in both βoxidation activity and peroxisome assembly (Chapter 15). In plants, glyoxysomes are crucial during seed germination, mobilizing stored carbon for growth by providing acetyl-CoA derived from the β-oxidation of stored fatty acids for gluconeogenesis. Most importantly, glyoxysomes partition the site of acetyl-CoA synthesis away from the mitochondrial tricarboxylic acid (TCA) cycle, such that, in plants like the castor bean, up to 75% of fatty-acid carbon can be diverted to sucrose production [9]. This diversion of carbon from fatty acids to sucrose is achieved by the integration of acetyl-CoA into the glyoxylate cycle with oxaloacetate to form citrate (Figure 1). Citrate is initially converted into isocitrate, then, through the activity of isocitrate lyase (an enzyme unique to glyoxysomes), into succinate and glyoxylate. Glyoxylate then reacts with fatty-acid-derived acetyl-CoA to form malate, which is eventually converted into sucrose in the cytoplasm via the gluconeogenic pathway. Succinate, the other product of the reaction catalysed by isocitrate lyase, is shuttled to mitochondria, where it feeds into the TCA cycle, ultimately re-forming oxaloacetate. Oxaloacetate is shuttled

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Figure 1

Schematic diagram of the glyoxylate cycle in plants

Fatty acids

3 Glulamate 3 2-Oxo glutarate

3 2-0x malate rather than malate —> oxaloacetate as required by a cyclic version of the glyoxylate pathway [10,37]. The equilibrium constant of the reaction favours the reaction in the oxaloacetate —» malate direction, especially in the presence of NADH 2 [57]. Glyoxysomal malate dehydrogenase from water melon has been studied extensively. Unusually for a peroxisomal enzyme, it is made as a higher-molecular-

Targetingofglyoxysomal proteins

mass precursor which is processed after import [58]. The N-terminal extension contains a PTS2 consensus sequence; mutagenesis and expression in the heterologous host Hansenula polymorpha has shown this to be a functional PTS2 [59], Interestingly, S. cerevisiae peroxisomal (glyoxysomal) malate dehydrogenase, the product of the MDH3 gene [60], is targeted by a PTSl SKL sequence [61]. To date, it appears that plants seem to make greater use of PTS2 signals than do fungi. The only known PTS2-targeted protein in S. cerevisiae is acetoacetylCoA thiolase. In Hansenula polymorpha, amine oxidase is also a PTS2 protein [62] and Perlp has both a PTSl and a PTS2 [63], However, these Hansenula peroxisomes are induced in response to growth on methanol and or amines, and so are not glyoxysomes as defined for the purposes of this review.

Other enzymes found in glyoxysomes Catalase Catalase is a constituent of all types of peroxisomes, not just glyoxysomes. Human catalase is targeted by the non-standard PTSl sequence ANL, but this requires the lysine residue which normally precedes the alanine in the third position in order to be functional [64]. Similarly, cotton seed catalase uses the non-standard C-terminal tripeptide PSI as a peroxisome targeting signal, but requires in addition the arginine residue normally preceding the proline in order to target the passenger protein CAT to tobacco peroxisomes. The nature of other residues immediately upstream of the PTSl also affects its function, indicating that the context of the PTS, as well as its specific sequence, is important [65]. Catalase from H. polymorpha is targeted to peroxisomes by the Cterminal sequence SKI [66]. S. cerevisiae catalase A has two independent targeting signals. The C-terminal hexapeptide -SSNSKF is sufficient for targeting a passenger protein to peroxisomes, and therefore constitutes yet another permutation of the PTSl sequence. However, this sequence can be deleted without affecting peroxisome protein import. Gene fusion experiments suggested a second targeting signal within the N-terminal third of the protein [67].

The glyoxysomal membrane Membrane proteins The uptake of glyoxysomal proteins from the cytosol must at some stage involve interaction(s) with as yet unidentified glyoxysomal membrane proteins; clearly, investigations into the identity of these components require the characterization of the glyoxysomal membrane. Yet the glyoxysomal membrane is little understood, owing to its fragility in most glyoxysome preparations and its low abundance. As pointed out earlier, electron micrographs show a tight association of glyoxysomes with other organelles; although the exact biological reason for this is currently unknown, such association in vivo may have consequences when glyoxysomes are

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isolated for studies in vitro, as preparations of these membranes are often contam­ inated with other membrane fractions. Efforts to isolate plant glyoxysome membranes by both differential and density-gradient centrifugation have also been hampered by the co-fractionation of the glyoxysomal core, the tendency of the membranes to aggregate and the lack of good markers for the membrane [68-70]. Further problems have also been observed when solubilizing glyoxysomal membrane proteins with non-ionic detergents [71,72]. Owing to these limitations, there is no clear indication to date of the exact composition of plant glyoxysomal membranes. Despite these difficulties, attempts have been made to purify glyoxysomal membranes from different plant tissues and to analyse their polypeptide composition (e.g. cotton [72], sunflower [73], pumpkin [74], various oil seeds [75,76], potato tuber peroxisomes [77] and pea leaf peroxisomes [78]). The most conclusive results from these studies have been the recent identification of a 31 kDa membrane-associated protein, ascorbate peroxidase [79,80], and of an Arabidopsis tbaliana orthologue of the mammalian 22 kDa peroxisomal membrane protein Pmp22 (B. Tugal, L.M. Sandalio, M.R. Pool, L.A. del Rio and A. Baker, unpublished work). Yeasts have proved more tractable to both genetic and biochemical analysis of microbody membranes. Several peroxisome assembly mutants are affected in genes encoding putative membrane proteins, some of which are candidates for components of the protein import machinery. Purification of membranes coupled with the microsequencing of abundant proteins has led to the cloning of a 27 kDa oleic acid-induced protein (Pmp27) from S. cerevisiae. Although it is a hydrophobic protein, Pmp27 has been shown to be peripherally associated with the membrane, but its orientation is currently unknown [81,82]. Another oleic acid-induced membrane protein is Candida boidinii Pmp47. This is a true integral membrane protein, as defined by its Triton X-114 phase separation and Na 2 CO 3 inextractability, and is a distant member of the mitochondrial solute carrier protein family, which have a six-transmembrane-spanning structure [83,84]. Pmp47, which comprises 423 amino acids, has both SKL at positions 320-322 and a C-terminal AKE; however, when these sequences are deleted, Pmp47 still sorts correctly and efficiently into microbodies [84], By constructing Pmp47-(dihydrofolate reductase) fusion proteins, McCammon et al. [84] investigated the targeting of Pmp47 in S. cerevisiae, and concluded that amino acids 199-267 (transmembrane regions 4-5 inclusive) are important for correct sorting. By constructing further Pmp47 internal deletions and by the use of fusion proteins incorporating sections of this region, Dyer et al. [85] were able to locate a cluster of basic amino acids important in the targeting of Pmp47. The amino acids present in this loop region were also found to be present in a number of other peroxisomal membrane proteins from yeast and humans. This is the first membrane-specific microbody targeting sequence to be identified.

Targeting of glyoxysomal proteins

Import receptors Receptors that recognize the PTSl and PTS2 targeting signals have been identified but are not, in the main, membrane associated. Current knowledge is consistent with a shuttling model whereby the PTSl receptor cycles between cytosol and membrane (or even the matrix). Receptors have not been isolated from higher plants, but one study investigated the peroxisomal targeting sequence binding 'activity' of glyoxysomal membranes by using an 1 2 5 I-Iabelled 12-amino-acid peptide terminating in the sequence SKL. The binding of this peptide to carbonatewashed glyoxysomal membranes was saturable, and was diminished by protease treatment of the membranes. By Scatchard analysis, two classes of binding site were identifiable with K i values of 160 nM and 1450 nM [86]. Postively charged residues adjacent to the SKL were required for binding to the high-affinity site [87], which is reminiscent of the results obtained with the non-standard catalase PTS [64,65]. It is not known if either of these binding sites corresponds to the plant PTSl receptor or if other membrane proteins, perhaps involved in later stages of import, also recognize the PTSl sequence.

Concluding remarks What conclusions can be drawn about signals involved in targeting proteins to glyoxysomes? The PTSl and PTS2 signals are used by plants, animals and yeasts to target proteins to peroxisomes in general and specifically to glyoxysomes, suggesting that the import machinery of the two types of organelle is similar if not identical. In higher-plant cotyledons, glyoxysomes undergo a developmentally programmed transition from glvoxysome to peroxisome during transition from heterotrophy to autotrophy, and in the reverse direction during senescence. When the glyoxysomal enzyme isocitrate lyase was expressed in green tissue (where only peroxisomes are present) it was correctly targeted, again pointing to a conservation of targeting mechanisms. Finally, numerous studies in which foreign proteins have been expressed and correctly targeted to peroxisomes in heterologous hosts point to a common mechanism for targeting and import between peroxisome-type organelles and across species. In contrast with this overall conservation of function, at the level of individual enzymes and species there are many variations. The same enzyme from different species can be targeted by completely different mechanisms; for example, malate dehydrogenase from S. cerevisiae uses a PTSl import mechanism, whereas that from plants uses a PTS2. A similar situation pertains with citrate synthase. There are also species-dcpcndent variations in what constitutes a functional PTSl. For example, GKI and AQI functioned as targeting signals for C. tropicalis tnfunctional enzyme when expressed in C. albicans, but not in S. cerevisiae. This may reflect differences in the specificities of the respective PTS1 receptors. It is also clear that there are 'context effects'; a non-standard PTS can be compensated for by

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adjacent residues [61,64,65,88]. However, the recent finding that peroxisomes can import oligomeric structures requires careful controls to be carried out. When a mutant protein is co-expressed with its wild-type counterpart (or with the endogenous protein of a closely related species), they may be able to assemble and be imported using the wild-type signal. This could lead to the erroneous scoring of some mutants as import-competent. The same caveat applies to studies in which sequences were found to be sufficient but not necessary for import. Nevertheless it is becoming clear that some peroxisomal proteins do have more than one targeting signal. In some cases, exemplified by carnitine acetyltransferase, there is redundant information within the protein specifying the same import pathway. In other cases, such as Perlp, there are signals for two different import pathways. Why this should be so is uncertain, although in Hansenula the PTS2 pathway appears to be inducible by nutritional conditions [89], so this may provide an additional method of modulating the uptake of particular proteins in response to environmental conditions. It is also likely that at least one additional import pathway exists for matrix proteins, as acyl-CoA oxidase from S. cerevisiae is still imported into peroxisomes in mutants with defects in either the PTSl or PTS2 import pathways, although the double mutant was not tested [24]. As there is at least one pathway for membrane protein insertion, this means that there are a minimum of four import pathways for peroxisomes, although the three pathways for matrix protein import may converge at a common membrane translocation step. This aspect of peroxisome protein import remains a black box. We acknowledge the Biotechnology and Biology Research Council and the Leverhulme Trust for finandal support.

References 1. 2.

de Duve, C. and Baudhuin, P. (1966) Physiol. Rev. 46, 323-357 Huang, A.C., Trelease, R.N. and Moore, T.S. (eds.) (1983) Plant Peroxisomes, Academic Press,

3.

New York Tolbert, N.E. (1981) Annu. Rev. Biochem. 50, 133-157

4. 5. 6. 7. 8. 9.

van den Bosch, H., Schutgens, R.B.H., Wanders, R.J.A. and Tager, J.M. (1992) Annu. Rev. Biochem. 61,157-197 Wanner, G. and Theimer, R.R. (1982) Ann. N.Y. Acad. Sci. 286,269-284

10. 11. 12.

Kionka 1 C. and Kunau, W.-H. (1982) J. Bacteriol. 161,153-157 Heupel, R., Markgraf, T., Robinson, D. and Heldt, H.W. (1991) Plant Physiol. 96, 971-979 Heupel, R. and Heldt, H.W. (1994) Eur. J. Biochem. 220,165-172 Baker, A., Halpin, C. and Lord, J.M. (1997) Plant Metabolism, 2nd edn. (Denis, D.T., Turnpin, D.H., Letebvre, D.D. and Layzell, D.B.), pp. 403^13, Longman, Harlow Mettler, I.J. and Beevers, H. (1980) Plant Physiol. 66, 555-560 Titus, D.E. and Becker, W.M.(1985)J. Cell Biol. 101,1288-1299 Gut, H. and MatiIe 1 P. (1988) Planta 176,548-550

13. 14.

De Bellis, L. and Nishimura, M. (1991) Plant Cell Physiol. 32, 555-561 Nishimura, M.,Takeguchi, Y., De Bellis, L. and Hara-Nishimura, I. (1993) Protoplasma 175,

15. 16.

131-137 Heinemann, P. and Just, W.W. (1992) FEBS Lett. 300, 179-182 Subramani, S (1993) Annu. Rev. Cell Biol. 9,445^78

Targeting of glyoxysomal proteins

17.

G o u l d , S.J., Keller, G . A . , H o s k e n , N „ Wilkinson, J. and S u b r a m a n i , S. (1989) J . Cell Biol. 108, 1657-1664

18.

Keller, G . - A . , K r i s a n s , S „ G o u l d , S.J., S o m m e r , J . M . , Wang, C . C . , S c h l i e b s , W., K u n a u , W„ B r o d y , S. and Subramani, S. (1991) J . Cell Biol. 114, 893-904 d e Z o y s a , P. and C o n n e r t o n , I.F. (1994) Curr. Genet. 2 6 , 4 3 0 - 4 3 7 M i y a z a w a , S., O s u m i , T „ H a s h i m o t o , T „ O h n o , K., M i u r a , S. and Fujiki, Y. (1989) M o l . Cell. Biol. 9 , 8 3 - 9 1

19. 20. 21. 22. 23. 24.

M i u r a , S., K a s u v a - A r a i , I., M o r i , H . , M i y a z a w a , S., O s u m i , T., H a s h i m o t o , T. and F u j i k i , Y. (1992) J. Biol. C h e m . 267, 14405-14411 O k a z a k i , K . , T a k e c h i , T., K a m b a r a , N . , F u k u i , S., K u b o t a , I. a n d K a m i r v o , T. ( 1 9 8 6 ) P r o c . N a t l . A c a d . Sci. U . S . A . 83, 1232-1236 Small, G . M . , S z a b o , L.J. and L a z a r o w , P.B. (1988) E M B O J. 7, 1167-1173 Zhang,J.W., Luckey, C . and L a z a r o w , P.B. (1993) M o l . Biol. Cell 4, 1351-1359

25. 26. 27.

H o o k s , M . A . , B o d e , K . and L o u e e , I. (1997) B i o c h e m . J . 3 2 0 , 6 0 7 - 6 1 4 Aitchison, J . D . , Murray, W.W. and Rachubinski, R . A . ( 1 9 9 1 ) J . B i o l . C h e m . 266, 2 3 1 9 7 - 2 3 2 0 3 H i l t u n e n , J . K . , Wenzel, B., Beyer, A., F o s s a , A. and K u a n u , W . - H . ( 1 9 9 2 ) J . Biol. C h e m . 267, 6646-6653

28.

A r a k a w a , H . , Takiguchi, M „ A m a y a , Y., N a g a t a , S., H a y a s h i , H . and Mori, M . (1987) E M B O J . 6,1361-1366 Hijikata, M „ Wen, J . K . , O s u m i , T. and H a s h i m o t o , T. (1990) J. Biol. C h e m . 265, 4 6 0 0 - t 6 0 6 Swinkels, B.W., G o u l d , S.J., Bodnar, A . G . , Rachubinski, R . A . and Subramani, S. (1991) E M B O J. 10, 3 2 5 5 - 3 2 6 2

29. 30. 31. 32. 33. 34.

Preisig-Mueller, R. and Kindl, H . (1993) Plant M o l . Biol. 22, 5 9 - 6 6 Glover, J . R . , A n d r e w s , D.W., S u b r a m a n i , S. and R a c h u b i n s k i , R . A . (1994) J . Biol. C h e m . 269, 7558-7563 T s u k a m o t o , T., H a t a , S., Y o k o t a , S., M i u r a , S., F u j i k i , Y., H i j k a t a , M . , M i y a z a w a , S., H a s h i m o t o , T. and O s n n , T. (1994) J. Biol. C h e m . 2 6 9 , 6 0 0 1 - 6 0 1 0 G l o v e r , J . R , A n d r e w s , D.W. a n d R a c h u b i n s k i , R . A . ( 1 9 9 4 ) P r o c . N a t l . A c a d . Sci. U . S . A . 91, 10541-10545

35. 36. 37.

M c N e w , J . A . and G o o d m a n , J . M . (1994) J. Cell Biol. 127, 1245-1257 Walton, P.A., Hill, P.E. and Subramani, S. (1995) M o l . Biol. Cell 6 , 6 7 5 - 6 8 3 van R o e r m u n d , C . W . T . , E l g e r s m a . Y., Singh, N . , W a n d e r s , R . J . A . and T a b a k , H . F . ( 1 9 9 5 ) E M B O J. 14,3480-3486

38.

E l g e r s m a , Y., van R o e r m u n d , C.W.T., W a n d e r s , R.J. A . and T a b a k , H . F . ( 1 9 9 5 ) E M B O J . 14, 3472-3479 R o s e n k r a n t z , M . , A l a m , T., K i m , K.S., C l a r k , B.J., Srere, P.A. and G u a r e n t e , L.P. ( 1 9 8 6 ) M o l . Cell. Biol. 9, 4509—1515 Lewin, A.S., H i n e s , V. and Small, G . M . (1990) M o l . Cell. Biol. 1 0 , 1 3 9 9 - 1 4 0 5 Singh, K.V., Small, G . M . and L e w i n , A . S . (1992) M o l . Cell. Biol. 12, 5 5 9 3 - 5 5 9 9 K a t o , A., H a y a s h i , M „ Mori, H . and N i s h i m u r a , M . (1995) Plant M o l . Biol. 27, 3 7 7 - 3 9 0 K a t o , A., H a y a s h i , M . , K o n d o , M . and N i s h i m u r a , M . (1996) Plant Cell 8, 1601-1611 U n g e r , E . A . , H a n d , J . M . , C a s h m o r e , A . R . and V a s c o n c e l o s , A . C . (1989) Plant M o l . Biol. 13, 411—418

39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51.

Courtois-Verniquet, F. and D o u c e , R. (1993) Biochem. J. 294, 103-107 O l s e n , L . J . , Ettinger, W.F., D a m s z , B „ M a t s u d a i r a , K „ Webb, A . and H a r a d a , J . J . (1993) Plant Cell 5 , 9 4 1 - 9 5 2 O n y e o c h a , I., Behari, R., Hill, D . and Baker, A. (1993) Plant Mol. Biol. 22, 385^396 L e e , M.S., Mullen, R.T. and Trelease, R . N . (1997) Plant Cell 9, 185-197 Behari, R. and Baker, A . (1993) J . Biol. C h e m . 2 6 8 , 7 3 1 5 - 7 3 2 2 G a o , X . , M a r r i s o n , J . L . , P o o l , M . R . , L e e c h , R . M . and Baker, A . ( 1 9 9 6 ) Plant P h y s i o l . 112, 1457-1464 Gainey, L . D . S . , C o n n e r t o n , I.F., Lewis, E . H . , Turner, G . and Ballance, D . J . (1992) Curr. Genet. 21, 4 3 - 4 7

52.

M c C a m m o n , M . T . , Veenhuis, M . , T r a p p , S . B . and G o o d m a n , J . M . ( 1 9 9 0 ) J . Bactcriol. 172,

53.

5816-5827 Taylor, K . M . , Kaplan, C.P., G a o , X . and Baker, A. (1996) Biochem. J. 319, 255-262

285

286

A. Baker and B.Tugal

54.

Kamada, Y , Ueda, M , Atomi, H , O d a , K , Kurihara, T , Naito, N , Ukita, R , Kamasawa, N , O s u m i , M . and Tanaka, A. ( 1 9 9 2 ) J . Ferment. Bioeng. 74 , 368-371

55.

H a y a s h i , M , A o k i , M , K a t o , A . and N i s h i m u r a , M . (1996) Plant J . 10, 2 2 5 - 2 3 4

56.

H a r t i g , A , S i m o n , M „ S c h u s t e r , T „ D a u g h e r t y , J . R . , Y o o , H . S . and C o o p e r , T . G . (1992) N u c l e i c Acids Res. 20, 5677-5686 Gietl, C . (1992) Biochim. B i o p h y s . Acta 1 1 0 0 , 2 1 7 - 2 3 4

57. 58. 59.

Gietl, C . (1990) Proc. N a t l . A c a d . Sci. U . S . A . 87, 5 7 7 3 - 5 7 7 7 Gietl, C , Faber, K . N , van der Klei, I.J. and Veenhuis, M . (1994) Proc. N a t l . A c a d . Sci. U . S . A . 91,3151-3155

60. 61.

Steffen, J.S. and McAllister-Henn, L. (1992) J . Biol. C h e m . 2 6 7 , 2 4 7 0 8 - 2 4 7 1 5 E l g e r s m a , Y , Vos, A , Van den B e r g , M , Van R o e r m u n d , C . W . T ; Van der Sluijs, P , Distel, B. and T a b a k , H . F . (1996) J. Biol. C h e m . 2 7 1 , 2 6 3 7 5 - 2 6 3 8 2

62.

Faber, K . N , K e i z e r - G u n n i c k , I , Pluim, D „ Harder, W „ A b , G . and Veenhuis, M . (1995) F E B S Lett. 357, 115-120

63.

Waterham, H . R . , T i t o r e n k o , V . I , H a i m a , P„ C r e g g , J . M , Harder, W. and Veenhuis, M . (1994) J. Cell Biol. 127, 7 3 7 - 7 4 9

64. 65. 66. 67.

Purdue, P E . and L a z a r o w , P.B. (1996) J. Cell Biol. 134, 8 4 9 - 8 6 2 Mullen, R.T., Lee, M.S. and Trelease, R . N . (1997) Plant J. 12, 3 1 3 - 3 2 2 D i d i o n , T. and R o g g c n k a m p , R. (1992) F E B S Lett. 303, 113-116 Kragler, F , L a n g e d e r , A , R a u p a c h o v a , J , B i n d e r , M . and H a r t i g , A . (1993) J . Cell Biol. 120, 665-673

68. 69. 70. 71.

B r o w n , R . H . , L o r d , J . M . and Merret, M.J. (1974) B i o c h e m . J . 144 , 559-566 Fang, T.K., D o n a l d s o n , R.P. and Vigil, E . L . (1987) Planta 1 7 2 , 1 - 1 3 C h a p m a n , K . D „ T u r l e y , R . B . and Trelease, R . N . (1989) Plant Physiol. 89, 3 5 2 - 3 5 9 L u s t e r , D . G , B o w d i t c h , M . I , E l d r i d g e , K . M . and D o n a l d s o n , R.P. ( 1 9 9 2 ) A r c h . B i o c h e m . B i o p h y s . 265 , 50-61

72.

C h a p m a n , K . D . and Trelease, R . N . (1992) Plant Physiol. B i o c h e m . 30, 1 - 1 0

73.

Jiang, L . W , B u n k e l m a n n , J . R , Towill, L „ Kleff, S. and Trelease, R . N . (1994) Plant Physiol. 106, 293-302 Yamaguchi, K „ Mori, H . and N i s h i m u r a , M . (1995) Plant Cell Physiol. 3 6 , 4 5 5 - 4 6 4

74. 75. 76. 77.

C o r p a s , F . J , Bunkelmann , J . and Trelease, R . N . (1994) Eur. J . Cell Biol. 65, 2 8 0 - 2 9 0 Bunkelmann, J . R , C o r p a s , F.J. and Trelease, R . N . (1995) Plant Sci. 1 0 6 , 2 1 5 - 2 2 6 S t r u g l i c s , A , F r e d l u n d , K . M , R a s m u s s o n , A . G . and M o l l e r , I . M . ( 1 9 9 3 ) P h v s i o l . Plant 88, 19-28

78.

L o p e z - H u e r t a s , E , S a n d a l i o , L . M . and del R i o , L . A . ( 1 9 9 5 ) Plant P h y s i o l . B i o c h e m . 33, 295-302 Yamaguchi, K , Mori, H . and N i s h i m u r a , M. (1995) Plant Cell Physiol. 36, 1 157-1162

79. 80. 81. 82.

Bunkelmann, J . R . and Trelease, R . N . (1996) Plant Physiol. 110, 589-598 E r d m a n n , R. and Blobel, G . (1995) J. Cell Biol. 128 , 5 0 9 - 5 2 3 M a r s h a l l , P . A , K r i m k e v i c h , Y . I , L a r k , R . H , D y e r , J . M , V e e n h u i s , M . and G o o d m a n , J . M . (1995) J. Cell Biol. 1 2 9 , 3 4 5 - 3 5 5

83.

M c C a m m o n , M . T , D o w d s , C . A , O r t h s , K , M o o m a w , C . R , Slaughter, C . A . and G o o d m a n , J . M . (1990) J . Biol. C h e m . 265 , 20098-20105 M c C a m m o n , M . T , M c N e w , J . A , Willy, P.J. and G o o d m a n , J . M . ( 1 9 9 4 ) J . C e l l B i o l . 124 , 915-925

84. 85. 86. 87.

D v e r , J . M , M c N e w , J . A . and G o o d m a n , J . M . (1996) J. Cell Biol. 1 3 3 , 2 6 9 - 2 8 0 Wolins, N . E . and D o n a l d s o n , R.P. (1993) J . Biol. C h e m . 269, 1149-1153 Wolins, N . E . and D o n a l d s o n , R.P. (1997) Plant Physiol. 113, 9 4 3 - 9 4 9

88. 89.

Mullen, R . T , Lee, M.S., Flvnn, C . R . and Trelease, R . N . (1997) Plant Phvsiol. 115, 8 8 1 - 8 8 9 Faber, K . N , H a i m a , P , G i e t l , C „ H a r d e r , W , A b , G . and Veenhuis^ M . ( 1 9 9 4 ) P r o c . N a t l . Acad. Sci. U . S . A . 91, 12985-12989

Subject index

acetoacetyl-CoA thiolase, 2 7 7 - 2 7 8

brefeldinA, 268

acid bristle, 235

Brownian Ratchet model,

aconitase, 279

bulk flow rate, 199

185

acyl-CoA oxidase, 2 7 7 adhesin, 143

calcium-sensitive protease,

Aeromonas salmonicida.

122, 125

AgfA, 161

198

Candida spp„ 2 6 2 , 2 7 7 cardiolipin, 243

Agrobacterium tumefaciens.

125

amino acid, 2 0 , 2 2 , 2 3 , 4 3 , 5 1 , 5 2 , 5 6 - 5 9 , 70,232,237,262,282 amphipathic

carnitine acetyl-transferase, 278 catalase, 281 cell cycle, 2 1 8 , 2 2 1 cellulase, 122

a-helix, 5 3 , 5 6 , 173

C F A / I . C S I and C S 2 fimbrial pathway,

P-strand, 52

147,161-164

amphiphilic

CfaB,

161,162

a-helix, 2 5 , 2 6

channel-forming peptide, 2 4 , 2 5

a-sheet, 20

chaperone, 8 0 - 8 1 , 8 8 , 115, 131, 135,

P-sheet, 20

147, 149,152, 155, 158, 177,

amphiphilicity, 19,20

198,223,251

annular lipid, 19

chaperone/usher pathway,

anterograde traffic, 197

chaperonin, 231

antifolding chaperone, 251

chloroplast biogenesis, 249

apocytochrome c, 7 , 2 3 7 , 2 4 0 - 2 4 1

cholera toxin, 122

A-protein, 122,128

cholesterol, 24, 195,203

ApsE, 128

cis-acting chaperone, 80

arm repeat, 218

c/s-Golgi network,

ATPase, 4 1 , 8 6 , 105, 127, 130, 178,234,

citrate synthase, 279

252,267

147

194

class L antibiotic, 24

ATPase-binding-cassette transporter, 128, 270

class L venom, 24 coat protein, 6 0 , 6 2

azide-resistant mutant, 94

coatamer, 196, 197

Bacillus subtilis, 125

coated vesicle, 196

coated protein,

196

bacterial membrane, 69

co-chaperonin, 239

bacterial type III protein, 71

compartmentalization, 261

band I (see also SecG), 92

concerted switch model, 173

fj-barrel, 5 5 , 1 4 9

conformational distortion,

binding energy, 2

conjugation,

BiP, 181, 185, 187, 198

conservative sorting, 240

8ordetella pertussis,

contact site,

147

10-12

125 113,239-240

287

288

Subject index

C o t A , 161 co-translation, 61, 176,231

F I - G I long/F I - G I short subfamilies, 153, 158

Cpn60, 2 5 2

FCL chaperone,

Cpr3, 242

ffh gene, 87

C s g protein, 160, 161

ffs gene, 87

158

C s o A , 161

Ffs, 87

cytochrome c haem lyase, 237

FGL/FGS subfamilies,

cytoskeletal protein, 205

filamentous phage, fimbria,

depth-weighted insertion hydrophobicity, 32 dibasic amino acid motif, 201 digitonin-permeabilized semi-intact cell, 216

153,158

125,132

143-145,147,152,161

fimbrial chaperone, pathway,

152

146,147

Fim protein,

145,155

Fth, 87

dipole moment, 4 0 , 4 3

FtsH, 93

disulphide bond, 20, 135, 149, 158,198

ftsY gene, 87

disulphide isomerase, 148,158

FtsY, 8 7

D N A transformation, 125, 132 DnaJ, 177,178,181 G DnaK, 88, 177, 178, 181 double-spanning membrane protein, 72-76

domain, 173 G 2 phase, 218 general insertion pore, 236 general secretion pathway,

121,147,159

DsbA, 158

general secretion pathway protein,

electochemical membrane potential, 70

germination, 274

electrophoretic insertion mechanism, 74

gluconeogenesis, 273

126-133

electrostatic interaction, 1 - 3 , 2 6 , 2 5 4

glycolate pathway, 275

endoplasmic reticulum

glycoprotein,

198

localization, 1 9 9 , 2 0 2

glycosome, 259

membrane, 50, 169

glycosylphosphatidylinositol anchor,

role in p e r o x i s o m e biogenesis, 2 6 7

198,202-206

pH, 195

glyoxylate pathway, 273

protein degradation, 198

glyoxysomal membrane protein, 281

quality control, 198,202

glyoxysome, 2 5 9 , 2 7 3

retrieval, 199

Golgi complex,

105,169,193-211

envelope transit signal, 251

G o u y - C h a p m a n relationship, 5

Eps protein, 1 2 2 , 1 2 8 - 1 3 0

GroEL, 8 8 , 2 4 2

Erd2p, 200

GroES, 242

Erwinia spp., 124,132

GTPase, 220-221

Escherichia coli, 1 1 2 , 1 2 4 , 1 4 7

GTPase-activating protein, 2 2 0

exe gene, 122

guanine-nucleotide-exchange factor, 220

Exe protein, 128, 131 exotoxin A, 134

Haemophilus influenzae,

e x p o r t signal, 224

hairpin loop, 170

extracellular nucleation/precipitation

Hansenula polymorpha,

pathway, 147

125,132,147 261

HDEL sequence signal, 200

289

Subject index

heat-shock protein (see also Hsp70), 177,222,235,242

lactose permease, 77,81 LamB,

helical hairpin, 4 2 , 5 4

113,114

large periplasmic loop, 75

a-helical membrane anchor, 56

leader peptidase, 3 9 , 4 0 , 7 4

helical-wheel-based algorithm, 23

leader sequence,

a-helix, 2, 1 3 , 2 0 - 2 2 , 2 5 , 2 6 . 3 9 . 4 3 , 4 9 ,

lipopolysaccharide, I 12

52-56,173,204

lipoprotein,

160

115-117,131

helix formation, 2 , 2 6

LolA, 116

3 1 0 helix, 55

lytic peptide, 23

helix-break-helix, 4 2 heterokaryon, 224

M domain, 173

H„ phase, 2 0 , 2 2

M phase, 218

HrcC, 132

M13 coat protein, 6 0 , 6 2 , 7 4

Hsc70, 223

malate dehydrogenase, 280

Hsp 10, 242

malate synthase, 280

Hsp60, 242

MalT, 131

Hsp70, 177, 178, 185, 186,222,223, 235,238,242,252

matrix stop-transfer signal, 233 matrix-processing peptidase, 232

hydrogen exchange, 12

matrix-processing protein, 240

hydrophilic translocation pore, 81

matrix-targeting signal, 2 3 2 , 2 3 4

hydrophobic moment, 2 0 , 2 3 , 2 8 , 3 1

membrane

hydrophobic region, 7 3 , 7 6

anchor, 56

hydrophobicity, 2 , 3 2 , 7 3 , 1 2 7

biosynthesis, 267

hydroxylited ami no acid, 232

insertion, 6 7 , 9 0 , 108

hypersensitive response, 132

lipid, 80-81

immunoglobulin fold, 149

potential,

import-competent peroxisome, 262

prokaryotic,

mitochondrial, 2 3 6 - 2 4 0

importin, 2 1 6 , 2 1 8 , 2 2 2

3,50,54,70,240,254 105,112,131,143,149,

152

insertion hydrophobicity, 32 integral membrane protein, 25 inter-helix region, 25

protein,

25,49,52,55,60,67-77,

202,281 sorting, 2 4 0 , 2 4 1

intermembrane space, 2 3 7 - 2 3 8 , 2 4 0 - 2 4 1 InvG, 132

membrane-catalysed a-helix formation, 26

isocitrate lyase, 279

membrane-spanning segment, 49 membrane-spanning 3-strand, 5 5 - 5 6

KAP60, 221

metastable trimer, 113

KAP95, 2 2 1 , 2 2 2

methylase, 127

KAR2, 181

microbody, 2 5 9 , 2 7 3

karyopherin, 2 1 6 , 2 1 8 , 2 2 2

mitochondrial

karyophilic protein, 215

Hsp70, 2 3 8 , 2 4 2

K.DEL receptor, 2 0 0 , 2 0 5

import-stimulating factor, 234

KDEL sequence signal, 200

membrane, 2 3 6 - 2 4 0

kin recognition, 204

precursor, 232

Klebsiella pneumoniae,

147

protein import, 2 3 1 - 2 3 2 targeting signal, 278 mitochondrial Mdj 1, 242

290

Subject index

molecular hydrophobic potential, 31

p i 0 / N T F 2 , 221

molten globular state, II

Pap-protein,

monolayer study, 4 2

passive diffusion, 213

144-145,147-149

multifunctional enzyme, 277

pectinase,

multi-spanning membrane protein, 7 6 - 7 7

pendulin, 217

MxiD, 132

122

penicillin-binding protein, 2 1 , 2 7 , 5 6

Myxococcus xanthus,

125

peptidase, 3 9 , 4 0 , 7 4 , 127,232 peptide—membrane binding, 2

nascent-polypeptide-associated complex, 174-176

peroxisomal targeting signal, 2 6 2 , 2 6 3 , 265,276

NBS-I/NBS-II, 89

peroxisome,

negative curvature strain, 24

pex mutant, 2 6 0 , 2 6 6

negatively charged amino acid residue, 2 2 , 5 8 - 5 9 , 7 0 , 232,237

259,260,267,271,273

Pex protein,

263,264,266,267

ApH-dependent system, 2 5 2 PhoA fusion protein, 133

membrane surface, 10,254

phosphatidylcholine, 6

phospholipid, 6 - 8 , 1 2 , 2 0 , 4 2 , 4 3 , 8 1 ,

phosphatidylglycerol, 5

94,243

phosphatidylinositol transfer protein,

Neisseria spp., 125,145

204

Neurospora crassa, 273

phosphoinositide, 204

N-glycosylation, 193,196

phospholipid

nuclear

affinity, 23

envelope, 2 1 3 , 2 6 7

annular, 19

export, 224-226

binding, 90

import, 2 1 3 - 2 2 3

monolayer, 27

localization signal, 2 1 3 , 2 1 5 - 2 1 7

negatively charged, 6 , 7 - 8 , 1 2 , 2 0 , 4 2 ,

pore complex, 213 protein, 214

43,81,90, 110,111,243 perturbation effect, 2

retention, 224

phospholipid-induced unfolding, 12

nuclear-pore-targeting complex, 216

phospholipid-protein complex, 12

nucleocytoplasmic shuttling, 224

photorespiratory glycolate pathway, 275

nucleolin, 2 2 4

photosystems I and II, 249

nucleoplasms, 214

Pil protein,

nucleoporin, 2 1 6 , 2 2 0

pilus, 1 2 4 - 1 2 7 , 1 3 0 , 132

126-130,132

nucleotide-binding site, 89

pl2 (see also SecG), 9 2

nucleotide-exchange factor, 2 3 9

Pmp protein, 270 polytopic membrane protein, 5 2 , 5 9

O-glycosylation, 196

pore-forming activity, 24

0 H 0 3 I , 217

porin, 2 0 , 5 5 , 1 14

oligomerization, 265

positive binding energy, 2

OmpA, 41,96

positive inside rule, 5 7 , 1 0 8

out gene, 124

positively charged

O u t protein, 1 2 6 , 1 2 9 - 1 3 3 outer membrane of Escherichia coli, 105, I 12 mitochondrial, 2 3 6 - 2 3 7 prokaryotic,

131,143,149,152

(i-oxidation, 274

amino acid residue, 2 0 , 2 2 , 4 3 , 5 1 , 5 2 , 56,70,232,262,282 signal sequence, 4 1 , 4 2 , 4 3 , 8 9 , 9 4

291

Subject index

post-translational

Rip I p, 226

import, 2 3 2 , 2 6 2

R N A processing, 221

protein assembly, 61

rose diagram, 30

translocation,

176

precursor protein, 41

Saccharomyces cerevisiae,

prefimbrin, 145

Salmonella typhimurium,

169, 170,277 147

preprotein, 8 6 , 8 9 - 9 2 , 9 8

Schiffer-Edmundson helical wheel, 29

prl protein, 4 0 , 8 8 , 9 3 , 9 4

Sec-dependent mechanism, 5 1 , 5 5 , 1 2 1

prl suppressor, 8 8 , 9 3

sec gene. 4 0 , 8 5 . 1 7 9 , 181

prokaryotic membrane, 6 7 , 6 9 , 105, 112, 131,143, 149,152 proline isomerase, 242

Sec-independent insertion pathway, 79 Sec pathway, 3 8 , 5 8 , 8 5 , 2 5 1 Sec protein

protease, 198

Sec61, 5 2 , 1 7 5 , 176,180, 181, 183,

protein

184,186

degradation, 198

Sec62p, 181

disulphide isomerase, 148,158

Sec63p, 181

folding, 2 , 1 3 , 4 3 , 5 0 , 1 3 5 , 1 4 9 , 2 4 2 - 2 4 3

SecA,

import, 231 - 2 3 2 , 2 8 2

37,41-43,50,78,86,89,95,

105, 147,252

localization suppressor (see also prl), 88,93

SecB, 3 7 , 4 3 , 5 0 . 8 6 , 105,252 SecD, 3 7 , 9 3 , 1 0 5

secretion, 50

SecE, 3 7 , 7 8 , 9 2 , 1 0 5

trafficking, 249

SecF, 3 7 , 9 3 , 105

translocase, 251

SecG,

protein-lipid complex, 12 proteolytic processing, 241

37,64,78,92,105

SecY, 3 7 , 9 2 , 105 SecY/E/G complex, 4 3 , 5 0 , 8 7 , 8 9

Proteus mirabilis, 147

Sec translocase, 7 8 , 8 0

protonmotive force, 3 7 , 4 0 , 4 1 , 4 3 , 8 6 ,

secretion pathway, 121, 193

9 7 , 9 8 , 1 0 5 , 112

seed germination, 2 7 4

Pseudomonas spp., 124, 145

senescence, 275

PTAC, 2 1 8 , 2 2 3

Sfas adhesin, 145

PTS protein, 2 6 3 , 2 6 5 , 2 7 6

S-fimbria, 144

PTSI receptor, 283

(3-sheet, 20

PTS2 receptor, 263

Shigella flexneri,

pul gene, i 2 2

signal peptidase, 131, 149, 170,242

132

Pul protein, 128, 130, 131, 133

signal peptide (see signal sequence)

pullulanase, 122

signal peptide-binding protein, 181 signal recognition particle, 5 0 , 8 6 , 8 7 ,

rab protein, 197 Ralstonia solanacearum,

170-179, 182, 183,256 132

Ran, 2 2 1 , 2 2 2 Ran/TC4, 220 Ras superfamily, 220 ratchet model, 136

signal recognition particle-dependent translocation, 68 signal recognition particle receptor, 87, 173, 178, 182, 183 signal sequence

R C C I , 221

domain, 3 9 , 4 0

reconstituted nucleus, 215

in endoplasmic reticulum,

redox potential, 195 retrograde traffic, 197

179,182,185 in the Golgi complex, 193

170-175,

292

Subject index

hydrophobic region, 73

channel, 5 0 , 5 5 , 9 8 , 2 3 3

membrane protein, 5 0 , 5 2

intermediate, 239

penicillin-binding protein, 27

mitochondrial membrane, 2 3 6 - 2 3 9

positively charged, 4 1 - 4 3 , 8 9 , 9 4

post-translational, 176

prokaryotic, 3 7 - 4 3 , 6 9 , 8 5 , 8 7 , 8 9 ,

in prokaryotes, 3 9 , 4 0 , 4 3 , 6 8 , 6 9 , 9 8

94-96, 105, I I I, 136, 147

translocation-incompetent domain, 59

receptor, 182,183

translocon-associated protein,

thylakoid protein, 251

transmembrane

simian virus 40T-antigen, 214

domain, 204

Singer-Nicholson model, 6

a-helix, 5 3 , 5 4

single-spanning membrane protein, 68-72

potential, 3 , 5 4

S-layer protein, 128

protein,

small GTPase, 220-221

signal sequence, 43

182-183

19,21,42,203,233

small GTP-binding protein, 197

tumour suppressor, 217

SNARE, 196

type I—IV membrane protein, 52,55,

snorkel model, 22

68,202

sorting signal, I 15 sphingolipid, 195,203

usher,

S-protein, 122

usher/chaperone pathway, 146

SRP protein,

147,156

172,173,175,179,216-218

Ssa I p, 177

venom, 24

start-stop transfer, 52

vesicle budding, 196

stop-transfer signal, 2 3 3 , 2 4 2

vesicle-mediated t r a n s p o r t 268

p-strand, 52

vesicular traffic, 196-197

P-structured acidic bristle, 88

Vibrio cholerae, 122, 132

suppressor mutation, 40,41

viral protein, 68

surface potential, 3 Walker box, 127 targeting signal, 2 7 6 , 2 7 8 TcpAsubunit, 145

Xanthmonas campestris,

thylakoid protein, 2 5 1 - 2 5 5

xcp gene,

125,132

122,124

Tim protein, 2 3 7 - 2 3 9

X c p protein,

128,129,130

Tom protein, 235,236

Xps protein,

122,133

topology determinant of, 5 7 , 6 0 , 6 8

Ydjlp, 178

inversion, 63, I 10

Yersinia enterocolitica, 132

membrane protein, 77,201

Yersinia outer protein virulence, 132

switching, 64

YscC, 131, 132

trans-Golgi network,

194,206

translocase, 7 1 , 7 8 - 7 9 , 8 5 , 9 2 , 9 5 , 1 0 5

Zellweger syndrome, 263

translocating-chain-associating protein,

zinc finger, 215

184-185 translocation

P-zipper motif, 155