seminars in
CELL BIOLOGY, Vol 3, 1992 : pp 301-307
Small GTP-binding proteins as compartmental markers Bruno Goud protein . Most of these proteins belong to the Sec4/ Ypt1/rab family, which is now considered to be a new branch of the p21ra' superfamily . Other small GTPbinding proteins which are candidates to regulate transport events are members of the ARF family .
Each intracellular compartment involved in the biosynthetic/ secretory pathway of eukaryotic cells bears at its surface at least one small GTP-binding protein . Most of them belong to a distinct branch of the p21'" superfamily, the Sec4/ Yptl /rab family . Other proteins are members of the ARF family . They play a key role in the regulation of budding and targeting/fusion events occurring during protein transport .
Proteins of the Sec4/Yptl/rab family
Key words : small GTP-binding proteins/ secretory pathway / intracellular transport
Sec4 and Yptl are two proteins involved in the secretory process of yeast Saccharomyces cerevisiae (in yeast, the secretory pathway is comparable to the constitutive biosynthetic or secretory pathway of mammalian cells-see review by A . Franzusoff, pp 309324 this issue) . SEC4 gene was identified in 1980 by Novick and Schekman when they characterized a series of mutants thermo-sensitive for secretion and cell surface growth . 1,2 SEC4 encodes a 23 .5 kDa protein that shares 32 % amino acid sequence identity with the product of the proto-oncogene H-ras . 3 YPT] gene was identified by Gallwitz and co-workers as an open reading frame between actin and tubulin genes . 4 It encodes a 23 kDa protein which shares 45 % amino acid identity with Sec4 (30 % with H-ras) . Evidence is now accumulating that Ypt 1 is primarily involved in protein transport between the endoplasmic reticulum and Golgi apparatus and between Golgi subcompartments (see below) . However, Ypt 1 has also been implicated in microtubule organization and functions as well as in the regulation of intracellular calcium levels . 6 The first rab genes were isolated in 1987 by A . Tavitian and co-workers from a rat brain cDNA library (rab stands for ras genes from rat brain), probed with an oligonucleotide corresponding to a conserved motif of ras and ras-related genes (the DTAGQE motif, see Figure 1) .% Subsequently, many other rab genes have been isolated from a variety of species including human and canine . 8-11 Rab homologues are also present in yeasts S. cerevisiae and Schizosaccharomyces pombe . 12-14 The rab gene family now comprises more than 20 members, but some are probably still missing . Most of the rab genes identified so far appear to be expressed ubiquitously, except rab3A (also called smg p25A) which is only expressed in neuronal and neuroendocrine cells . 1 s ,16
PROTEINS MOVING along the biosynthetic/secretory
pathway from the endoplasmic reticulum to their final destination (plasma membrane, endosomes/ lysosomes . . .) must sequentially cross a series of membraneous compartments or organelles, including the intermediate compartment (IC), the Golgi subcompartments and the trans-Golgi network (TGN) . A current view of this process is that inter-organelle transport is mediated by carrier vesicles which bud from one compartment (becoming the donor compartment) and fuse with the following compartment (becoming the acceptor compartment) . A number of laboratories are currently investigating the molecular mechanisms underlying the formation and specific targeting of transport vesicles . Small GTP-binding proteins (or GTP-ases) are now recognized to play a central role in these events . We will focus in this review on those involved in protein transport and membrane traffic along the secretory/biosynthetic pathway . Small GTP-binding proteins along the biosynthetic/secretory pathway Within the past few years, each intracellular compartment involved in transport has been shown to bear on its surface at least one small GTP-binding From the Unite de Genetique Somatique (URA CNRS 361), Institut Pasteur, 2 .5 rue du Dr . Roux, 75724 Paris Cedex 15, France ©1992 Academic Press Ltd 1043-4682/.92/050301 + 07$8 .00/0
301
30 2
B . Goud
N-terminus
C-terminus
L
Z% GI
G2
G3
Effector domain
G4
G5
Hy
Membrane binding domain
Figure 1 . Structure of Sec4/Yptl/rab proteins . ∎ , Domains involved in GTP/GDP binding and GTP hydrolysis . These domains are highly conserved in all Ras and Ras-related GTP binding proteins ; 0 , Domains conserved in Sec4/Yptl/rab proteins ; 0 , Hypervariable region involved in targeting to distinct intracellular compartments . The average amino acid identity between each rab protein is about 30 01o, but some of them share > 90 % identity and are called A, B, and C . We know for example rab1A, rab1B, rab3A, B, and C . Whether they represent isoforms of the same protein or fulfill different functions is not yet understood . Rab gene products share 30-70% amino acid identity with Sec4/Yptl . The closest one is rablA (70% identity with Yptl) which can functionally replace this protein in yeast . 17 The Sec4 homologue, if it exists, has not yet been identified in mammalian cells . Figure 1 shows a diagrammatic representation of the Sec4/Yptl/rab proteins . All Ras and Ras-like GTP-binding proteins have the same basic structure . Some domains, in addition to the one involved in GTP/GDP binding, are highly conserved between rab proteins and Sec4/Yptl . They include the region identified as the effector domain (G2 loop) of Ras proteins . 18,19 This domain, which changes its conformation according to the binding of Ras proteins either to GTP or GDP, also interacts with GAP proteins (GTP-ase activating proteins) that stimulate the very low intrinsic GTPase activity of Ras and Ras-related small GTP-binding proteins . 20 The conservation of the putative effector domain suggests that Sec4/Yptl/rab proteins interact with closely related effectors, different from those of other families of Ras-like GTP-binding proteins . Peptides
mimicking the effector region of rab3A have been recently shown to inhibit in vitro several transport steps . 21 Sec4 and Yptl have been localized to compartments of the yeast secretory pathway . Sec4 resides on the surface of post-Golgi secretory vesicles and on the inner surface of the plasma membrane . 22 Yptl has been found associated with Golgi-like structures . 23 Rhyl, the yeast homologue of rab6, could be present on Golgi membranes . 13 Only a few rab proteins have been localized by immunofluorescence and immunoelectron microscopy in mammalian cells (reviewed in ref 24) . Rab proteins distributed along the biosynthetic/secretory pathway are rablA (J . Saraste, B . Goud, unpublished results), rablB, 25 rab2, 26 rab3A, 27,211 and rab6 29 (see Figure 2) . Preliminary evidence indicates that rab8 is associated with basolateral vesicles in polarized cells (Kai Simons, personal communication) . At the steady state, Sec4/Yptl/rab proteins are also found in the cytosol . Evidence exists that this pool, which might represent up to 50% of the total protein, is in equilibrium with the membrane-bound form . 22,30 Sec4/Yptl/rab proteins are synthesized as soluble precursors which are post-translationally modified in order to bind to membranes . All the proteins terminate with the amino-acid residues CC or CXC (C = cysteine and X = any amino acid), except rab8
Small GTP-binding proteins as compartmental markers
Sarll
O O ER
IYpt
I
Rhy 191
000
303
Set 4
O
Golgi
A
PM
0
could be the product of the BET2 gene, initially identified as a gene required for transport from endoplasmic reticulum to Golgi apparatus and needed for the membrane attachment of both Yptl and Sec4 . 36 The fact that rab proteins are associated with distinct organelles indicates that they contain structural information responsible for their targeting . In addition, overexpression of rab proteins does not lead to their mislocalization . Chavrier et al have recently provided evidence that the targeting information resides in the hypervariable region located upstream from the carboxy-terminus . 37 It is not clear yet whether a specific rab receptor exists on the membrane of each compartment . This receptor could in fact recognize rab proteins complexed with GDI and serve as a GDS protein (see discussion below and Figure 3) .
Other small GTP-binding proteins HI arf
I rob 6
rab 3A
I
rob IA, rob 2 I I I rob IB B
Figure 2 . Localization of small GTP-binding proteins along the biosynthetic/secretory pathway in yeast (A) and in mammalian cells (B) . In yeast Saccharomyces cerevisiae, Sarl is found on ER membranes, Yptl on Golgi and Sec4 on post-Golgi secretory vesicles and on the inner face of the plasma membrane . Rhy 1, the yeast homologue of rab6 could be associated with Golgi membranes . In mammalian cells, rab1B is associated with ER and Golgi, rablA and rab2 with intermediate compartment (IC) and Golgi . Membrane-bound ARF is found on cis-Golgi . Rab6 is found on medial and trans-Golgi cisternae as well as on TGN and post-Golgi vesicles . Rab3A is localized to synaptic vesicles and chromaffin granules .
which terminates with CVLL . Removal of one or two of the cysteines impairs both membrane association and function . 31,32 Recent data indicates that one (in the CC motif) or two (in the CXC motif) of these cysteines are modified by the addition of a geranyl-geranyl moiety (C20 isoprene) . 33,34 Lipid modification is catalyzed by a type II geranyl-geranyl transferase (GGTase) which has not been yet purified but is different from the type I GGTase that modifies another protein of the Ras superfamily . 35 These enzymes consist of two subunits called alpha and beta . In yeast, the ,l3-subunit of the GGTase
Recent evidence indicates that another family of small GTP-binding proteins, the ARF family, is also involved in vesicular transport . ARF was first identified as the cofactor required for in vitro ADPribosylation of the Gs a subunit by Vibrio cholerae toxin . 38 ARF was subsequently shown to be a GTPbinding protein . 39 The domains involved in GTP binding and hydrolysis differ significantly from those of other Ras and Ras-like proteins and appear to be more similar to those found in the heterotrimeric (signal transducing) G proteins . At least six ARF genes have been now identified in mammals and two ARF genes (ARFI and ARF2) exist in S . cerevisiae . 40 They encode highly conserved, ubiquitous proteins . ARF proteins lack cysteine residues near or at the carboxyterminus and membrane attachment occurs through myristoylation of the N-terminus . ARF proteins display a very low or undetectable GTP-ase activity . In contrast to Sec4/Yptl/rab proteins, ARFs are abundantly expressed in eukaryotic cells (1 % of total brain proteins) . The localization to the Golgi apparatus of ARF (using an antibody against a peptide identical in all mammalian ARFs except ARF6) has recently provided the first clue for the physiological role of ARF proteins . 41 In addition, it has been shown that yeast ARFI null mutants display a secretory defect . Very recently, Serafini et al reported that ARF is highly enriched in Golgi-derived vesicles and is a structural component of these vesicles . 42
304
B. Goud
Acceptor compartment
Fusion
-*
exchange/ binding
Figure 3 . Role of a small GTP-binding protein (rab) in a targeting/fusion event . In this model, the small GTP-binding protein monitors the accuracy of transport vesicle targeting to the acceptor membrane (see text) . GDI : GDP dissociation inhibitor ; GDS : GDP dissociation stimulator (exchange factor) ; GAP : GTPase activating protein . The family of ARF proteins may also include the product of yeast SARI gene . This gene, originally identified as a suppressor of the SEC12 gene involved in ER to Golgi transport encodes a 21 kDa GTP-binding protein . 43 Although the aminoacid identity between Sari and ARF is only 34 .5 To, the two proteins display significant structural similarities .
Role of the small GTP-binding proteins The first role which has been attributed to small GTP-binding proteins is to control targeting/fusion events occurring during intracellular transport . This observation is mainly based on studies of Sec4 and Yptl . A thermo-sensitive mutation in SEC4 gene results in a dramatic accumulation of post-Golgi vesicles blocked in their transport or attachment to the plasma membrane at the restrictive
temperature . 3 The same phenotype is observed when a mutation which locks Sec4 in its active conformation is introduced in the protein (Sec4-Ileu 133 ) . 32 YPT1 mutants display a block in transport at an early stage of the secretory pathway . In vitro studies indicate that Yptl functions in a step after the formation of transport vesicles from the ER most likely in targeting/fusion of these vesicles with the Golgi apparatus . 44-46 However, Yptl might also function within the Golgi apparatus . 44,46 Two rab proteins associated with the biosynthetic/ secretory pathway appear to be involved in targeting/ fusion events : rabI B which might fulfill in mammalian cells a role similar to that of Ypt 1 in yeast 25 and rab3A which participates in the targeting/fusion of synaptic vesicles to the plasma membrane . i n nerve terminals . 47 Figure 3 shows a plausible model for the role of a small GTP-binding protein in a targeting/ fusion event . The basic hypothesis, originally
305
Small GTP-binding proteins as compartmental markers
formulated by H . Bourne, 48 proposes that the small GTP-binding protein functions in a cycle between the cytoplasm, transport vesicles and the target membrane and facilitate the accurate targeting/fusion of the transport vesicles with the acceptor membrane . In its inactive (GDP-bound) cytoplasmic state, the small GTP-binding protein is complexed with a GDI (GDP-dissociation inhibitor) protein that both prevents GDP dissociation and membrane binding . A rab3A/smg p25A-GDI protein, also active on Sec4, has been identified by Takai and co-workers . 49,50 GDI probably interacts with the isoprenylated form of rab3A . Exchange reaction (replacement of GDP by GTP) could take place on the membrane of the transport vesicle and be catalyzed by a GDS protein (GDP dissociation stimulator protein) . A tentative hypothesis is that GDS may serve itself as the receptor for the small GTP-binding protein . Once the small GTP-binding protein is activated and anchored to the vesicle (through its lipid moiety), GDS could dissociate from the vesicle . GTP hydrolysis occurs when the small GTP-binding protein interacts with a GAP protein . If GAP is a component of the machinery responsible for docking/ fusion of the transport vesicle with the acceptor membrane, GAP would thus monitor the accuracy of the targeting event . GDP-bound small GTPbinding protein is then released from the membrane into the cytosol . The release process could be stimulated by GDI, as documented by Takai and co-workers for smg p25A/rab3A . 51 The above model fits with the localization of the small GTP-binding protein on both the transport vesicle and the acceptor compartment (as shown for example in the case of Sec4) . However, a lot of uncertainty remains about its validity . First, and related to one of the most crucial points of the model no evidence exists so far showing that GAP is associated with the target membrane . Sec4/Yptl/ rab-GAPs are still largely unknown . However, a rab3A-GAP activity has been recently detected in both crude cytosol and membrane fractions . 52 A rab1/Yptl-GAP has also been identified, but seems to be mostly cytosolic . 53 It is also unclear whether interaction with GAP (GTP hydrolysis) induces a series of reactions that result in vesicle/acceptor compartment fusion or, alternatively, targeting and fusion are two independent events . In the latter case, one can imagine that another rab protein directly controls the fusion event . Finally, no exchange factors (GDS) active on Sec4/ Yptl/rab proteins have been reported so far .
Another role for a small GTP-binding protein could be to control budding or formation of transport vesicles from a donor compartment . GTP-'y-S, a non-hydrolyzable analog of GTP, has been shown to inhibit vesicle formation from ER in yeast 54 and from TGN in mammalian cells 55 (see review by Tooze and Stinchcombe, this issue, pp 357-366) . A role in budding has been attributed to Sarl . This protein would bind to Sec 12 on ER membranes and promote formation of transport vesicles . 56,57 A similar role at a later stage of secretion in mammalian cells has been recently proposed for ARF by Rothman and co-workers . 42 These authors propose that cytosolic ARF is recruited to Golgi membranes through a specific ARF-GDS . ARF-GTP then regulates binding of cytosolic coat proteins (including (3COP) to Golgi membranes and nascent vesicles . GTP hydrolysis could provoke the release of ARF into the cytosol and promote coat disassembly . This event would take place during vesicle transport or at the target membrane . 42
Are GTP-binding proteins true compartmental markers? Two striking facts have emerged from studies performed within the past few years on small GTPbinding proteins : they are numerous in eukaryotic cells and their remarkable properties make them good candidates to ensure specificity of multiple transport steps occurring along the biosynthetic/ secretory and endocytic pathways . A simple view would be thus that each compartment possesses its own, unique set of small GTP-binding proteins : as a donor compartment, one small GTP-binding protein involved in vesicle formation and, as an acceptor compartment, one small GTP-binding protein involved in targeting/fusion of the transport vesicles coming from another compartment . However, the reality might not be so simple . Evidence now exists that the same small GTPbinding protein of the Sec4/Yptl/rab family might function at several transport steps . One example is rab1B, which seems to be required for initial steps in protein export from the ER (including the formation or early transport of ER-derived vesicles), but also in transport between successive cis- and medial-Golgi compartments . 25 These data are consistent with the distribution of rab1B, found to be associated with both the ER and Golgi apparatus . 25 Evidence also exists that yeast Yptl
306 plays a role in targeting/fusion of ER-derived vesicles as well as in intra-Golgi transport . 44 ' 46 Another example is rab6p : although the function of this protein is still unknown, rab6 is found on medial- and trans-cisternae of the Golgi apparatus as well as on the TGN . 58 In addition, rab6 is associated with post-Golgi vesicles in at least some cell types such as neuronal cells . (A . Tixier-Vidal et al, unpublished results) and the electrocyte of Torpedo marmorata . 59 It is then possible that rab6 could function at different steps of intra-Golgi and eventually post-Golgi transport . The interpretation of these results is still unclear and they raise many intriguing questions . One possibility could be that some small GTP-binding proteins have a relatively broad function, such as in the regulation of formation or targeting of transport vesicles at different steps of the secretory pathway, or even in the regulation of both formation and targeting of transport vesicles . This does not rule out, of course, the possibility that some small GTPbinding proteins can fulfill more specific functions . Clearly, a better understanding of upstream and downstream components which interact with all these GTP-binding proteins will help to unravel their exact role in transport . However, it may be a long way to go .
References 1 . Novick P, Field C, Schekman R (1980) Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway . Cell 21 : 205-215 2 . Novick P, Ferro S, Schekman R (1981) Order of events in the yeast secretory pathway . Cell 25 :461-469 3 . Salminen A, Novick P (1987) A ras-like protein is required for a post-Golgi event in yeast secretion . Cell 47 :527-538 4 . Gallwitz D, Donath C, Sander C (1983) A yeast gene encoding a protein homologous to the human c-has/bas protooncogene product . Nature 306 :704-707 5 . Schmitt H, Wagner P, Pfaff E, Gallwitz D (1986) The rasrelated YPTI gene product in yeast : a GTP-binding protein that might be involved in microtubule organization . Cell 47 :401-412 6 . Schmitt H, Puzicha M, Gallwitz D (1988) Study of a temperature-sensitive mutant of the ras-related YPTI gene product in yeast suggest a role in the regulation of intracellular calcium . Cell 53 :635-647 7 . Touchot N, Chardin P, Tavitian A (1987) Four additional members of the ras gene superfamily isolated by an oligonucleotide strategy : molecular cloning of YPT-related cDNAs from a rat brain library . Proc Natl Acad Sci USA 84 :8210-8214 8 . Zahraoui A, Touchot N, Chardin P, Tavitian A (1987) The human rab genes encode a family of GTP-binding proteins related to yeast YPTI and SEC4 products involved in secretion . J Biol Chem 264 :12394-12401
B. Goud
9 . Viehl E, Touchot N, Zahraoui A, Tavitian A (1989) Nucleotide sequence of a rat cDNA : RABIB, encoding a RABI-YPT related protein . Nucleic Acid Res 17 :1770 10 . Chavrier P, Vingron M, Sander C, Simons K, Zerial M (1990) Molecular cloning of YPT1/SEC4-related cDNAs from an epithelial cell line . Mol Cell Biol 10 :6578-6585 11 . Chavrier P, Simons K, Zerial M . The complexity of the rab and rho GTP-binding protein family revealed by a PCR cloning approach . Gene, in press 12 . Fawell E, Hook S, Sweet D, Amstrong J (1991) Novel YPT1-related genes from Schizosaccharomyces pombe . Nucleic Acid Res 18 :4264 13 . Hengst L, Lehmeier T, Gallwitz D (1990) The rhyl gene in the fission yeast Schizosaccharomyces cereaisiae encoding a GTP-binding protein related to ras, rho and ypt : structure, expression and identification of its human homologue . EMBO J 9 :1949-1955 14 . Miyake S, Yamamoto M (1990) Identification of rasrelated, YPT family genes in Schizosaccharomyces pombe . EMBO J 9 :1417-1422 15 . Olofsson B, Chardin P, Touchot N, Zahraoui A, Tavitian A (1988) Expression of the ras-related ralA, rho and rab genes in adult mouse tissues . Oncogene 3 :231-234 16 . Sano K, Kikuchi A, Matsui Y, Teranishi Y, Takai Y (1989) Tissue-specific expression of a novel GTP-binding protein (smg p25A) mRNA and its increase by nerve growth factor and cyclic AMP in rat pheochromocytoma PC 12 cells . Biochem Biophys Res Com 158 :377-385 17 . Haubruck H, Prange R, Vorgias C, Gallwitz D (1989) The ras-related mouse yptl protein can functionally replace the YPTI gene product in yeast . EMBO J 8 :1427-1432 18 . Barbacid M (1987) Ras genes . Annu Rev Biochem 56 :779-828 19 . Bourne H, Sanders D, McCormick F (1991) The GTPase superfamily : conserved structure and molecular mechanism . Nature 349 :117-127 20 . McCormick F (1989) Ras GTPase activating protein : signal transmitter and signal terminator . Cell 56 :5-8 21 . Plutner H, Schwaninger R, Pind S, Balch W (1990) Synthetic peptides of the Rab effector domain inhibit vesicular transport through the secretory pathway . EMBO J 9 :2375-2383 22 . Goud B, Salminen A, Walworth N, Novick P (1988) A GTP-binding protein required for secretion rapidly associates with secretory vesicles and the plasma membrane in yeast . Cell 53 :753-768 23 . Segev N, Mulholland J, Botstein D (1988) The yeast GTPbinding YPTI protein and a mammalian counterpart are associated with the secretion machinery . Cell 52 :915-924 24 . Goud B, McCaffrey M (1991) Small GTP-binding proteins and their role in transport . Curr Opin Cell Biol 3 :626-633 25 . Plutner H, Cox A, Pind S, Khosravi-Far R, Bourne J, Schwaninger R, Der C, Balch W (1991) Rablb regulates vesicular transport between the endoplasmic reticulum and successive Golgi compartments . J Cell Biol 115 :31-43 26 . Chavrier P, Parton R, Hauri H . P, Simons K, Zerial M (1990) Localization of low-molecular weight GTP-binding proteins to exocytic and endocytic compartments . Cell 62 :317-329 27 . Fisher von Mollard G, Mignery G, Baumert M, Perin M, Hanson T, Burger P, Jahn R, Sudhorf T (1990) Rab3 is a small GTP-binding protein exclusively localized to synaptic vesicles . Proc Nail Acad Sci USA 87 :1988-1992 28 . Darchen F, Zahraoui A, Hammel F, Monteils M-P, Tavitian A, Scherman D (1990) Association of the GTPbinding protein Rab3A with bovine adrenal chromaffin granules . Proc Nail Acad Sci USA 87 :5692-5696 29 . Goud B, Zahraoui A, Tavitian A, Saraste J (1990) Small GTP-binding protein associated with Golgi cisternae . Nature 345 :553-556
Small GTP-binding proteins as compartmental markers 30 . Bailly E, McCaffrey M, Touchot N, Zahraoui A, Tavitian A, Goud B, Bornens M (1991) Phosphorylation of two small GTP-binding proteins of the rab family by p34c d 2 Nature 350 :715-718 31 . Molenaar C, Prange R, Gallwitz D (1988) A carboxyterminal cysteine residue is required for palmitic acid binding and biological activity of the ras-related yeast YPT1 protein . EMBO J 7 :971-976 32 . Walworth N, Goud B, Kabcenell A, Novick P (1989) Mutational analysis of SEC4 suggests a cyclical mechanism for the regulation of vesicular traffic . EMBO J 8 :1685-1693 33 . Khosravi-Far R, Lutz R, Cox A, Conroy L, Bourne J, Sinensky M, Balch W, Buss J, Der C (1991) Isoprenoid modification of rab proteins terminating in CC or CXC motifs . Proc Natl Acad Sci USA 88 :6264-6268 34 . Farnsworth C, Kawata M, Yoshida Y, Takai Y, Gelb M, Glomset J (1991) C terminus of the small GTP-binding protein smg p25A contains two geranylgeranylated residues and a methyl ester . Proc Natl Acad Sci USA 88 :6186-6200 35 . Seabra M, Reiss Y, Casey P, Brown M, Goldstein J (1991) Protein farnesyltransferase and geranylgeranyltransferase share a common cx subunit . Cell 65 :429-434 36 . Rossi G, Jiang Y, Newman A, Ferro-Novick S (1991) Dependence of Ypt l and Sec4 membrane attachment on Bet2 . Nature 351 :158-161 37 . Chavrier P, Gorvel J-P, Stelzer E, Simons K, Gruenberg J, Zerial M (1991) Hypervariable C-terminal domain of rab proteins acts as a targeting signal . Nature 353 : 769-772 38 . Kahn R, Gilman A (1984) Purification of a protein cofactor required for ADP-ribosylation of the stimulatory regulatory component of adenylate cyclase by cholera toxin . J Biol Chem 259 :6228-6234 39 . Kahn R, Gilman A (1986) The protein cofactor necessary for ADP-ribosylation of G, by cholera toxin is itself a GTP-binding protein . J Biol Chem 261 :7906-7911 40 . Moss J, Vaughan M (1991) Activation of cholera toxin and Escherichia coli heat-labile enterotoxins by ADP-ribosylation factors, a family of a 20 kda guanine nucleotide-binding proteins . Mol Microbiol 5 :2621-2627 41 . Stearns T, Willingham M, Botstein D, Kahn R (1990) ADP-ribosylation factor is functionally and physically associated with the Golgi complex . Proc Natl Acad Sci USA 87 :1238-1242 42 . Serafini T, Orci L, Amherd M, Brunner M, Kahn R, Rothman J (1991) ADP-ribosylation factor is a subunit of the coat of Golgi-derived COP-coated vesicles : a novel role for a GTP-binding protein . Cell 67 :239-253 43 . Nakano A, Muramatsu M (1989) A novel GTP-binding protein, Sarl p, is involved in transport from the endoplasmic reticulum to the Golgi apparatus . J Cell Biol 109 :2677-2691 44 . Bacon R, Salminen A, Ruohola H, Novick P, Ferro-Novick S (1989) The GTP-binding protein YTP1 is required for transport in vitro : the Golgi apparatus is defective in YPT1 mutants . J Cell Biol 109 :1015-1022
307 45 . Baker D, Wuestehube L, Schekman R, Botstein D, Segev N (1990) GTP-binding Yptl protein and Cat, function independently in a cell-free protein transport reaction . Proc Nat] Acad Sci USA 87 :355-359 46 . Segev N (1991) Mediation of the attachment or fusion step in vesicular transport by the GTP-binding Yptl protein . Science 252 :1553-1556 47 . Fischer von Mollard G, Sudhof T, Jahn R (1991) A small GTP-binding protein dissociates from synaptic vesicles during exocytosis . Nature 349 :79-81 48 . Bourne H (1988) Do GTP-ases direct membrane traffic in secretion? Cell 53 :669-671 49 . Sasaki T, Kikuchi A, Araki S, Hata Y, Isomura M, Kuroda S, Takai Y (1990) Purification and characterization from bovine brain cytosol of a protein that inhibits the dissociation of GDP from and the subsequent binding of GTP to smg p25A, a ras p21-like GTP-binding protein . J Biol Chem 265 :2333-2337 50 . Sasaki T, Kaibuchi K, Kabcenell A, Novick P, Takai Y (1991) A mammalian inhibitory GDP/GTP exchange protein for smg p25A is active on the yeast Sec4 protein . Mol Cell Biol 11 :2909-2912 51 . Araki S, Kikuchi A, Hata Y, Isomura M, Takai Y (1990) Regulation of reversible binding of smg p25A, a ras p21-like GTP-binding protein, to synaptic plasma membranes and vesicles by its specific regulatory protein, GDP dissociation inhibitor . J Biol Chem 265 :13007-13015 52 . Burstein E, Linko-Stentz-K, Lu Z, Macara 1 (1991) Regulation of the GTPase activity of the ras-like protein p25`ab'A . J Biol Chem 266 :2689-2692 53 . Becker J, Tan T, Trepte H-H, Gallwitz D (1991) Mutational analysis of the putative effector domain of the GTP-binding Yptl protein in yeast suggests specific regulation by a novel GAP activity . EMBO J 10 :785-792 54 . Rexach M, Schekman R, (1991) Distinct biochemical requirements for the budding, targeting, and fusion of ERderived transport vesicles . J Cell Biol 114 :219-229 55 . Tooze S, Weiss U, Huttner W (1991) Requirement for GTP hydrolysis in the formation of secretory vesicles . Nature 347 :207-208 56 . D'Enfert C, Wuestehube L, Lila T, Schekman R (1991) Sec 12p-dependent membrane binding of the small GTPbinding protein Sarlp promotes formation of transport vesicles from the ER . J Cell Biol 114 :663-670 57 . Oka T, Nishikawa S, Nakano A (1991) Reconstitution of GTP-binding Sarl protein function in ER to Golgi transport . J Cell Biol 114 :671-679 58 . Antony C, Cibert C, Geraud G, Santa Maria A, Maro B, Mayau V, Goud B (1992) The small GTP-binding protein rab6p is distributed from medial Golgi to the trans-Golgi network as determined by a confocal microscopy approach . J Cell Sci, in press 59 . Jasmin C, Goud B, Camus G, Cartaud J (1992) Association of rab6p with assymetrically distributed post-Golgi vesicles in a polarized cell . Neuroscience, in press
CELL BIOLOGY, Vol 3, 1992 : pp 301-307
Small GTP-binding proteins as compartmental markers Bruno Goud protein . Most of these proteins belong to the Sec4/ Ypt1/rab family, which is now considered to be a new branch of the p21ra' superfamily . Other small GTPbinding proteins which are candidates to regulate transport events are members of the ARF family .
Each intracellular compartment involved in the biosynthetic/ secretory pathway of eukaryotic cells bears at its surface at least one small GTP-binding protein . Most of them belong to a distinct branch of the p21'" superfamily, the Sec4/ Yptl /rab family . Other proteins are members of the ARF family . They play a key role in the regulation of budding and targeting/fusion events occurring during protein transport .
Proteins of the Sec4/Yptl/rab family
Key words : small GTP-binding proteins/ secretory pathway / intracellular transport
Sec4 and Yptl are two proteins involved in the secretory process of yeast Saccharomyces cerevisiae (in yeast, the secretory pathway is comparable to the constitutive biosynthetic or secretory pathway of mammalian cells-see review by A . Franzusoff, pp 309324 this issue) . SEC4 gene was identified in 1980 by Novick and Schekman when they characterized a series of mutants thermo-sensitive for secretion and cell surface growth . 1,2 SEC4 encodes a 23 .5 kDa protein that shares 32 % amino acid sequence identity with the product of the proto-oncogene H-ras . 3 YPT] gene was identified by Gallwitz and co-workers as an open reading frame between actin and tubulin genes . 4 It encodes a 23 kDa protein which shares 45 % amino acid identity with Sec4 (30 % with H-ras) . Evidence is now accumulating that Ypt 1 is primarily involved in protein transport between the endoplasmic reticulum and Golgi apparatus and between Golgi subcompartments (see below) . However, Ypt 1 has also been implicated in microtubule organization and functions as well as in the regulation of intracellular calcium levels . 6 The first rab genes were isolated in 1987 by A . Tavitian and co-workers from a rat brain cDNA library (rab stands for ras genes from rat brain), probed with an oligonucleotide corresponding to a conserved motif of ras and ras-related genes (the DTAGQE motif, see Figure 1) .% Subsequently, many other rab genes have been isolated from a variety of species including human and canine . 8-11 Rab homologues are also present in yeasts S. cerevisiae and Schizosaccharomyces pombe . 12-14 The rab gene family now comprises more than 20 members, but some are probably still missing . Most of the rab genes identified so far appear to be expressed ubiquitously, except rab3A (also called smg p25A) which is only expressed in neuronal and neuroendocrine cells . 1 s ,16
PROTEINS MOVING along the biosynthetic/secretory
pathway from the endoplasmic reticulum to their final destination (plasma membrane, endosomes/ lysosomes . . .) must sequentially cross a series of membraneous compartments or organelles, including the intermediate compartment (IC), the Golgi subcompartments and the trans-Golgi network (TGN) . A current view of this process is that inter-organelle transport is mediated by carrier vesicles which bud from one compartment (becoming the donor compartment) and fuse with the following compartment (becoming the acceptor compartment) . A number of laboratories are currently investigating the molecular mechanisms underlying the formation and specific targeting of transport vesicles . Small GTP-binding proteins (or GTP-ases) are now recognized to play a central role in these events . We will focus in this review on those involved in protein transport and membrane traffic along the secretory/biosynthetic pathway . Small GTP-binding proteins along the biosynthetic/secretory pathway Within the past few years, each intracellular compartment involved in transport has been shown to bear on its surface at least one small GTP-binding From the Unite de Genetique Somatique (URA CNRS 361), Institut Pasteur, 2 .5 rue du Dr . Roux, 75724 Paris Cedex 15, France ©1992 Academic Press Ltd 1043-4682/.92/050301 + 07$8 .00/0
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B . Goud
N-terminus
C-terminus
L
Z% GI
G2
G3
Effector domain
G4
G5
Hy
Membrane binding domain
Figure 1 . Structure of Sec4/Yptl/rab proteins . ∎ , Domains involved in GTP/GDP binding and GTP hydrolysis . These domains are highly conserved in all Ras and Ras-related GTP binding proteins ; 0 , Domains conserved in Sec4/Yptl/rab proteins ; 0 , Hypervariable region involved in targeting to distinct intracellular compartments . The average amino acid identity between each rab protein is about 30 01o, but some of them share > 90 % identity and are called A, B, and C . We know for example rab1A, rab1B, rab3A, B, and C . Whether they represent isoforms of the same protein or fulfill different functions is not yet understood . Rab gene products share 30-70% amino acid identity with Sec4/Yptl . The closest one is rablA (70% identity with Yptl) which can functionally replace this protein in yeast . 17 The Sec4 homologue, if it exists, has not yet been identified in mammalian cells . Figure 1 shows a diagrammatic representation of the Sec4/Yptl/rab proteins . All Ras and Ras-like GTP-binding proteins have the same basic structure . Some domains, in addition to the one involved in GTP/GDP binding, are highly conserved between rab proteins and Sec4/Yptl . They include the region identified as the effector domain (G2 loop) of Ras proteins . 18,19 This domain, which changes its conformation according to the binding of Ras proteins either to GTP or GDP, also interacts with GAP proteins (GTP-ase activating proteins) that stimulate the very low intrinsic GTPase activity of Ras and Ras-related small GTP-binding proteins . 20 The conservation of the putative effector domain suggests that Sec4/Yptl/rab proteins interact with closely related effectors, different from those of other families of Ras-like GTP-binding proteins . Peptides
mimicking the effector region of rab3A have been recently shown to inhibit in vitro several transport steps . 21 Sec4 and Yptl have been localized to compartments of the yeast secretory pathway . Sec4 resides on the surface of post-Golgi secretory vesicles and on the inner surface of the plasma membrane . 22 Yptl has been found associated with Golgi-like structures . 23 Rhyl, the yeast homologue of rab6, could be present on Golgi membranes . 13 Only a few rab proteins have been localized by immunofluorescence and immunoelectron microscopy in mammalian cells (reviewed in ref 24) . Rab proteins distributed along the biosynthetic/secretory pathway are rablA (J . Saraste, B . Goud, unpublished results), rablB, 25 rab2, 26 rab3A, 27,211 and rab6 29 (see Figure 2) . Preliminary evidence indicates that rab8 is associated with basolateral vesicles in polarized cells (Kai Simons, personal communication) . At the steady state, Sec4/Yptl/rab proteins are also found in the cytosol . Evidence exists that this pool, which might represent up to 50% of the total protein, is in equilibrium with the membrane-bound form . 22,30 Sec4/Yptl/rab proteins are synthesized as soluble precursors which are post-translationally modified in order to bind to membranes . All the proteins terminate with the amino-acid residues CC or CXC (C = cysteine and X = any amino acid), except rab8
Small GTP-binding proteins as compartmental markers
Sarll
O O ER
IYpt
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Rhy 191
000
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Set 4
O
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A
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0
could be the product of the BET2 gene, initially identified as a gene required for transport from endoplasmic reticulum to Golgi apparatus and needed for the membrane attachment of both Yptl and Sec4 . 36 The fact that rab proteins are associated with distinct organelles indicates that they contain structural information responsible for their targeting . In addition, overexpression of rab proteins does not lead to their mislocalization . Chavrier et al have recently provided evidence that the targeting information resides in the hypervariable region located upstream from the carboxy-terminus . 37 It is not clear yet whether a specific rab receptor exists on the membrane of each compartment . This receptor could in fact recognize rab proteins complexed with GDI and serve as a GDS protein (see discussion below and Figure 3) .
Other small GTP-binding proteins HI arf
I rob 6
rab 3A
I
rob IA, rob 2 I I I rob IB B
Figure 2 . Localization of small GTP-binding proteins along the biosynthetic/secretory pathway in yeast (A) and in mammalian cells (B) . In yeast Saccharomyces cerevisiae, Sarl is found on ER membranes, Yptl on Golgi and Sec4 on post-Golgi secretory vesicles and on the inner face of the plasma membrane . Rhy 1, the yeast homologue of rab6 could be associated with Golgi membranes . In mammalian cells, rab1B is associated with ER and Golgi, rablA and rab2 with intermediate compartment (IC) and Golgi . Membrane-bound ARF is found on cis-Golgi . Rab6 is found on medial and trans-Golgi cisternae as well as on TGN and post-Golgi vesicles . Rab3A is localized to synaptic vesicles and chromaffin granules .
which terminates with CVLL . Removal of one or two of the cysteines impairs both membrane association and function . 31,32 Recent data indicates that one (in the CC motif) or two (in the CXC motif) of these cysteines are modified by the addition of a geranyl-geranyl moiety (C20 isoprene) . 33,34 Lipid modification is catalyzed by a type II geranyl-geranyl transferase (GGTase) which has not been yet purified but is different from the type I GGTase that modifies another protein of the Ras superfamily . 35 These enzymes consist of two subunits called alpha and beta . In yeast, the ,l3-subunit of the GGTase
Recent evidence indicates that another family of small GTP-binding proteins, the ARF family, is also involved in vesicular transport . ARF was first identified as the cofactor required for in vitro ADPribosylation of the Gs a subunit by Vibrio cholerae toxin . 38 ARF was subsequently shown to be a GTPbinding protein . 39 The domains involved in GTP binding and hydrolysis differ significantly from those of other Ras and Ras-like proteins and appear to be more similar to those found in the heterotrimeric (signal transducing) G proteins . At least six ARF genes have been now identified in mammals and two ARF genes (ARFI and ARF2) exist in S . cerevisiae . 40 They encode highly conserved, ubiquitous proteins . ARF proteins lack cysteine residues near or at the carboxyterminus and membrane attachment occurs through myristoylation of the N-terminus . ARF proteins display a very low or undetectable GTP-ase activity . In contrast to Sec4/Yptl/rab proteins, ARFs are abundantly expressed in eukaryotic cells (1 % of total brain proteins) . The localization to the Golgi apparatus of ARF (using an antibody against a peptide identical in all mammalian ARFs except ARF6) has recently provided the first clue for the physiological role of ARF proteins . 41 In addition, it has been shown that yeast ARFI null mutants display a secretory defect . Very recently, Serafini et al reported that ARF is highly enriched in Golgi-derived vesicles and is a structural component of these vesicles . 42
304
B. Goud
Acceptor compartment
Fusion
-*
exchange/ binding
Figure 3 . Role of a small GTP-binding protein (rab) in a targeting/fusion event . In this model, the small GTP-binding protein monitors the accuracy of transport vesicle targeting to the acceptor membrane (see text) . GDI : GDP dissociation inhibitor ; GDS : GDP dissociation stimulator (exchange factor) ; GAP : GTPase activating protein . The family of ARF proteins may also include the product of yeast SARI gene . This gene, originally identified as a suppressor of the SEC12 gene involved in ER to Golgi transport encodes a 21 kDa GTP-binding protein . 43 Although the aminoacid identity between Sari and ARF is only 34 .5 To, the two proteins display significant structural similarities .
Role of the small GTP-binding proteins The first role which has been attributed to small GTP-binding proteins is to control targeting/fusion events occurring during intracellular transport . This observation is mainly based on studies of Sec4 and Yptl . A thermo-sensitive mutation in SEC4 gene results in a dramatic accumulation of post-Golgi vesicles blocked in their transport or attachment to the plasma membrane at the restrictive
temperature . 3 The same phenotype is observed when a mutation which locks Sec4 in its active conformation is introduced in the protein (Sec4-Ileu 133 ) . 32 YPT1 mutants display a block in transport at an early stage of the secretory pathway . In vitro studies indicate that Yptl functions in a step after the formation of transport vesicles from the ER most likely in targeting/fusion of these vesicles with the Golgi apparatus . 44-46 However, Yptl might also function within the Golgi apparatus . 44,46 Two rab proteins associated with the biosynthetic/ secretory pathway appear to be involved in targeting/ fusion events : rabI B which might fulfill in mammalian cells a role similar to that of Ypt 1 in yeast 25 and rab3A which participates in the targeting/fusion of synaptic vesicles to the plasma membrane . i n nerve terminals . 47 Figure 3 shows a plausible model for the role of a small GTP-binding protein in a targeting/ fusion event . The basic hypothesis, originally
305
Small GTP-binding proteins as compartmental markers
formulated by H . Bourne, 48 proposes that the small GTP-binding protein functions in a cycle between the cytoplasm, transport vesicles and the target membrane and facilitate the accurate targeting/fusion of the transport vesicles with the acceptor membrane . In its inactive (GDP-bound) cytoplasmic state, the small GTP-binding protein is complexed with a GDI (GDP-dissociation inhibitor) protein that both prevents GDP dissociation and membrane binding . A rab3A/smg p25A-GDI protein, also active on Sec4, has been identified by Takai and co-workers . 49,50 GDI probably interacts with the isoprenylated form of rab3A . Exchange reaction (replacement of GDP by GTP) could take place on the membrane of the transport vesicle and be catalyzed by a GDS protein (GDP dissociation stimulator protein) . A tentative hypothesis is that GDS may serve itself as the receptor for the small GTP-binding protein . Once the small GTP-binding protein is activated and anchored to the vesicle (through its lipid moiety), GDS could dissociate from the vesicle . GTP hydrolysis occurs when the small GTP-binding protein interacts with a GAP protein . If GAP is a component of the machinery responsible for docking/ fusion of the transport vesicle with the acceptor membrane, GAP would thus monitor the accuracy of the targeting event . GDP-bound small GTPbinding protein is then released from the membrane into the cytosol . The release process could be stimulated by GDI, as documented by Takai and co-workers for smg p25A/rab3A . 51 The above model fits with the localization of the small GTP-binding protein on both the transport vesicle and the acceptor compartment (as shown for example in the case of Sec4) . However, a lot of uncertainty remains about its validity . First, and related to one of the most crucial points of the model no evidence exists so far showing that GAP is associated with the target membrane . Sec4/Yptl/ rab-GAPs are still largely unknown . However, a rab3A-GAP activity has been recently detected in both crude cytosol and membrane fractions . 52 A rab1/Yptl-GAP has also been identified, but seems to be mostly cytosolic . 53 It is also unclear whether interaction with GAP (GTP hydrolysis) induces a series of reactions that result in vesicle/acceptor compartment fusion or, alternatively, targeting and fusion are two independent events . In the latter case, one can imagine that another rab protein directly controls the fusion event . Finally, no exchange factors (GDS) active on Sec4/ Yptl/rab proteins have been reported so far .
Another role for a small GTP-binding protein could be to control budding or formation of transport vesicles from a donor compartment . GTP-'y-S, a non-hydrolyzable analog of GTP, has been shown to inhibit vesicle formation from ER in yeast 54 and from TGN in mammalian cells 55 (see review by Tooze and Stinchcombe, this issue, pp 357-366) . A role in budding has been attributed to Sarl . This protein would bind to Sec 12 on ER membranes and promote formation of transport vesicles . 56,57 A similar role at a later stage of secretion in mammalian cells has been recently proposed for ARF by Rothman and co-workers . 42 These authors propose that cytosolic ARF is recruited to Golgi membranes through a specific ARF-GDS . ARF-GTP then regulates binding of cytosolic coat proteins (including (3COP) to Golgi membranes and nascent vesicles . GTP hydrolysis could provoke the release of ARF into the cytosol and promote coat disassembly . This event would take place during vesicle transport or at the target membrane . 42
Are GTP-binding proteins true compartmental markers? Two striking facts have emerged from studies performed within the past few years on small GTPbinding proteins : they are numerous in eukaryotic cells and their remarkable properties make them good candidates to ensure specificity of multiple transport steps occurring along the biosynthetic/ secretory and endocytic pathways . A simple view would be thus that each compartment possesses its own, unique set of small GTP-binding proteins : as a donor compartment, one small GTP-binding protein involved in vesicle formation and, as an acceptor compartment, one small GTP-binding protein involved in targeting/fusion of the transport vesicles coming from another compartment . However, the reality might not be so simple . Evidence now exists that the same small GTPbinding protein of the Sec4/Yptl/rab family might function at several transport steps . One example is rab1B, which seems to be required for initial steps in protein export from the ER (including the formation or early transport of ER-derived vesicles), but also in transport between successive cis- and medial-Golgi compartments . 25 These data are consistent with the distribution of rab1B, found to be associated with both the ER and Golgi apparatus . 25 Evidence also exists that yeast Yptl
306 plays a role in targeting/fusion of ER-derived vesicles as well as in intra-Golgi transport . 44 ' 46 Another example is rab6p : although the function of this protein is still unknown, rab6 is found on medial- and trans-cisternae of the Golgi apparatus as well as on the TGN . 58 In addition, rab6 is associated with post-Golgi vesicles in at least some cell types such as neuronal cells . (A . Tixier-Vidal et al, unpublished results) and the electrocyte of Torpedo marmorata . 59 It is then possible that rab6 could function at different steps of intra-Golgi and eventually post-Golgi transport . The interpretation of these results is still unclear and they raise many intriguing questions . One possibility could be that some small GTP-binding proteins have a relatively broad function, such as in the regulation of formation or targeting of transport vesicles at different steps of the secretory pathway, or even in the regulation of both formation and targeting of transport vesicles . This does not rule out, of course, the possibility that some small GTPbinding proteins can fulfill more specific functions . Clearly, a better understanding of upstream and downstream components which interact with all these GTP-binding proteins will help to unravel their exact role in transport . However, it may be a long way to go .
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