The diversity of Rab proteins in vesicle transport

496

The diversity of Rab proteins in vesicle transport Peter Novick* and Marino Zerialt Rab

proteins

as regulators

have been primarily implicated of SNARE

in vesicle docking

pairing. Recent findings, however,

indicate that their function in vesicle trafficking can go beyond this role, and a number of proteins, unrelated to each other, have been identified as putative Rab effecters. GTPase

Although the

switch of Rab proteins is highly conserved,

mechanisms

functional

may be highly diversified among members

of the

Rab family.

Addresses ‘Department of Cell Biology, Yale University School of Medicine, PO Box 208002, New Haven, CT 06520-8002, USA; e-mail: [email protected] tEuropean Molecular Biology Laboratory, Meyerhofstrasse 10.2209, 69012 Heidelberg, Germany; e-mail: [email protected] Current

Opinion

in Cell Biology

1, Postfach

1997, 9:496-504

http:llbiomednet.comlelecref/0955067400900496 0 Current

Biology Ltd ISSN 09550674

Abbreviations ER GAP GDI

endoplasmic reticulum GTPase-activating protein GDP-dissociation inhibitor

GEF NSF REP SNAP SNARE TGN XTP

guanine nucleotide exchange factor N-ethylmaleimide-sensitive factor Rab escort protein soluble NSF-attachment protein SNAP receptor frans-Golgi network xanthosine 5’-triphosphate

Introduction Maintaining the integrity of the organelles in the cell requires the function of molecules that selectively regulate the generation of transport vesicles and the targeting and fusion of the vesicles with the appropriate acceptor membrane. Two classes of proteins have emerged as specific and essential players in many vesicle transport processes. One class is represented by integral membrane proteins, SNARES (soluble NSF-attachment protein [SNAP] receptors), which serve as receptors for soluble factors that are necessary for docking and fusion (see Hay and Scheller, this issue, pp 505-512). The second class comprises proteins of a branch of the Ras superfamily of small GTPases, called Rab proteins. Rab proteins have been primarily implicated in vesicle docking as regulators of SNARE pairing. However, recent findings, which are the focus of this review, suggest that the function of Rab proteins in vesicle trafficking can go beyond this role. Rab proteins clearly constitute the largest family of small GTPases. The first Rab proteins to be identified, Sec4p and Yptlp, were found in yeast [1,2]; there is a total of 11 proteins in the yeast Rab family (see Table 1; the

other nine proteins are YptSlp, Ypt32p, Ypdlp, Ypt52p, Ypt53p, Ypt6p, Ypt7p, and two novel members encoded by genes called YBR264 and YNL304W). In mammalian cells, however, the number is even larger and about 40 proteins have been identified (the last one being Rab30 [3]), including isoforms. The complexity of the Rab protein family in mammalian cells correlates with the diversity of vesicle transport routes displayed in a variety of mammalian cell types [4]. Some Rab proteins ‘are cell-type specific and regulate transport events that are not shared by all mammalian cells; for example, Rab3A is involved in regulated secretion in neurons and neuroendocrine cells. A summary of the involvement of Rab proteins in membrane traffic is shown in Table 1.

The Rab cycle The proposed functional cycle of Rab proteins is illustrated in Figure 1. The model assumes that the Rab protein associates with the transport vesicle budding from the donor compartment, moves to the acceptor compartment where docking and fusion take place, and finally is recycled back to the donor membrane via a cytosolic intermediate. In the cytosol, Rab proteins are maintained in the GDP-bound inactive conformation by Rab GDP-dissociation inhibitor (GDI), an important co-factor in the Rab functional cycle. Although there is only one Rab GDI protein in yeast (Gdilp) [S], mammalian cells express several GDI isoforms [6-81. No remarkable functional differences between the GDI molecules have been detected, but the relative proportion of Rab proteins complexed to a given GDI varies depending on the cell type. Perhaps this reflects a further mechanism for regulating the function of Rab proteins. Rab GDI has the ability to extract GDP-bound Rab proteins from the membrane and, in the complexed form, to mediate their membrane attachment [9,10]. In vitro studies have helped to uncover the molecular details of this process. First, one or more factors recognize the Rab protein complexed to Rab GDI, disrupt the complex, thus releasing Rab GDI into the cytosol, and allow Rab.GDP to bind the membrane. Second, after a certain delay GDP/GTP exchange occurs on Rab [9,10]. Nucleotide exchange is, therefore, not a prerequisite for membrane binding but serves to activate the Rab protein, rendering it resistant to removal by Rab GDI. Displacement of GDI and GDP/GTP exchange could be catalyzed by distinct factors [9,10]. Recent results suggest that this assumption may be correct. A protein with the property of a GDI-displacement factor for endosomal Rab proteins (RabS, Rab7 and Rab9) has been identified [ll’]. This molecule lacks nucleotide exchange activity. Similarly, a membrane protein that specifically mediates the recruitment of Rab4 to endosomes has been found

The diversity of Rab proteins in vesicle transport Novick and Zerial

497

Table 1 Locallzatlon

and functional

properties

of Rab proteins in (a) the yeast Sac&aromyces

and (b) mammalian

cells.

Function

Localization

Protein

cerevisiae

(a) ER; Golgi Post-Golgi

ER+Golgi

transport; intra_Golgi transport

Yptl P Sec4p Ypt31 p, Ypt32p

Golgi membranes

Ypt51 pNps21

Endosomes

Intra-Golgi transport; transport to vacuole? Transport in the early endocytic pathway;

Ypt6p

n.d. n.d.

transport to vacuole 154-561 Similar to Ypt51 p? Transport from Golgi to vacuole; intra-Golgi transport?

YPt7P YBR264 YNL304W

n.d. n.d. n.d.

Transport from late endosomes

(b) Rabl A, Rabl B Rab2 Rab3A

ER-Golgi intermediate compartment ER-Golgi intermediate compartment Synaptic vesicles; chromaffin granules

ER-tGolgi

transport; intra-Golgi transport

ER+Golgi Regulated

transport exocytosis in pancreatic

Ypt52p,

p

Ypt53p

Golgi+plasma

secretory vesicles

membrane

transport 1401

147,481

to vacuole 152,531

n.d. n.d.

acinar cells,

adrenal chromaffin cells and mast cells Tight junction region in polarized

Rab3B Rab3C Rab3D Rab4A, Rab5A,

Rab4B RabBB, Rab5C

Rab7 Rab8

Middle Golgi-TGN Late endosomes Post-Golgi exocytic vesicles; tight junction in epithelial cells

RabQ RablO Rabll

Late endosomes and TGN Golgi complex TGN; constitutive secretory vesicles;

Rab6

n.d.

epithelial cells Synaptic vesicles Zymogen granules in pancreatic acinar cells 1781 Early endosomes Plasma membrane; clathrin-coated vesicles; early endosomes

n.d. n.d. Recycling pathway from endosomes to plasma membrane Plasma membrane+early endosome transport and homotypic fusion between early endosomes Intra-Golgi retrograde transport? [491 Transport from early to late endosomes and lysosomes 158,591 Golgi+plasma

membrane

transport

1701

Transport from late endosomes to TGN n.d. Transport through recycling endosomes

[461

secretory granules; recycling endosomes References

refer to papers published in 1996

and others discussed

in the text. For the other proteins that are not specifically referenced,

see [4].

nd.. not determined.

and is distinct from a guanine nucleotide exchange factor (GEF) [12’]. Conversely, the GEF for Rab3 is inactive if Rab3 is complexed with Rab GDI [13], suggesting that the activity of the GEF follows displacement of Rab GDI from Rab3. Rab GDI prevents indiscriminate membrane binding and contributes selectivity to the process of recruitment to the membrane [14,15]. Although the two activities are tightly coupled, it is not clear whether recognition of the Rab-Rab GDI complex and release of Rab GDI are mediated by the same protein. Clearly, these factors are very important elements of the transport machinery because they specifically determine the site on the membrane where the Rab protein is recruited and exerts its activity. GEFs can either have a broad substrate specificity [16] or be highly specific [13]. Their function can also transcend the GDP/GTP-exchange activity. For example, Se&p has been shown to act as a GEF for Sec4p [17*]. As SEC2 mutations cause random rather than polarized accumulation of vesicles in yeast, Se&p may somehow couple the activation of Sec4p to the polarized delivery of vesicles to the site of exocytosis. Like

GEFs, GTPase-activating proteins (GAPS) can also be specific for some Rab family members [18]. GEF, effector molecules and GAP determine which nucleotide is bound to Rab proteins on the membrane, thereby modulating the accessibility of Rab proteins to Rab GDI. Together these factors keep Rab proteins in a dynamic equilibrium between the cytosol and the membrane.

Rab proteins are isoprenylated inactive

while kept

To bind to membranes, Rab proteins must be prenylated. The enzyme catalyzing this reaction is Rab geranylgeranyltransferase (GGTase), an heterodimer of a and p subunits which transfers a geranylgeranyl group to (usually) two cysteines present at the carboxy-terminal region of the Rab molecule [19]. An additional component, called Rab escort protein (REP), binds nonprenylated precursors and presents them to the catalytic component. REP can stably form complexes with unprenylated, monoprenylated and diprenylated Rab proteins [ZO], thereby ensuring that Rab proteins stay bound to REP until both cysteines are modified. Rab GDI was proposed to serve as an acceptor for prenylated Rab proteins that are complexed

498

Membranes and sorting

Figure 1

GD,

'

displacement

(b)

~

GTP

GDP

an~ehn:n~e°tide

,

>

(d)

(a)

GDI~ Nucleotide hydrolysis

S 0

(_

f

x/ C N I R T E L

E A R T

~, Acceptor compartment

© 1997 CurrentOpen/onin Cell Biology

The proposed functional cycle of Rab proteins. Rab.GDP is shown as a black circle labelled GDP with zigzagged lines indicating the attached isoprenyl lipid moieties; Rab.GTP is shown as a black oval labelled GTP with zigzagged lines attached; and Rab proteins that are in a state of nucleotide exchange/hydrolysis are shown as unlabelled black circles with zigzagged lines attached. (a) In the cytosol, Rab proteins are maintained in the GDP-bound inactive conformation by Rab GDI. Rab GDI prevents indiscriminate membrane binding by Rab. (b) As Rab.GDP binds to the membrane of the donor organelle compartment or vesicle, GDI is displaced by a GDl-displacement factor (GDF). (c) Exchange of GDP for GTP on Rab is catalyzed by a GEE Nucleotide exchange serves to activate the Rab protein and render it resistant to removal from the membrane by Rab GDI. (d) The transport vesicle buds from the donor compartment. (e) Binding of transport vesicles to acceptor compartments is mediated by Rab.GTP on the transport vesicle and probably an effector on the acceptor compartment. GTP hydrolysis is mediated by GAP. The release of vesicle contents (represented by different upper case letters) into the acceptor compartment is shown. (f) GDI can release Rab.GDP from the acceptor compartment, and the cycle can begin again.

with REP and to target them to the membrane. Although evidence in favour of this possibility has been obtained in vitro [21,22°], Rab G D I is not an obligatory intermediate. This is because REP itself behaves biochemically as a G D I [23,24]. REP bears sequence homology with its yeast counterpart, Mrs6p, but also with mammalian and yeast Rab GDI. REP binds only the GDP-bound form of both unprenylated and prenylated Rab proteins, inhibits GDP release and delivers Rab proteins directly

to the membrane following geranylgeranylation [23]. T h e biochemical properties of REP explain why prenylation occurs with 10-50-fold higher efficiency for GDP-bound Rab compared with GTP-bound Rab [24]. That Rab G D I is not necessary in the initial membrane targeting is demonstrated by an elegant study using a RablB effector mutant (RablB Asp44---~Asn) which fails to complex with Rab G D I in vitro but is prenylated and efficiently associates with the membrane in vivo [22"]. These

The diversity of Rab proteins in vesicle transport Novick and Zerial

results are consistent with REP mediating the membrane delivery of newly synthesized Rab proteins and Rab GDI mediating the subsequent recycling between membrane and cyrosol. The crystal structure of bovine Rab GDI has been determined and two of the sequences that are conserved between Rab GDI and REP have been shown to be clustered at one face of the molecule [ZS”]. This domain is presumably involved in the nucleotide-dependent binding of Rab proteins. Despite the sequence conservation, however, REP cannot be replaced by Rab GDI in the prenylation reaction. REP is therefore a specialized GDI that plays a dual role in Rab prenylation and membrane association. It is likely that, shortly after their synthesis, Rab proteins are first the substrates of GAPS in the cytosol that make them competent to bind REP Together, GAPS, Rab GDI and REP ensure that Rab proteins are maintained in the inactive GDP-bound conformation in the cytosol so that activation occurs exclusively on the membrane. Two REPS have been identified, REP1 and REP2 Defects in the REP1 gene result in choroideremia (CHM), an X-linked form of retinal degeneration, REP& which can assist in the prenylation of most Rab proteins, cannot overcome this defect in the retina. The molecular explanation for the retinal degeneration is suggested by recent studies showing that in CHM lymphoblasts the majority of Rab proteins are prenylated with the exception of one, Ram/RabZ7 [26]. Consistently, this particular Rab protein is expressed at high levels in the retinal pigment epithelium and choriocapillaries, the two sites of earliest degeneration in CHM. It remains to be shown whether the deficiency in prenylation of Ram/Rab27 is directly responsible for the specific phenotype in retinal degeneration. If this were true, it would provide another example of the cell type specificity of some Rab family members.

GTP hydrolysis The nonhydrolyzable GTP analogue GTPyS has been extensively used to explore the involvement of GTPases in transport reactions in vitro. In most cases, GTP@ inhibits vesicle budding or fusion. Endosome fusion in vitro, however, is an interesting example in which GTPyS can be either stimulatory or inhibitory depending on the concentration of cytosol in the assay, suggesting that multiple GTPases may be affected [27,28]. As GTP$S does not discriminate between different GTPases, the inhibited proteins are unknown. In the case of dynamin, the conformational change that converts the protein from the active GTP-bound form to the inactive GDP-bound form is necessary for clathrin-coated-vesicle formation at the plasma membrane [29,30]. In the case of Rab proteins, however, the expression of hydrolysis-deficient mutants has shown that GTP hydrolysis is not always essential for vesicle fusion. In yeast, the hydrolysis-defective Leu79

499

mutant allele of Sec4 results in a partial loss of function [31]. In mammalian cells, Rab3A mutants defective in GTP hydrolysis inhibit exocytosis from neuroendocrine cells [32,33]. In contrast, expression of a GTPase-deficient mutant of RabS stimulates rather than inhibits endosome fusion [34]. It is possible that hydrolysis may not be required for fusion per se but instead allows the protein to recycle efficiently between membrane and cytosol (Figure 1). Mutant Rab proteins with altered nucleotide specificity that preferentially bind xanthosins 5’-triphosphate (XTP) have recently emerged as interesting tools with which to study nucleotide exchange and hydrolysis [35,36’]. In the case of Rab5, the Rab5 Asp136+Asn mutant protein stimulates endosome fusion even in the presence of the nonhydrolyzable analogue XTP@ [36*]. In this case, the inhibition of endosome fusion caused by GTPyS at high cytosolic concentration can be attributed not to Rab5 but to other small GTPases, most likely ADP-ribosylation factor (ARF). Interestingly, hydrolysis of [32P]XTP by membrane-bound Rab5 has been shown to occur constitutively; that is, hydrolysis is uncoupled from membrane fusion [36*]. This suggests that, at least in the case of RabS, hydrolysis serves as a timer to switch off the protein and prevent its prolonged activation. Hydrolysis, however, is slowed down upon effector binding (see below), indicating that a pool of Rab.5 is trapped in its GTP-bound state. Thus, the clock is modified once the timer can be committed to membrane docking and fusion. It will be interesting to see if this mechanism can be generalized to other Rab proteins. More information on the kinetics of hydrolysis by Rab proteins is necessary and the XTP-binding mutants are expected to be particularly useful for these measurements.

Diverse functions

of Rab proteins

The findings that Rab proteins are compartmentalized and regulate specific membrane transport events lend support to the hypothesis that a different Rab protein may be required for each step of vesicular transport. In particular, Rab proteins have been assigned a role at the docking/fusion stage. For example, Sedp appears to be exclusively required for the delivery of Golgi-derived secretory vesicles to the plasma membrane in yeast [l]. Ypt7p has been recently demonstrated to act specifically at the docking step in homotypic yeast vacuole fusion in vitro [37*]. There are, however, exceptions. In some cases, Rab proteins have been shown to participate in more than one transport step. For example, Yptlp functions in the first two stages of transport along the secretory pathway in yeast [38] as does Rabl in mammalian cells [39]. It is not known whether Yptlp may function in both anterograde and retrograde traffic or whether other Ypt proteins are involved. In mammalian cells, endoplasmic reticulum (ER)+Golgi transport requires Rab2, which is not expressed in yeast, as well as Rabl. This indicates that the control of Rab proteins at the same transport step

500

Membranes and sorting

has become evolutionarily more complicated from yeast to mammals, and raises questions as to the precise role of Rab2. A number of observations are consistent with an involvement of Rab proteins in vesicle budding as well. Depletion of both Ypt3lp and Ypt32p in yeast induces the accumulation of stacks of membranes that resemble the typical Golgi cisternae of mammalian cells, suggesting a role for these proteins in intra-Golgi transport and in the formation of transport vesicles at the exit point of the Golgi complex [40]. As Rab mutants that preferentially bind GDP appear to block vesicle formation from the ER [41] and from late endosomes [42], the recruitment of Rab proteins to the donor membrane has been proposed to be necessary to ensure that the vesicles are already supplied with the correct delivery machinery. It is intriguing, however, that transport vesicles can also acquire their Rab proteins after budding in vitro [15,43] and, therefore, carry the necessary recruitment machinery. A second possibility is that Rab proteins do regulate vesicle budding in addition to docking and fusion. This possibility is supported by the recent finding that a complex of RabS-Rab GDI appears to be required for coat invagination and receptor sequestration in an in vitro budding assay (E Smythe, personal communication). It is interesting in this respect that overexpression of Rab5 stimulates endocytosis and decreases the number of coated pits on the plasma membrane in viva [44]. Clearly, this principle cannot be generalized as not all Rab proteins appear to be required at the budding step. In yeast strains carrying various dominant-negative alleles of SEC4, vesicle formation proceeds normally [1,45]. Perhaps these differences reflect the differing needs for recycling of some rate-limiting components required for budding. At any rate, it is important to realize that, directly or indirectly, vesicle budding appears to be coupled to vesicle docking and fusion. This mechanism could be important to ensure that vesicle formation and consumption are correctly balanced. Disruption of several YPT genes (YPT31, YPT32, YPTSI, YPT6 and YPT7) affects the vacuolar sorting pathway (see below). The precise role of these proteins is not clear yet and the phenotypes observed do not always match those of the mammalian homologues. YpQp GTPases appear to be associated, at least partially, with Golgi membranes and, in addition to playing a role in intra-Golgi transport, are required for transport to the vacuole (401. Their closest mammalian homologue, Rabll, is localized to the perinuclear recycling endosome and is required for receptor recycling through this compartment [46]. However, as Rabll is also associated with the trans-Golgi network (TGN) it may additionally play a role in transport between the Golgi complex and the endosomes. Mutant strains defective in Ypt6p are viable but display defects in the maturation of vacuolar hydrolases [47,48]. This

phenotype is difficult to reconcile with the presumed function of mammalian Rab6, which complements a YPT6 null strain [47], but is implicated in retrograde traffic between the Golgi and the ER [49]. These differences may be explained by the different experimental strategies used (gene deletion compared with expression of mutants) but may also reflect functional diversity between yeast and higher eukaryotes. In keeping with the view that transport from the Golgi complex to the vacuole involves one or more prevacuolar endosomal intermediates [SO], as in mammalian cells [Sl], it is conceivable that these Ypt proteins may be required at different stages along the pathway to the vacuole. Some proteins may act primarily in the endocytic pathway. Disruption of YPT7 affects vacuole biogenesis [37*,52,53]. Ypt5lp has been identified, together with Ypt52p and Ypt53p, in a search for a yeast homologue of Rab5 [54] and, independently, as VpsZlp, a vacuolar protein sorting mutant [55]. It is possible that Ypdlp plays a different role in yeast to that which Rab5 plays in mammalian cells [50]. Alternatively, given that in AYptSlp mutants both a-factor degradation and endocytic transport to the vacuole are inhibited, Ypdlp could regulate access to an endosome/prevacuolar compartment where the vacuolar secretory and endocytic pathways converge [56,57]. The sequential functions of Ypdlp and Ypt7p in endocytic transport [52-56) resemble those of Rab5 and Rab7 in mammalian cells [44,58,59]. However, tracing a parallel between the yeast and mammalian traffic routes remains difficult at this stage and more work needs to be done to characterize these prevacuolar compartment(s) and to understand how the link between the biosynthetic and endocytic pathways is regulated.

Rab effecters Understanding the cellular role of the Rab proteins will require the definition of their direct effector molecules and the biochemical pathways that are regulated by them. Two main approaches have been followed towards this end, namely, the identification of proteins, so called Rab effecters, that preferentially bind to GTP-bound Rab proteins, and the isolation of genetic suppressors that can bypass partial or complete loss of Rab protein function, thus delineating the elements of the downstream pathways. The first Rab effector to be identified was rabphilin [60]. This protein is expressed in neurons and specifically recognizes the GTP-bound form of Rab3. The aminoterminal region constitutes the Rab3-binding site, while the carboxy-terminal region contains two C&like domains that will bind Caz+ and phospholipid [61]. Expression or microinjection of either the amino- or the carboxy-terminal region of rabphilin will block Caz+-dependent exocytosis in several different systems [62]. Rabphilin localizes to the cytoplasmic surface of synaptic vesicles [63], and several lines of evidence indicate that rabphilin is recruited

The diversity of Rab proteins in vesicle transport Novick and Zerial

there by Rab3 [64,65], but another report suggests chat it is targeted to synaptic vesicles independently of Rab3 [66]. One clue to the mechanism of action of rabphilin in regulated secretion is its interaction with the actin-bundling protein a-actinin [67]. Rabphilin promotes the bundling activity of a-actinin, but this function is blocked by Rab3A in its GTP-bound form. This suggests a model in which Rab3A acts through rabphilin to trigger the remodelling of the actin-based cytoskeleton in preparation for vesicle docking and fusion, although other roles for Rab3A and rabphilin are certainly possible. An unrelated protein, termed rabaptin-5, was identified by its interaction with activated Rab5 in a yeast two-hybrid screen [68]. Rabaptin-5 is recruited from the cycosol to endosomes by activated Rab5. There it plays a role in the homotypic fusion of early endosomes. Depletion of rabaptin-5 from the cytosol inhibits endosomal fusion in vitro while overexpression of rabaptin-5 in vivo promotes the formation of enlarged endosomes by stimulating endosomal fusion. These results indicate a role for rabaptin-5 as the Rab5 effector in the endocytic pathway. The sequence of rabaptin-5 predicts a coiled-coil structure which could mediate interactions with other proteins. Recent findings indicate chat rabaptin-5 is complexed to other proteins in the cytosol (M Zerial, unpublished data). Proteins interacting with the activated forms of Rab6, RabS and Rab9 have also been identified through twohybrid screens. The Rab8-interacting protein (Rab8ip) is a serine/threonine protein kinase that is activated by cell stress [69]. Like Rab8, this kinase is localized to the Golgi apparatus where it may regulate vesicular transport to the cell surface in response to Rab8. Rab8ip shares sequence homology with Saccharomyces cemisiae SteZOp and mammalian p65PAK, protein kinases that are involved in the signalling function of small GTPases of the Rho family in the organization of the actin cycoskeleton. Interestingly, Rab8 is able to promote the reorganization of actin and microtubules and the formation of cellular processes to which secretory proteins are preferentially delivered [70]. A Rab6-interacting protein is a member of the kinesin family (B Goud, personal communication). Rab6 may control retrograde transport from the Golgi by regulating the function of this microtubule-based motor. The Rab9-interacting protein is associated with endosomes and shows synergy with Rab9 in its ability to stimulate transport of the mannose 6-phosphate receptor from endosomes to the TGN in an in vitro transport assay [71]. Genetic studies in yeast have suggested a role for the Rab proteins in the formation of a complex of proteins that are involved in vesicle docking and fusion and which are known as SNARES (see Hay and Scheller, this issue, pp 505-512). Each class of carrier vesicles is thought to be tagged with one or more specific members of a family of integral membrane proteins that is typified

501

by the synaptic vesicle protein synaptobrevin/VAMP (vesicle-associated membrane protein). These proteins are termed Vesicle-SNARES (V-SNARES). Each target membrane is tagged with a member of a family of integral membrane proteins that are related to syntaxin, a neuronal protein on the presynaptic membrane. These proteins are termed target-SNARES (t-SNARES). The interaction of the cytoplasmic domains of a v-SNARE and a t-SNARE results in the formation of a complex and this is thought to be a key step in the reaction leading to vesicle fusion. The specificity of this protein-protein interaction may underlie the specificity of vesicle targeting. Although Yptlp is an essential Rab protein that controls the early stages of the yeast secretory pathway, the complete loss of Yptlp can be tolerated if certain components of the relevant SNARE complex are overproduced. While overexpression of either SecZZp or Beclp will weakly suppress a deletion of YPTI [72], the co-overexpression of either Sec22p and Boslp [73] or Boslp and Betlp [74] will very efficiently bypass the need for Ypclp. These are all SNARE proteins that are required for ER+Golgi traffic. The loss of Yptlp can also be efficiently suppressed by a dominant point mutation in SLY1 [72] which encodes a member of the Seclp protein family that may serve to regulate the formation of SNARE complexes by interacting with the t-SNARE, in this case SedSp. At the final stage of the yeast secretory pathway, a cold-sensitive mutation in the effector domain of the Rab protein Sec4p can be efficiently suppressed by overexpression of SEC9 [75]. Sec9p is a member of the SNAP25 (synaptosome-associated protein of 25 kDa) family of proteins that serves to promote SNARE-complex formation. In total, the evidence strongly suggests that these Rab proteins act, either directly or indirectly, to promote SNARE-complex formation. In confirmation of this model, the ER+Golgi SNARE complex fails to form in yptl mutants 1761. The mechanism by which Rab proteins promote SNAREcomplex formation has not yet been resolved. In yeast, the V-SNARES Boslp and Sec22p form a hetero-oligomer on the carrier vesicles and formation of this hetero-oligomer requires Yptlp [76]. This has led to the suggestion that Rab proteins function by activating their respective V-SNARES. Rab proteins are not stable components of SNARE complexes. However, this does not preclude a transient interaction. In fact, Yptlp has recently been shown to interact with the t-SNARE Sed5p and, to a lesser extent, with the v-SNARE SecZ2p, although the nucleotide dependence of the interaction has not yet been addressed [77]. The interaction with Sed5p may serve to displace Slylp and thereby promote interaction with the V-SNARES Sec22p and Boslp.

Conclusions Further studies will be needed to understand the relationship between the proposed role of Rab proteins

502

Membranes and sorting

in the promotion of SNARE-complex formation and the biochemical activities of the specific proteins identified to date as Rab effecters. The effecters could serve as intermediaries on a linear pathway leading to SNAREcomplex formation, but a more plausible interpretation is that the Rab proteins control several distinct activities in parallel. These activities may include motors needed for vesicle movement, vesicle-binding proteins needed for vesicle docking, kinases needed for additional levels of coordinate regulation and, ultimately, the formation of SNARE complexes for fusion. This would place the Rab proteins in the position of the master regulators of membrane traffic, a lofty title for these small GTPases.

References

and recommended

13.

Wada M, Nakanishi H, Satoh A, Hirano H, Obaishi H, Matsuura Y, Takai Y: Isolation and characterization of a GDP/GTP exchange protein specific for the Rab3 subfamily small G proteins. J Biol Chem 1997, 272:3875-3878.

14.

Dirac-Svejstrup AB, Soldati T, Shapiro AD, Pfeffer SR: RabGDI presents functional RabQ to the intracellular transport machinery and contributes selectivity to Rab9 membrane recruitment J Biol Chem 1994, 269:15427-l 5430.

15.

Horiuchi H, Giner A, Hoflack BA, Zerial M: A GDP/GTP exchange stimulatory activity for the RabS-RabGDI complex on clathrincoated vesicles from bovine brain. J Biol Chem 1995, 271 :11257-l 1262.

16.

Burton JL, Burns ME, Gatti E, Augustine GJ, De Camilli P: Specific interactions of Mss4 with members of the Rab GTPase subfamily. EM60 J 1994, 13:5547-5558.

reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

. ..

ceptor for Rab4. Treatment of endosome membranes with elastase released the receptor activity and rendered endosomes incompetent for Rab4 recruitment. Membrane association could be rescued by supplementing the endosomes with the elastase supernatant. By this clever experimental approach, the authors further demonstrated that the putative Rab4 receptor lacks nucleotide exchange activity. This study also addressed the mechanism whereby phosphorylation of Rab4 by cdc2-cyclin B kinase causes redistribution of Rab4 into the cytosol. The authors could show that this is not due to removal of Rab4 from the membrane but rather to inhibition of Rab4 binding to its receptor. These results, thus, provide important clues as to the mechanisms of membrane association of Rab4 in interphase and during mitosis.

of special interest of outstanding interest

1.

Salminen A, Novick PJ: A ras-like protein is required for a postGolgi event in yeast secretion. Cell 1987, 4Q:527-538.

2.

Gallwitz D, Donath C, Sander C: A yeast gene encoding a protein homologous to the human c-hadf~es proto-oncogene product Nature 1983, 306:704-707.

3.

Chen D, Guo J, Miki T, Tachibana M, Gahl WA: Molecular cloning of two novel rab genes from human melanocytes. Gene 1996, 174:129-134.

4.

Simons K, Zerial M: Rab proteins and the road maps for intracellular transport Neuron 1993, 11:789-799.

5.

Garrett MD, Zahner JE, Cheney CM, Novick PJ: GDll encodes a GDP dissociation inhibitor that plays an essential role in the yeast secretory pathway. EM60 J 1994, 13:1718-l 728.

6.

Shisheva A, Siidhof TC, Czech MP: Cloning. characterization, and expression of a novel GDP dissociation inhibitor isoform from skeletal muscle. MO/ Cell Biol 1994, 14:345Q-3468.

7.

Nishimura N, Nakamura H, Takai Y, Sano K: Molecular cloning and characterization of two rab GDI species from rat brain: brain-specific and ubiquitous types. J Biol Chem 1994, 269:14191-14198.

18.

Fukui K, Sasaki T, lmazumi K, Matsuura Y, Nakanishi H, Takai Y: Isolation and characterization of a GTPase activating protein specific for the Rab3 subfamily small G proteins. J Biol Chem 1997,272:4655-4658.

8.

Yang C, Slepnev VI, Goud B: Rab proteins form in viva complexes with two isoforms of the GDI-dissociation inhibitor protein (GDI). J Biol Chem 1994, 269:31891-31899.

19.

Casey PJ, Seabra MC: Protein prenyltransferases. 1996, 271:5289-5292.

20. 9.

Ullrich 0. Horiuchi H, Bucci C, Zerial M: Membrane association of Rab5 mediated by GDP-dissociation inhibitor and accompanied by GDPIGTP exchange. Nature 1994, 368:157-l 60.

Shen F, Seabra MC: Mechanisms of digeranylgeranylation Rab proteins. J Biol Chem 1996, 271:3692-3698.

21.

Sanford JC, Yu J, Pan JY, Wessling-Resnick M: GDP dissociation inhibitor serves as a cytosolic acceptor for newly synthesized and prenylated rabl. J Biol Chem 1995,270:26904-26909.

10.

Soldati T, Shapiro AD, Dirac Svejstrup AB, Pfeffer SR: Membrane targeting of the small GTPase Rab9 is accompanied by nucleotide exchange. Nature 1994, 369:76-78.

Dirac-Svejstrup AB, Sumizawa T, Pfeffer SR: Identification of a GDI displacement factor that releases endosomal Rab GTPases from Rab-GDI. EM60 J 1997, 16:465-472. Biochemical fractionation procedures were used to search for proteins that increase the binding of [ssSIGTP@ to prenylated Rab9 complexed with Rab GDI. Rab GDI inhibits nucleotide exchange on Rab proteins because it stabilizes them in the GDP-bound conformation. The authors succeeded in identifying a factor that relieves this inhibition and provided an elegant demonstration that this protein is a GDI-releasing factor rather than a nucleotide exchange factor. These results lend direct support to the hypothesis that release of GDI and GDPlGTP exchange are catalyzed by distinct factors. Furthermore, this paper shows that the GDI-displacement factor here identified acts on endosomal Rab proteins (Rab5, Rab7 and Rab9) but is inactive on Rabl, a Rab protein of the secretory pathway. 11. .

Ayad N, Hull M, Mellman I: Mitotic phosphoryletion of rab4 prevents binding to a specific receptor on endosome membranes. EM60 J 1997, in press. The authors reconstituted the Rab GDI mediated binding of Rab4 to early endosomes and demonstrated the presence of a specific and saturable re-

1 7. .

Walch-Solimena C, Collins RC, Novick PJ: SecPp mediates nucleotide exchange on Sec4p and is involved in polarized delivery of post-Golgi vesicles. J Cell Biol 1997, in press. S. cerevisiae grows in a polarized fashion by the insertion of secretory vesicles into the plasma membrane of the bud on one pole of the cell. The activity of the small GTPase Sec4p is required for the docking and fusion of Golgi-derived secretory vesicles with the plasma membrane. Mutations in the SEC4 gene cause accumulation of these vesicles in the forming bud. This paper reports that mutations in SEC2, which encodes an essential protein that is also required at the same stage of the secretory pathway, cause random rather than polarized accumulation of Sec4p-containing vesicles. Sec2p acts in Golgi+plasma membrane traffic at an earlier step than the majority of the other SEC gene products implicated in this process. Furthermore, SecPp interacts directly with Sec4p and acts as a nucleotide exchange factor, catalyzing the dissociation of GDP from Sec4 and promoting the binding of GTP. The proposal of these authors is that the task of SecZp is not only to enhance nucleotide exchange on Sec4p but also to couple activation of Sec4p to polarized vesicle delivery to the site of exocytosis.

J B/o/ Chem of

Wilson AL, Erdman RA,.Maltese WA: Association of RablB with GDP-dissociation inhibitor (GDI) is required for recycling but not initial membrane targeting of the rab protein. J Biol Chem 1996,271:10932-l 0940. Can REP itself deliver prenylated Rab proteins to membranes or is Rab GDI required as an intermediate? The solution lo this problem was found using an ingenious mutagenesis approach. The authors exploited a Rabl B effector mutant (Asp44+Asn) that fails lo form a complex with Rab GDI. This defect is not due lo the inability of the mutant protein to undergo prenylation, which occurs normally. The mutant protein was also shown to be efficiently delivered to the membrane but was absent from the cytosol. This study therefore demonstrates that newly synthesized RablB can be prenylated and delivered to the membrane by REP without requiring a Rab GDI bound intermediate. In contrast, Rab GDI is absolutely required for the recycling of Rabl B from the membrane to the cytosol. 22. .

23.

Alexandrov K, Horiuchi H, Steele-Mortimer 0, Seabra M, Zerial M: Rab escort protein-l is a multifunctional protein that accompanies newly prenylated rab proteins to their target membranes. EM60 J 1994, 13:5262-5273.

24.

Seabra MC: Nucleotide dependence of rab geranylgeranylation. J Biol Chem 1996, 271 :14398-l 4404.

12. .

The diversify

25. ..

Schalk I, Zeng K, Wu S-K, Stura EA, Matteson 1, Huang M, Tandon A, Wilson IA, Balch WE: Structure and mutational analvsis of Rab GDP-dissociation inhibitor. Nature 1996. 381:42-48. This paper reports the crystal structure of the first described bovine Rab GDI (Rab GDI-I or a isoform) to a resolution of 1.8A. Rab GDI is organized into two domains, one of which shares structural similarity with FADdependent flavoproteins. The sequences that are conserved between Rab GDI and REP form a comoact structure at the aoex of the molecule. This domain is imolicated in the’interaction with Rab proteins and mutations in these sequences consistently cause a drastic reduction in the binding of Rab proteins and in the ability of Rab GDI to extract Rab proteins from membranes. Once the structure- of a Rab protein is solved, ihwill be interesting to determine how Rab proteins can interact with Rab GDI in a nucleotide-dependent manner. Presumablv. the eftector reoion of Rab oroteins is imolicated in this interaction. The a&lability of the structure is expected to provide important insights into the molecular mechanism whereby Rab GDI interacts with Rab proteins and controls their association with the membrane.

of Rab proteins

in vesicle

transport

Novick and Zerial

uoles in vitro. Prior to docking both vacuole membranes reauire the activitv of Secl7p (a-SNAP) and Seciep (NSF). The small GTPaso Ypt7p instead is required at the docking stage. This is the first demonstration that a member of the Rab family is directly involved in the docking process itself. 38.

Jedd G, Richardson C, Litt R, Segev N: The Yptl GTPase is essential for the first two steps of the yeast secretory pathway. J Cell Biol 1995, 131:583-590.

39.

Peter F, Nuotfer C, Pind SN, Batch WE: Guanine nucleotide dissociation inhibitor is essential for Rabl function in budding from the endoplasmic reticulum and transport through the Golgi stack. J Cell Biol 1994, 126:1393-l 406.

40.

Benli M, Doring F, Robinson DG, Yang X, Gatlwitz D: Two GTPase isoforms, Ypt31 p and Ypt32p, are essential for Golgi function in yeast EM60 J 1996, 15:6460-6475.

41.

Nuoffer C, Davidson HW, MattesoqJ, Meinkoth J, Batch WE: A GDP-bound form of rebl inhibits protein export from the endoplasmic reticulum and transport between Golgi compartments. J Cell Bioll994, 125:225-237.

26.

Seabra MC, Ho YK, Anant JS: Deficient garanylgeranylation of Ram/Rab27 in choroideremia. J Biol Chem 1995, 270124420-24427.

42.

27.

Mavoraa LS. Diaz R. Colombo MI. Stahl PD: GTPvS stimulation of &ndosome fusion suggests a role for a GTPIbinding protein in the priming of vesicles before fusion. Cell Regulation 1989, 1 :113-l 24.

Riederer MA, Soldati T, Shapiro AD, Lin J, Pfeffer S: Lysosome biogenesis requires Rab9 function and receptor recycling from endosomes to the bans Golgi network J Cell Bioll994, 125573-582.

43.

28.

Lenhard JM, Kahn RA, Stahl PD: Evidence for ADP-ribosylation factor @RF) as a regulator of in vitro endosome-endosome fusion. J Biol Chem 1992, 267:13047-l 3052.

29.

Takei K, McPherson PS, Schmid SL, De Camilli P: Tubular membrane invaginations coated by dynamin rings are induced by GTP-@ in nerve terminals. Nature 1995, 374:186-l 90.

Barlowe C, Orci L, Yeung T, Hosobuchi M, Hamamoto S, Salama N, Rexach MF, Ravazzola M, Amherdt M, Schekman R: COPII: a membrane coat formed by Set proteins that drive vesicle budding from the endoplasmic reticulum. Cell 1994, 77:895-907.

44.

Bucci C, Parton RG, Mather IH, Stunnenberg H, Simons K, Hoflack B, Zerial M: The small GTPase rabl functions as a regulatory factor in the early endocytic pathway. Cell 1992. 70:715-720.

45.

Walworth NC, Goud B, Kabcenell AK, Novick P: Mutational analysis of SEC.4 suggests a cyclical mechanism for the regulation of vesicular traffic. EM60 J 1989, 6:1685-l 693.

46.

Ullrich 0. Reinsch S. Urbs S. Zerial M. Parton RG: Rabll regulates recycling ‘through the pericentriolar recycling endosome. J Cell Bioll996, 135:913-924.

30.

Hinshaw JE, Schmid SL: Dynamin self-assembles into rings suggesting a mechanism for coated vesicles budding. Nature 1995, 374:190-l 92.

31.

Walworth NC, Brennwald P, Kabcenell AK, Garrett M, Novick P: Hydrolysis of GTP by sec4 protein plays an important role in vesicular transport and is stimulated by a GTPase-activating protein in Saccharomyces cerevisiae. Mol Cell Biol 1992, 12:2017-2026.

503

32.

Holz R, Brondyk WH, Senter RA, Kuzion L, Macara IG: Evidence for the involvement of Rab3A in Caa+-dependent exocytosis from adrenal chromaffin cells. J Biol Chem 1994, 269:10229-l 0234.

47.

Tsukada M, Gallwitz D: Isolation and characterization of SYS genes from yeast, multicopy suppressors of the functional loss of the transport GTPase Ypt6p. J Cell Sci 1996, 109:2471-2481.

33.

Johannes L, Lledo P-M, Roa M, Vincent J-D, Henry J-P, Darchen F: The GTPase Rab3a negatively controls calciumdependent exocytosis in neuroendocrine cells. EM60 J 1994, 13:2029-2032

48.

Li B, Warner JR: Mutation of the Rab6 homologue of Saccharomyces cerevisiae, YPTG, inhibits both early Golgi function and ribosome biosynthesis. J Biol Chem 1996, 271 :16813-l 6819.

34.

Stenmark H, Parton RG, Steele-Mortimer 0, Liitcke A, Gruenberg J, Zeriai M: Inhibition of rab5 GTPase activity stimulates membrane fusion in endocytosis. EM60 J i 994, 13:1267-l 296.

49.

Martinez 0, Schmidt A, Salamsro J, Hoflack B, Roa MA, Goud B: The small GTP-binding protein rab6 functions in intra-Golgi transport J Cell Biol 1994, 127:1575-l 588.

50.

35.

Jones S, Litt RJ, Richardson CJ, Segev N: Requirement of nucleotide exchange factor for Yptl GTPase mediated protein transport J Cell Bioll995, 130:1051-l 061.

Horazdovsky BF, DeWald DB, Emr SD: Protein transport to the yeast vacuole. Corr Opin Cell Bioll995, 7:544-551.

51.

Ludwig T, LeBorgne R, Hoflack B: Roles for mannose 6phosphate receptors in lysosomal enzyme sorting, IGF-II binding and clathrin-coat assembly. Trends Cell Bioll995, 5:202-205.

52.

Wichmann H, Hengst L, Gallwitz D: Endocytosis in yeast: evidence for the involvement of a small GTP-binding protein (ypt7p). Cell 1992, 71 :1131-l 142.

53.

Schimmoller A, Riezman H: Involvement of YpDp, a small GTPbinding protein, in traffic from late endosome to the vacuole in yeast J Cell Sci 1993,106:823-830.

54.

Singer-Kruger B, Stenmark H, Diisterhoft A, Philippsen P, Yoo J-S, Gallwitz D, Zerial M: Role of three RabS-like GTPases, Yptsl p, Ypt62p. and Ypt63p. in the endocytic and vacuolar protein sorting pathways of yeast J Cell Biol 1994, 125:283-298.

55.

Horazdovsky BF, Busch GR, Emr SD: Yeast Ypt6lp and mammalian Rab6: counterparts with similar function in the early endocytic pathway. J Cell Sci 1994, 13:1297-l 309.

56.

Singer-Kruger B, Stenmark H, Zerial M: Yeast Ypt61 p and mammalian Rab5: counterparts with similar function in the early endocytic pathway. J Cell Sci 1995, 108:3509-3521.

57.

Vida TA, Huyer G, Emr SD: Yeast vacuolar proenxymes are sorted in the late Golgi complex and transported to the vacuole via a prevacuolar endosome-like compartment J Cell Biol 1993, 121:1245-l 256.

36. .

Rybin V, Ullrich 0, Rubino M, Alexandrov K, Simon I, Seabra MC, Goody R, Zerial M: GTPase activity of rabl acts as a timer for endocytic membrane fusion. Nat&e 1996, 383:266-269. Is the GTPase activity of Rab proteins required for, and coupled to, membrane docking and fusion? To address these questions a Rab5 mutant (Asp1 36+Asn) that preferentially binds XTP was engineered and the kinetics of [szP]XTP hydrolysis by the protein bound on the endosome membrane were measured. The Rab5 mutant stimulated early endosome fusion in the presence of XTP but also in the presence of XTpIs, a nonhydrolyzable anatogue of XTP, indicating that nucleotide triphosphate hydrolysis by Rab5 is not obligatory for early endosome fusion. Kinetics analysis indicated that nucleotide hydrolysis occurred in the presence but also in the absence of membrane fusion. This means that membrane-bound Rab5 undergoes futile cycles of XTP (and consequently GTP for wild-type Rab5) binding and hydrolysis. Binding of the Rab5 effector, rabaptin-5, however, stabilized Rab5 in the nucleotide triphosphate bound form. The authors propose that GTP hydrolysis by Rab5 acts as a timer that determines the frequency of membrane docking/fusion events in the early endocytic pathway. GTP hydrolysis by Rab5 would limit the activity of Rab5 so that endocytic transport occurs but excessive homotypic fusion between endosomes is avoided. 37. .

Mayer A, Wickner W: Docking of yeast vacuoles is catalped by the Ras-like GTPase Ypt7p after symmetric priming by Secl8p (NSF). J Cell Biol 1997, 136:307-317. Using biochemical and morphological assays, the authors of this paper analyzed the biochemical requirements in the docking and fusion of yeast vac-

504

Membranes and sorting

58.

Feng Y, Press B, Wandinger-Ness A: Rab7: an important regulator of late endocytic membrane traffic. I Cell Biol 1995, 131:1435-1452.

68.

Stenmark H, Vitale G, Ulrich 0, Zerial M: Rabaptin-5 is a direct effector of the small GTPase Rab5 in endocytic membrane fusion. Cell 1995, 83:423-432.

59.

M&esse S, Gorvel J-P, Chavrier P: The rab7 GTPase resides on a vesicular compartment connected to lysosomes. J Cell Sci 1995, 108:3349-3358.

69.

60.

Shirataki l-l, Zkaibuchi K, Sakoda T, Kishida S, Yamaguchi K, Wada K. Mivazaki M. Takai Y: Rabohilin-3A a outative taraet protein for ;mg pP&/rabBA smail GTP-binding protein related to synaptotagmin. MO/ Cell Biol 1993, 13:2061-2068.

Ren M, Zeng J, De Lemos-Chiarandini C, Rosenfeld M, Adesnik M, Sabatini DD: In its active form, the GTP-bindina protein Rab8 interacts with a stress-activated protein kinas;. -froc Nat/ Acad Sci USA 1996,93:5151-5155.

70.

Periinen J, Auvinen P, Virta H, Wepf R, Simons K: Rab8 promotes polarized membrane transport through reorganization of actin and microtubules in fibroblasts. J Cell Biol 1996, 135:153-l 67.

71.

Diaz E, Schimijller F, Pfeffer SR: A novel Rab9 effector required for endosome to TON transport, J Cell Bioll997, in press.

72.

Dascher C, Ossig R, Gallwitz D, Schmitt HD: The yeast SLY gene products, suppressors of defects in the essential GTPbinding Yptl protein, may act in endoplasmic reticulum-toGolgi transport MO/ Cell Biol 1991, 11:872-885.

73.

Lian JP, Stone S, Jiang Y, Lyons P, Ferro-Novick S: Yptl p implicated in V-SNARE activation. Nature 1994, 372:698-701.

74.

Stone S, Sacher M, Mao Y, Carr C, Lyons P, Quinn AM, FerroNovick S: Bet1 p activates the V-SNARE, Bosl p. MO/ Biol Ce// 1997, in press.

75.

Brennwald P, Kearns B, Champion K, Ker$inen S, Bankaitis V, Novick P: Sec9 is a SNAP-25like component of a yeast SNARE complex that may be the effector of Sec4 function in exocytosis. Cell 1994, 79:245-258.

76.

Sogaard M, Tani K, Ye RR, Geromanos S, Tempst P, Kirchhausen T, Rothman JE, S6llner T: A rab protein is required for the assembly of SNARE complexes in the docking of transport vesicles. Cell 1994, 78:937-948.

77.

Lupashin W, Waters MG: t-SNARE activation through transient interaction with a Rab-like GTPase. Science 276:1255-l 258.

61.

Yamaguchi T, Shirataki H, Kishida S, Miyazaki M, Nishikawa J, Wada K, Numata S, Kaibuchi K, Takai Y: Two functionally different domains of rabphillin-3A. rab3A p25/smg p25Abinding and phospholipid- and Cal+-binding domains. J Biol Chem 1993, 268:27164-27170.

62.

Matsumoto N, Sasaki T, Tahara M, Mammoto A, lkebuchi Y, Tasaka K, Tokunaga M, Takai Y, Miyake A: Involvement of rabphilin-3A in c&tical granule eiocytosis in mouse eggs. J Cell Biol 1996, 135:1741-l 747.

63.

Mizoguchi A, Yano Y, Hamaguchi H, Yanagida H, Ide C, Zahraoui A, Shirataki H, Sasaki T, Takai Y: Localization of rabphillin-3A on the synaptic vesicle. Biochem Biophys f?es Commun 1994, 202:1235-l 243.

64.

Li C, Takei K, Geppert M, Daniel1 L, Stenius K, Chapman ER, Jahn R. DeCamilli R Sudhof TC: Synaptic taraeting of rabphilin3A, a iynaptic vesicle Ca2+/phosbhdlipid-biding protein, depends on RabSA/C. Neuron 1994,13:885-898.

65.

Stahl B. Chou JH. Li C. Sudhof TC. Jahn R: Rab3 reversiblv recruit; rabphilin to &aptic vesicles by a mechanism ’ analogous to raf recruitment by ras. EMBO J 1996, 15:1799-l 809.

66.

Shirataki H, Yamamoto T, Hagi S, Miura H, Oishi H, Jin-no Y, Senbonmatsu T, Takai Y: Rabphilin-3A is associated with synaptic vesicles through a vesicle protein in a manner independent of Rab3A. J Biol Chem 1994,269:32717-32720.

67.

Kato M, Saaaki T, Ohya T, Nakanishi H, Nishioka H, lmamura M, Takai Y: Physical and functional interaction of rabphilin-3A with a-actinin. J Biol Chem 1996, 271:31775-31778.

78.

1997,

Valentiin JA. Senauota D. Gumkowski FD. Tana LH. Konieczko EM. Jamiesbn Jb: Rai3b lo&es to secret&y g;anuies in rat pancreatic acinar cells. Eur J Cell Biol 1996, 70:33-41.