Rab GTPases in vesicular transport

Rab GTPases in vesicular Marino European

Zerial and Harald

Molecular

Biology

Laboratory,

transport Stenmark

Heidelberg,

Germany

Specificity and directionality are two features shared by the numerous steps of membrane transport that connect cellular organelles. By shuttling between specific membrane compartments and the cytoplasm, small GTPases of the Rab family appear to regulate membrane traffic in a cyclical manner. The restriction of certain Rab proteins to differentiated cell types supports a role for these GTPases in defining the specificity of membrane trafficking. Current

Opinion

in Cell Biology

Introduction

Eukaryotic cells are divided into distinct membranebound organelles. While this functional compartmentalization has the advantage of both efficiency and regulation, it also requires controlled interactions between the various organelles. Exchange of constituents between organefles is thought to occur through vesicular or tubular intermediates that selectively deliver proteins and lipids from one compartment to another [ 10-3.1. Specific molecules control this complex trafficking network. Over the past few years, a large number of small GTPases of the Rab family have been described and localized to various intracellular compartments. By alternating between two distinct conformations in a nucleotide-dependent fashion, these proteins function as molecular switches. Through this mechanism they have been proposed to ensure fidelity in the process of docking and/or fusion of vesicles with their correct acceptor compartment [4]. The cycle of GTP binding and hydrolysis would allow multiple rounds of vesicular transport and confer vectorial@. This raiew focuses on the involvement of Rab proteins in individual trafficking routes between intracellular compartments.

Function

of Rab proteins

in membrane

traffic

The role of Rab GTRases in membrane traffic was first demonstrated by studies on Sec4p and Yptlp, which function in the exocytic pathway of Saccbaromyces cere ~r&ae [2*]. Rab proteins, the mammalian counterparts of yeast Sec4p and Yptlp [ 51, are localized to distinct intracellular compartments (Table 1) and a combination of in vitro and in vivo studies has emphasized their role in the regulation of different exocytic and endocytic transport processes.

1993, 5~613-620

In the exocytic pathway, in vitro studies have shown that the Rablb protein is involved in endoplasmic reticulum (ER) to Golgi transport [6] and work in intact cells has indicated that this transport process requires also Rabla and Rab2 proteins [ 71. The participation of three different Rab proteins in ERGolgi transport might reflect sequential steps of vesicular transport or, alternatively, the concerted function of several Rab GTPases at the same step. Rab proteins also function in regulated secretion. Synthetic peptides corresponding to the effector region of Rab3a stimulate regulated exocytosis [S-lo]. In the endocytic pathway, both Rab4 and Rab5 proteins are associated with early endosomes (Rab5 is also on the plasma membrane) [ 11,121. However, they have distinct activities. An increase in the level of Rab5 stimulates endocytosis and expands the size of the early endosomes, whereas expression of a Rab5 GTP-binding defective mutant has the opposite effect [13*]. These morphological alterations are consistent with a role of Rab5 in the fusion between early endosomes in vitro [ 141. In contrast, overexpression of Rab4 increases the re&x of endocytic markers to the cell surface and induces the accumulation of endocytic tubules and tubule clusters that could correspond either to structurally altered early endosomes or even to a distinct recycling compartment [IS*]. Another example of distinct function of Rab proteins in the same organelle is provided by Rab7 and Rab9. Both proteins have been local@ed to late endosomes [12,16], but only Rab9 spectically functions in transport from this compartment to the tram&olgi network (TGN) in vitro [16]. While the role of mammalian Rab7 remains unknown, functional studies on a structural homologue in S. cerevzs&e, Ypt7p, suggest that this protein may function at a late step of the endocytic pathway [17’]. Ypt7 null mutants are viable and exhibit normal exocytosis and u-factor internalization, but a-factor degradation is inhibited and the vacuole is fragmented. By analogy

Abbreviations CHM-choroideremia; CDL-GDP

DC&-dominant suppressor of Set+ ER-endoplasmic reticulum; CAP-GTPase activating dissociation inhibitor; W-mammalian suppressor of Sec4; NSF-N-ethylmaleimide-sensitive fusion SNAP-soluble NSF attachment protein; SNARE-SNAP receptor; TCN-trans-Colgi network.

@ Current

Biology

Ltd ISSN 0955+674

protein; protein;

613

614

Membranes

.

Table 1. Summary and

of the cells.

mammalian

Protein

Localization

Yptl (yeast) Sec4

ER-Golgi

localization,

functional

ER-Golgi ER-Golgi intermediate compartment Synaptic vesicles Chromaffin granules

Rab3a

RaMa

Early

Rab5a

Rab7 Rab8

Rab17

Apical basolateral

‘Regulatory most

clathrin-

Late endosomes Post-Golgi basolateral secretory vesicles Late endosomes, TGN

Rab9

factors

Rab proteins.

active

of Yptl,

on different

Sec4

and

Interacting

DSSC,

Rab proteins

in yeast

components

MSSC

Rabphilin-3A

Not determined Golgi-plasma membrane transport Transport from late endosomes to TGN Not determined

dense tubules, plasma membrane

See text

components

Regulated exocytosis in pancreatic acinar cells, adrenal chromaffin cells and mast cells Early endosomes-plasma membrane recycling pathway Plasma membrane-early endosome transport and homotypic fusion between early endosomes Not determined Transport in endocytic pathway

endosomes

Plasma membrane, coated vesicles, early endosomes Middle Colgi-TGN Not determined

Rab6 Ypt7

interacting

Fusion of post-ER vesicles with plasma membrane Fusion of post-Golgi vesicles with plasma membrane ER-Golgi transport in yeast and mammalian cells ER-cis-Colgi and c&medial Colgi transport ER-Goigi transport

dabla

Rab2

and

Function

Secretory vesiclesplasma membrane

Rabl b

properties

Rab proteins.

Rab GDI

is not

listed

among

the

interacting

components,

as it interacts

with

for references.

with mammalian endocytic pathway organization and localization of Rab7 to late endosomes, Ypt7p is thought to control transport between early and late endosomal compartments in yeast [ 17’1. This interpretation is consistent with the existence of kinetically and biochemically distinct endqtic organelles in yeast [ 181. Certainly, these observations suggest that the yeast endocytic machinery shares features with the mammalian endocytic apparatus. A vesicle transport process can be typically divided into several steps: budding, uncoating, docking and fusion [1~3*]. While Rab proteins are implicated in the regulation of distinct membrane traffic events, the exact step controlled by each protein is not clear. Both Sedp and Rab5 appear to control vesicle docking/fusion. However, while yeast Yptlp is thought to function in the same step [2*], its mammalian-related protein, Rablb, is thought to be essential for vesicle formation [ 61. The interpretation of in vivo studies is complicated by the fact that transport between compartments may occur in both directions, forwards and backwards, and affecting one direction is likely to influence the other. Overexpression

of Rab5 stimulates endocytosis but also accelerates recycling from the early endosomes to the plasma membrane [WI. Likewise, inhibition of retrograde transport could, in principle, indirectly affect anterograde movement between the ER and the Golgi, and vice versa Clearly, these studies have shown that changes in the balance between vesicles coming from and returning to a compartment have profound effects on organelle organization. Similar effects have also been observed with other components of the traf3cking machinery. For example, the sbi6ire mutation in a dynamin-related protein of Drosophila mehnogaster is thought to block coated vesicle budding from the plasma membrane [W-21 1. This causes an increase in its surface area, an accumulation of elongated coated pits and a decrease in the number of endocytic tubular elements [ 191. Thus, similar to the overexpression of Rab4 and Rab5, the mutation induces drastic morphological changes. Altogether, these biochemical and morphological studies only represent a first step towards a more detailed dissection of Rab protein function.

Rab CTPases in vesicular

Complexity

of the Rab protein

family

The large number of Rab GTPases identiiied in mammalian cells (30 different members) 122-241 led to the hypothesis that each step of vesicular traffic is regulated by at least one Rab protein [4]. Nevertheless, the implications of this complexity are not clear. First, the picture is complicated by the existence of subgroups of Rab proteins sharing high sequence identity (e.g. Rabla, Rablb, Rab3a, Rab3b, Rab3c, Rab3d). The high sequence conservation suggests that proteins belonging to one subgroup may have a similar function, as supported by the finding that both Rabla and Rablb regulate ER-Golgi transport [7]. However, this simple interpretation is countered by the fact that members of a subgroup may have different biochemical properties: both the exocytlc Rabla and the endocytlc RaMa proteins contain a phosphotylation site for p34u kinase [25,26*] whereas Rablb and Rab4b do not [27]. Anti-Rab5a specific antibodies block early endosome fusion in vitro, despite the presence of the Rab5b and Rab5c proteins [28]. Different members of the Rab3 subgroup carry out distinct functions (see below). Altogether, these differences question the idea that Rab isofonns are functionally redundant. Instead, the diversity might be responsible for hne tuning intracellular transport by the coordinated activity of several Rab isoforms. Second, recent morphological data indicate that several Rab proteins localize to the same organelle. Early endosomes provide a striking example: they contain at least three additional Rab proteins besides the Rab4 and Rab5 subgroups (VM Olkkonen, P Dupree, 1 Killisch, A Lutcke, M Zerial, et al. and A Liitcke, A Valencia, VM Olkkonen, G Griffiths, RG Patton, et al., unpublished data). What are the roles played by these different Rab proteins? One possibility is that they function in either of the transport routes that meet in this compartment: transport from and to the cell surface, and from the TGN and to late endosomes [28]. Alternatively, several distinct Rab proteins might be required in a given transport process. These proteins might carry out either the same function (e.g. vesicle docking) or different functions (e.g. budding, uncoating, docking or fusion).

transport

Zerial

and Stenmark

indicate that epithelial cells, which have distinct apical and basolateral transport pathways, and tmnscymtic routes &vkewed in [33,34]), also express specific Rab proteins once they become polarized. In the developing kidney, Rab17 is absent from the mesenchymal precursors but is induced upon their differentiation into epithelial cells. In the adult organ, the protein is associated with the basolateral plasma membrane and with apical tubules [35*]. Both expression and localization suggest that Rabl7 may function in transcytosis, an epithelial cell specilic transport process [ 331. Rab8, a ubiquitously expressed protein sharing high sequence homology with Sec4 [27], functions in tratfic from the TGN to the basolateral surface in polarized MDCK cells and to the somatodendrltic plasma membrane in hippocampal neurons [36,37]. These two domains are thought to correspond to tile plasma membrane of non-polarized cells [ 381. This suggests that Rab8 does not function in an epithelial cell speciIic pathway. In contrast, another as yet unidenti6ed GTP-binding protein is found on immuno-isolated apical exocytic vesicles and is implicated in apical exocytosis, the epithelial cell specific transport step (361. Collectively, these data suggest that specialized Rab proteins are part of the machinery regulating cell type specilic transport processes. The localization and function of the Rab proteins must depend on other cell type specific regulatory components, for example, factors that regulate their GTP-GDP cycle.

Regulatory

factors

for Rab proteins

Rab proteins

The cycle of GTP binding and hydrolysis of Rab proteins has been postulated to ensure directionality in the vesicle docking/fusion process [ 41. According to a common view [4,39], a speciiic Rab protein is recruited on the donor membrane or on nascent transport vesicles in its GTP-bound form. Hydrolysis of GTP by the Rab protein is thought to trigger fusion of the vesicle with the acceptor membrane [4,40*]. Subsequently, the GDP-bound Rab protein is released into the cytosol and returned to the donor membrane. There, a GDP-GTP exchange protein catalyzes GDP release and GTP binding and allows the cycle to continue.

The notion that Rab proteins control distinct intracellular transport processes suggests that cell type speciiic steps in membrane traific also require specific Rab proteins. Several Rab proteins that are cell type or tissue-specific have been identiIied. For instance, neurons, exocrine and endocrine cells, which display regulated secretion, speciIically express the Rab3a protein [ 29-311. The expression of cell type specific Rab proteins is regulated during differentiation. Expression of Rab3d increases during ditferentiation of 3T3-Ll cells into adipocytes. This protein might control the insulindependent exocytosis of vesicles containing glucose transporter in these specialized cells [32]. Recent data

Proteins that promote GDP-GTP exchange by members of the Rab protein family have been characterized in yeast and mammalian cells [41,42*,43m]. Do these factors regulate the activity of speciiic Rab proteins? In vitro, yeast Dss4 stimulates GDP dissociation of both Sec4p and Yptlp [42*]. Mammalian suppressor of Sec4, Mss4 protein, that acts on Sec4p, also stimulates GDP release from Yptlp and mammalian Rab3a [43’]. Thus, exchange proteins appear to be active on multiple Rab proteins. If Rab proteins associate with their respective compartments in the GTP-bound form [ 391, then exchange factors might function in association with organelle-specific components. In contrast, GTPase-activating proteins (GAPS) appear to be more selective as the recently identified GAP

Cell type specific

615

616

Membranes,

of Ypt6p (GYP6) does not act efficiently 0; other Ypt proteins [44*]. Association of Rab proteins with membranes requires gemnylgeranyl groups on the carboxyl-terminal cysteine motif [45,46]. This process is reversed by a cytosolic factor, Rab GDI, originally puriiied as a GDP dissociation inhllitor for Rab3a 1471. Rab GDI is able to recognize several geranylgeranylated Rab proteins and extract them from membranes in vitro [48-521. These combined properties suggest that Rab GDI could function to remove Rab proteins from membranes after GTP hydrolysis and to return them to the donor membrane. As predicted from such a function, cytosolic Rab proteins are found in complex with Rab GDI [50-521 and a small but significant fraction of Rab GDI is associated with various organelles [50]. Thus, Rab GDI appears to be an essential component that ensures the cyclical and vectorial function of Rab proteins. Is the function of GDI regulated? Analysis of the quartet mutation in D. mekznogaster suggests that it might be. Mutations in the quartet locus are associated with pleiotropic developmental defects in the larvae and distinguished by the presence of a basic shift in the isoelectric point of three abundant proteins, one of which has been identihed as a Drosopbilu homologue of Rab GDI [ 531. This suggests that Drosophila Rab GDI is post-translationally mod&d or demodiiied by the wildtype quartet gene product. Although there is no evidence that quurtetencodes a kinase, it is interesting to note that the cytosolic form of mammalian GDI seems to be highly phosphorylated (J Gruenbetg, personal communication). Phosphorylation or other post-translational modifications of Rab GDI might be necessary to inhibit reassociation of Rab proteins with the target membrane and direct them to the donor compartment. Other components may also be involved in the Rab protein cycle. Geranylgeranylation of Rab proteins is catalyzed by geranylgeranyl transferase II, an enzyme that, in contmst to other known isoprenyl transferases [54], requires sequences amino-terminal to the target cysteine motif [55-571. This peculiarity may be due to a third enzyme subunit, component A [ 541. Perhaps component A recognizes Rab proteins and presents them to component B, the catalytic part of the transferase. Intriguingly, the gene products of human and mouse choroideremia (CI-IM), a disease associated with degeneration of the retina and the underlying vasculature, the choroid, share sequence homology with rat component A 158,591. Two additional iindings support the possibility that CHM could be a member of a family of A components. First, in cytosol of lymphoblasts from CHM patients, geranylgeranylation activity is reduced and isoprenylation of Rab3a is more a&c&d than that of Rabla [&I. Second, another CHM-like gene, CHML, has been identiiied [ 611. Interestingly, CHM shares sequence similarity with Rab GDI [62]. This sequence conservation might reflect the ability of both GDI and component A to interact with geranylgeranylated Rab proteins. However, unlike Rab GDI, component A can also recognize the non-prenylated form of Rab proteins, and it interacts

with both the GTP- and the GDP-bound forms [59’]. Therefore, it is thought that component A would be more likely to act as a chaperone for Rab proteins during the initial process of membrane association, than to remove F&b proteins from membranes. This function might be necessary to prevent interactions of the hydrophobic geranylgeranyl moiety with non-target membranes.

Rab proteins machinery

and the vesicle

transport

If Rab proteins are implicated in the regulation of vesicular transport, the real challenge now is to understand how they function. For this task it is important to study the relationship between Rab proteins and other elements of the transport machinery. Different vesicle fusion processes in mammalian cells and in yeast require N-ethylmaleimide-sensitive fusion protein (NSF) and the soluble NSF attachment proteins (SNAPS) [ 1*,2*]. More recently, a search for SNAP receptors (SNARES) has yielded four different factors from bovine brain, all associated with the synapse [63**,64*]. VAMP/synaptobrevin-2 is found on transport vesicles (vSNARE), while syntaxin A and B are on target membranes (t-SNARES). It is not clear where the fourth protein, SNAP-25 (synaptosome-associated protein of 25 kDa) is localized. These findings have led to the hypothesis that, in general, specificity in the process of vesicle fusion with its target compartment might be mediated by the interaction of a specific v-SNARE with its complementary t-SNARE through the NSF-SNAPS complex (Fig. 1). Where do Rab proteins fit into this scheme? The answer is not there yet. However, recent progress suggests possible connections between Rab proteins and the vesicle fusion apparatus. Rabphilin-$4, a protein that interacts with Rab3a, has recently been puriiied and characterized [65,66*]. Structural analysis of the deduced amino acid sequence from the corresponding cDNA revealed high homology with synaptotagmin, a synaptic vesicle integral membrane protein [67]. However, unlike synaptotagmin, rabphilin3A contains neither a signal peptide nor a putative transmembrane domain. The precise function of synaptotagmin is unknown, although it has been postulated to be a regulatory component of calcium-dependent exocytosis in the presynaptic terminal [67,68]. The identification of rabphilin3A provides a potential link between Pab proteins and t-SNARES.In fact, syntaxin (a t-SNARE) can be co-immunoprecipitated with synaptotagmin suggesting an association of the two proteins [67], and rabphilin3A is a synaptotagmin-like molecule. In addition, the SLK’Z/SEC22 and SLY12IBETl genes, which are multicopy suppressors of functional loss of YPTl in S. cerevtie, encode two members of the synaptobrevin (v-SNARE) family [69-711. Therefore, it is possible to envisage interactions between a rabphilin and a t-SNARE and between a Rab protein and a v-SNARE (Fig. 1). The speciiicity of interactions between V-SNARESand t-SNARESin a complex with NSF and SNAPSmight thus require further regulation by a Rab protein. This regula-

Rab

(a) z

GTPases

in vesicular

transport

Zerial

and

Stenmark

617

lated by GAPS and exchange proteins) control the interactions between Rab proteins and their target molecules? The synaptotagmin-like protein rabphilin-3A is certainly an interesting candidate for a Rab target [66*]. Complicated molecular machineries are often regulated by a number of different GTPases, and Rab proteins are not the only GTPases implicated in intracellular transport. Compelling evidence indicates that members of other subfamilies of GTPases function in regulating different aspects of membrane traffic, such as vesicle budding and vesicle coat assembly and disassembly [28,39]. Future studies will reveal whether there are links between Rab proteins and these other GTPases. The identification of several tissue- and cell type specific Rab proteins suggests that the complexity of this fanily of GTPases could still increase, when one considers the high heterogeneity of cell types from different organs. Learning more about the distribution of Rab proteins in various cell types will contribute to understanding the relationship between structural features of organelles and their specialized functions, and between sub-compartments and transport routes.

Fig. 1. Model for possible interactions between Rab proteins and known components of the membrane traffic machinery. Proper docking and fusion of a vesicle with its correct target membrane depends on interactions between specific components present on both compartments. (a) NSF and SNAPS interact with vSNARE- and t-SNARE-type molecules 1671. (b) interaction between the vesicle-specific v-SNARE and its target-specific t-SNARE is still hypothetical. Cc) The vesicle-specific, CTP-bound Rab protein interacts with its corresponding rabphilin, and Cd) possibly, to a vSNARE. (e) Rabphilin might interact with a t-SNARE as suggested by the interaction of syntaxin with synaptotagmin [671. In this chain of connections, the CTPase activity of Rab proteins could be utilized to proof-read I41 the interaction between the correct pair of SNARES.

tory mechanism might be used to proof-read the correct interactions between these components. As rabphilin3A can only recognize the GTP-bound form of Rab3A [66*], GTP hydrolysis would confer unidirectional@ to the docking/fusion process.

Conclusions A comprehensive analysis of the molecules implicated in membrane trticking is beginning to establish which components are common to many transport processes, and which are specific. The data obtained so far suggest that, whereas NSF and SNAPS belong to the first cathegory, Rab proteins belong to the latter one. Although a number of studies have demonstrated the role of these GTPases as regulators of membrane traffic, their precise function remains unknown. Proteins that regulate the membrane association and nucleotide state of Rab proteins have now been identified, and further mechanistic studies will undoubtedly focus on three key questions. First, what brings about the organelle-specific localization of Rab proteins? Second, which are their target molecules? Third, how does the nucleotide state (regu-

Acknowledgements We thank

Jean

phy, Rob Parton, suggestions.

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Whereas normal Chinese hamster ovaq cells contained only 20% of their transferring receptors on their surface, this number was raised to 80 % In stable transfectants overexpressing Rab4. Overexpression also prevented delivery of intemalized transferrin to acidic endosomes, and a large fraction of internalized transferrin was associated with small, tubular dusters incapable qf efficient acidI6cation. Overexpression of Rab4 did not affect initial rates of transfenin internalization, but the rate of transferrin recycling and a fluid phase marker to the plasma membrane were increased. The results suggest that Rab4 controls the rate of recycling, possibly by controlling the rate of transport between the early endosome and a putative recycling compartment. 16. ~MBARDI D, SOIDATI T, R~EDERERw GODA Y, ZERVU.M, PF’EFFERSR: Rab9 Functions in Transport Between Late Endosomes and the Trans Golgi Network. EMBO J 1993, 12:677-682. 17. WKHMANN H, HENGST L, GALLWITZD: Endocytosis in Yeast: . Evidence for the InvoIvement of a SmslI GTP-Binding Protein (Ypt7p). Cell 1992, 71:1131-1142. A S. cerevtiegene encoding Ypt7, a protein with 63 % sequence identity to mammalian Rab7, was cloned. Ypt7 and gab7 have identical effector regions. Gene disruption of Ypt7 had essentially no effect on the growth properties of yeast ceUs at various temperatures. However, the Ypt7 null mutants were characterized by a highly fragmented vacuole. while uptake of the pheromone alpha factor occurred at a normal rate, its degradation was severely inhibited in the null mutants. 18.

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Rab CTPases in vesicular Cloning of a mouSe cDNA for a novel Rab protein, Rabl7, is reported. Northern blot analysis revealed that Rab17 is expressed exclusively in polarized epitheiial cells and is thus the first example of an epitheiiai ceii speciUc Rab protein. In situ hybridization on murine developing kidneys showed that rabl7 expression was turned on concomittantIy with the onset of epitheiiai differentiation. In the adult kidney, Rab17 was found to be associated with the basolaterai plasma membrane and with apical tubules. Localization data suggest that Rabl7 might be involved in reguiating transcytosis. 36.

HUBER IA, PIMPUKAR S, PARTON RG, VIRTA H, ZERIAL M, SIMONS K: Rab8, a SmaU GTpase Involved In Vesicular Traffic be-

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42. .

BU~~TEINES, MAC~RAIG: Characterization of a Guanlne Nucleotide Releasing Factor and GTPase Activating Protein that Are Specitic for the Ras-Related Rotein p25 Rab3a Proc Nat1 Acad Sci USA 1992, 89:1154-1158. MOYA

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SOIDATI T, REIDERERMA, PFEFFERSR Rab GDI: a Solubiliaing and Recycling Factor for Rab9 Protein. Mel Bid Cell 1993, 4:425434.

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SEABRA MC, GOLDSTEINJI, SUDHOF TC, BROWN MS: Rab Geranylgeranyl Transferase. A Multisubunit Enzyme that Prenylates GTP-Binding Proteins Terminating in Cys-X-Cys or Cys-Cys. J Bid Cbem 1992, 26714497-14503.

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P: A Mammalian Guanine-Nucleotide Releasing Protein Enhances Function of Yeast Secretory Protein Sec4. Nature 1993, 361:464-467. A rat-brain cDNA library was expressed in temperature-sensitive se&8 yeast ceUs,and screened for suppression of the growth defect at the restrictive temperature. A temperature-resistant clone was recovered, and the mss4 cDNA was isolated. The Mss4 protein has a molecular mass of 14kDa and has sequence homology with Dss4-lp [41]. Ms.&p stimulated GDP release from Sec4p, and to a lesser extent from Rab3a and Yptl, but was inactive on Ras2. The mss4 gene is expressed in a variety of tissues, suggesting that its natural target is one or more widely expressed Sec4-iike proteins. .

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Yeast cells were transformed with a yeast genomic library on a multicopy plasmid, and extracts from the individual transformants wem assayed for their ability to stimulate the GTPase activity of Ypd. A single, positive colony was isolated, and from this the Cl?6 (GAP of Ypt6) gene was cloned. The gene product, Gyp6, has no sign&ant homology to other known proteins, Including other GTPase-activating proteins. Purified Gyp6 strongly stimuiated the GTPase activity of Ypt6, and had a weak activity on Ypt7. It did not stimulate the GTPase activ ity of Yptl, Sec4, YpUA, H-ras or Rab2. Whereas @tideletion mutants are temperature-sensitive, disruption of gyp6resulted in no phenotypic alterations.

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Cit!ZMERS FPM, VAN DE POL DJR, VAN KERXHOFFLPM, WtElUNGA B, ROPERS H-H: Cloning of a Gene that Is Rearranged In Patients with Choroideremia Nuhtre 1990, 347~674-677.

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SEABRA MC, BROWN MS, SIAUGHTER

(2% SUDHOF TC,

JL

A of Rab

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of

Component

of J Cd

GOID!%IN

GeranyIgeranyI

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Membranes Transferase: Possible Identitv with the Choroideremia Gene Product. Cell 1992, 70:104P-1057. Partial peptide sequences of purllied rat component A of geranylgetanyl transferase II showed high similarity to the human and mouse CHM gene products. 60. SEABRAMC, BROW MS, GOIDSTEINJL: Retinal Degeneration . in Choroideremia: Deficiency of Rab Geranylgeranyl Transfemse.

Science

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SNAPS.a- and Y-SNAPS are ubiquitously expressed and act synerglstially, whereas B-SNAP is bralnspeclftc. B-SNAP competes for the same biding site on SNAP receptors as a-SNAP, but unlike a-SNAP it cannot restore cell-free Golgl transport in cytosol from secl7 mutant yeast. It is most abundant in areas of high neuron density and may be involved in neurosecretion. 65.

259:377+X.

Cytosol prepared from lymphoblasts of patients suiTering from the Xlinked eye disease CHM exhibited reduced ability to geranylgeranylate RabIa and Rab3a in c&n The ability to geranylgeranylate Rab3a was more alfected than that of Rabla The results are consistent with the idea that the CHM gene product may be member of a family of A components of Rab getanylgeranyl transferases. 61.

CREMERsFPM, MOUOY CM, VAN DE POL DJR, VAN DEN HURK JAJM, BACH I, GEURT~VAN KERNELAHM, ROPERSH-H: An Autosomal Homologue of the Choroideremia Gene Colocalizes with the Usher Syndrome ‘Qpe II Locus on the Distal Part of Chromosome lq. Hum Mol Gene1 1992, 1:71-75.

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SOMR T, WHI?EHEARTSW, BRUNNERM, ERDJUMEN~-BROMAGE H, GEROMANOS S, TEMPST P, ROTHMANJE: SNAP Receptors Implicated in Vesicle Targeting and Fusion. Nahtre 1993, 362:318324. SNAPSare soluble receptors for the general fusion protein NSF. In this paper, SNAP receptors (SNARES) from bovine brain were isolated by the use of al??nity columns containing NSF/SNAP complexes. Remarkably, of the four SNARESidentified in this manner, all are proteins that have been previously found to be associated with the synapse. One of them, VAMP/synaptobrevln 2, is a synaptic vesicle protein, whereas two others, syntaxin A and syntaxln B, are found on the synaptic plasma membrane. The fourth SNARE, SNAP-25, is also thought to be associated with synaptic vesicles or plasma membrane. The results thus indicate that SNAP/NSF can intemct with SNARESin the vesicular membrane (V-SNARES)as well as with SNARESin the target membrane (t.SNARFs). Whereas NSF and SNAPS appear to be universal components of the vesicle fusion apparatus, it is possible that families of V-SNARESand tSNARESensure speciiic interactions between vesicles and their target membranes. 64. Ww SW, Gatrr IC, BRUNNERM, CIAR( DO, MAYERT, . B~JHROWSA, ROTHMANJE: SNAP Family of NSF Attachment FToteins Includes a Brain-Specific Isoform. Nufure 1993, 63.

..

SH~RATAJCI H, KA~~UCHIK, YAMAGUCHIT, WA~A K, HO~UUCHI H, TAKAI Y: A Possible Target Protein for smg p25A/Rab3A Protein. J Biol Ckm 1992, Small GTP-Biding 267:10946-10949.

SHIRATAKIH, KAIBUCHI K, SAKODA T, KISHIDA S, YAMAGUCHI . T, WADA K, MNM, TAKA~ Y: RabphiIin-3A, a Putative Target Protein for Smg p25A/Rab3A ~25 Small GTP-Biiding Protein Related to Synaptotagmin. Mol Cell Biol 1993, 13:2061-2068. This paper reports the cloning of a cDNA encoding tabphilin3A a protein isolated from bovine brain crude membranes by chemical crosslinking to Rab3a. The carboxyl-terminal part of rabphilln3A shows significant sequence homology to the synaptic vesicle protein synaptotagmln, and contains two Cz domains like those found in protein klnase C and synaptotagmin. Unlike synaptotagmin, rabphilin3A does not contain any putative transmembrane region. Rabphliin3A was found to form a complex with the GTPyS form of Rab3a, but not with Rab3a-GDP and not with the GTPyS-form of Rabll. 66.

67.

BENNET MK, SCHELUR RI-I: The Molecular Machinery for Secretion Is Conserved from Yeast to Neurons. Proc Nat1 Acud

Sci USA

1993, 90:25592563.

68.

SHOJ~-KAQIY, YOSH~DAA SATO K, HOSHINO T, OGURA A, KONDO S, FUJIMOTOY, Kuw~u~a~ R KATO R, T~HI M: Neurotransmitter Release from Synaptotagmin-Deficient Clonal Variants of PC12 CelIs. Science 1992, 256:182&1823.

69.

NEWMANAP, SHIMJ, FERRO-NOVICK S: BETf, BOSI, and SEC22 are Members of a Group of Interacting Yeast Genes Required for Transport from the Endoplasmic Reticulum to the Golgl Cotnlex. Mof Cell Biol 1990, 10:3405-3414.

70.

DASCHERC, Osstc R G~uwrtz D, SCHM~I-~HD: Identiiication and Structure of Four Yeast Genes (SLY) that Are Able to Suppress the Functional Loss of KPTI, a Member of the RAS Supedamily. Mol Cell Biol 1991, 11:872+385.

71.

NEWMANAP, GIW J, MANctm P. ROSSI G, UAN JP, FEattoNO~~CKS: SEC22 and SLY2 are Identical. Mol Cell Biol 1992, 12:36633664.

362:353355.

This paper reports the cloning and sequencing of cDNAs encoding a., g. and ~-SNAPS. a- and B-SNAPSwere found to be 83% identical to each other, whereas -+SNAP showed about 25% identity to the other

M Zerial and H Stenmark, European laboratoty of Molecular Biology, Postfach 10.229, Meyerhofstrasse 1,6900 Heidelberg, Germany.