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466

The role of ARF and Rab GTPases in membrane transport Philippe Chavrier* and Bruno Goudt

Two key events of intracellular transport and membrane

trafficking in eukaryotic cells, the formation of transport

vesicles and their specific delivery to target membranes, are

controlled by small GTPases of the ADP-ribosylation factor

(ARF) and Rab families, respectively. The past 18 months have

seen the identification of proteins that regulate ARF and Rab

GDP/GTP cycle, as well as the characterization of their

effecters, shedding light on the molecular mechanisms of ARF

and Rab function.

Addresses *Centre d’lmmunologie INSERMlCNRS de Marseille-Luminy, Case 906, 13288 Marseille Cedex 9, France; e-mail: [email protected] tlaboratoire ‘Mecanismes moleculaires du transport intracellulaire: lnstitut Curie, CNRS UMR 144, 26 rue d’Ulm, 75248 Paris Cedex 5, France; e-mail: [email protected]

Current Opinion in Cell Biology 1999, 11:466-475

http://biomednet.comlelecref/0955067401100466

0 Elsevier Science Ltd ISSN 0955-0674

Abbreviations ARF ADP ribosylation factor ARNO ARF nucleotide binding site opener BFA brefeldin A ER endoplasmic reticulum GAP GTPase-activating protein GDF GDI-displacement factor GDI GDP-dissociation inhibitor GEF guanine nucleotide exchange factor myrARF1 myristoylated ARFl NSF N-ethylmaleimide sensitive fusion factor PA phosphatidic acid PI3-kinase phosphoinositide 3’-kinase PIP, phosphatidylinositol 4,5-bisphosphate PIP, phosphatidylinositol 3,4,5-triphosphate PLD phospholipase D SNARE soluble NSF-attachment protein receptor

Introduction In eukaryotic cells, the vectorial transport of macromole- cules between two compartments and homeostasis of these compartments depend on a bi-directional flux of vesicles or of vesiculo-tubular structures. From each organelle, specific mechanisms allow selective packaging into vesicles of proteins and lipids (‘cargo’) that are destined for another compartment. These vesicles can then specifically recog- nize the membrane of the acceptor compartment and fuse with it to deliver their cargos.

Both formation and targeting/docking of vesicles are based on a complex network of interactions between regulatory molecules and mechanical components. The cargos destined to leave a compartment are incorporated into vesicles via an interaction, direct or indirect, with cytoplasmic proteins, which assemble on the membrane of the donor organelle to form a coat. Coat assembly promotes a change in shape of the donor membrane compartment and induces

the formation of transport vesicles. The targeting, docking and fusion of vesicles with the acceptor membranes require an interaction with the actin and microtubule cytoskeleton, recruitment of cytosolic docking complexes and specific recognition between integral membrane proteins known as v- and t-SNARES (soluble NSF attachment protein receptors). Within the past ten years, small GTPases of the ARF and Rab family have emerged as master regulators of these events.

ADP-ribosylation factor proteins ADP-ribosylation factors (ARFs) form a group of six, small (20 kDa), ubiquitous GTP-binding proteins, related to Ras, which are required for maintaining the integrity of organelle structure and intracellular transport. ARFl, the best-characterized ARF, is localized to the Golgi complex and is a common regulator of nonclathrin (coatomer for COP1 vesicles) and clathrin (AP-1 and AP-3 adaptor complexes) coat recruitment [l-3]. A number of studies have led to a model for the mechanism of ARFl activation and function in coat recruitment (for reviews see [ 1,2]). GDP-bound ARF 1 is inactive in the cytosol. As for all Ras-like proteins, spontaneous GDP release from ARF (a prerequisite for GTP binding) is very slow and must be catalyzed by a guanine nucleotide exchange factor (GEF). In addition, in the case of ARFl the GDP to GTP transition induces a conformational change, which allows the amino-terminal myristoylated amphipathic helix of ARFl to interact with the lipid bilayer ([4] and references therein). Consequently, the location of the GEF is thought to be a determinant for the definition of the appropriate membrane sites of ARFl activation. Membrane-bound ARFl*GTP then triggers the recruitment of the coat protein complex to the donor compartment. Assembly of coat subunits at the membrane deforms the donor membrane and captures cargo molecules in the forming vesicle. A vesicle-associated GTPase-activating protein (GAP) stimulates the hydrolysis of bound GTP to release ARFl*GTP and to trigger coat dissociation prior to vesicle fusion.

Guanine nucleotide exchange factors A genetic approach in Sacchammyces cermisiae has been used to clone two related ARF GEF genes, called GEAl and GEAZ [S]. ARF nucleotide binding site opener (ARNO), a human ARFl GEF has been discovered based on sequence conservation with GEAl [6] and several ARF GEFs have now been cloned (Figure 1). They all share a domain of approximately 200 amino acids, termed the Sec7 domain, which is sufficient for guanine nucleotide exchange activity [6]. This domain is conserved in Sec7p, a yeast ARF1/2 GEF [7] that was initially identified as a protein required for transport at different stages of the yeast secretory pathway.

The role of ARF and Rab GTPases in membrane transport Chavrier and Goud 467

Figure 1

MW (kDa) Substrate BFA

Sec7

p200

Geal

ARNO

Cytohesin-1

GRPl/ARNOS

EFA6

230 yARF1, yARF2 +

207 ARFl, ARF3 +

160 ARFl +

47 ARFl,ARFG -

47 ARF1,3 -

47 ARFl -

77 ARFG -

Current Opinion in Cell Biology

The Sec7 domain ARF GEF family. Each factor has a conserved Sec7 domain (light grey), which is responsible for guanine nucleotide

BFA-resistant). The percentage identity of the Sec7 domain of each factor with the domain of yeast Sec7p is indicated. Dark grey boxes

exchange. The sensitivity of the different guanine nucleotide exchange factors to brefeldin A (BFA) is indicated (+, BFA-sensitive; -,

indicate PH domains. Black boxes indicate regions with predicted coiled-coil structure.

ARNO is a soluble 47 kDa protein that contains, in addition to a central Sec7 catalytic domain, a predicted amino-terminal coiled-coil region and a carboxy-terminal pleckstrin homology (PH) domain [6]. Two highly related proteins (85% identity at the amino acid level) with the same structural organization have also been identified, and are called Cytohesin-1 [B] and ARNOS (GRP-1) [9,10]. ARNO was initially characterized as a GEF acting on ARFl and optimal activity was observed using GDP- bound myristoylated ARFl (myrARF1) as a substrate in the presence of negatively charged phospholipid vesicles supplemented with phosphatidylinositol 4,5-bisphosphate (PIP,) [6]. By binding to the PH domain, PIP,, might attract ARNO to specialized membrane subdomains, where it interacts with GDP-bound myrARF1, which has low affinity for lipids [4].

The crystal structure of the Sec7 domain of ARNO has been determined by two groups and reveals a helical domain with a hydrophobic groove [ll’,lZ’]. The edges of this groove are formed by two short sequences of amino acids, which are conserved in all Sec7 domains identified so far. Site-directed mutagenesis [13’] and determination of the three-dimen- sional structure of a complex between nucleotide-free A17ARFl (a mutant lacking the amino-terminal myristoylated helix) and the Sec7 domain of yeast GeaZp [14”] show that the switch 1 and switch 2 regions of ARFl penetrate the groove. How is GDP released from the nucleotide-binding pocket? A glutamate residue (Glu156) located at the edge of the ARNO Sec7-domain groove appears critical for the exchange reaction [13’]. The long glutamate carboxylic sidechain group exerts a strong steric and repulsive effect on bound GDP that expels GDP from the nucleotide-binding site [14”]. Interestingly, this glutamate residue is conserved in all Sec7 domains, indicating a common mechanism for all Sec7-domain-containing GEFs. Comparison of the crystal structures of ARFl bound to GDP or to the nonhydrolysable

GTP analog GppNHp reveals that during the GDP to GTP transition - and as already described in the case of Ras - the two switch regions adopt a conformation allowing effector recognition. But, unique to ARFl, the GDP/GTP transition induces changes that are propagated to the myristoylated amino terminus, which becomes exposed for membrane binding [ 14”].

The Sec7-domain-containing GEFs identified so far can be separated into two classes depending on their sensitivity to brefeldin A (BFA) (Figure 1). BFA is a fungal metabolite that blocks protein secretion and severely per- turbs the structure of the Golgi complex by inhibiting a Golgi-associated GEF activity specific for ARFl [Z]. ARNO-like GEFs are BFA-insensitive [6,8,10], whereas the exchange activity of a group of large (-200 kDa) ARF GEFs - including yeast Sec7p [7] and Gealp [S] as well as ~200 (a bovine homologue of yeast Sec7p) [15] - is inhibited by BFA. Two groups have recently identified specific residues within the Sec7 domain that confer BFA- sensitivity to Gea/Sec7 GEFs [16’,17”]. These residues map to a region overlapping with the ARF binding site and are not conserved in the Sec7 domain of ARNO family GEFs, providing a molecular basis for the specificity of BFA. Unexpectedly, BFA does not compete with ARF for binding to BFA-sensitive Sec7 domains but rather acts by stabilizing an abortive ARF.GDP-Sec7 domain complex [17”]. These findings, together with the fact that Geal/Zp and Sec7p are required for secretion in yeast, support the idea that member(s) of the Gea/Sec7 family might mediate the profound effect of BFA on Golgi structure and function. The situation might not be this simple, however, as overexpression of ARNO and ARNOS in mammalian cells induces disassembly of the Golgi complex and inhibition of secretion [lO,lB]. This indicates that BFA-insensitive ARNO-related GEFs might also regulate ARFl activity in mammalian cells.

468 Membranes and sorting

Figure 2

Lumen

t Vesicle formation

Current Op1n10n in Cell &dog:

A model for how ARFl might trigger COPI coat assembly. ARFl *GDP,

which has a low affinity for membrane phospholipids through its myristoyl group (zigzag), binds to Golgi membranes and interacts with a

membrane-associated Sec7-domain-containing GEF that promotes

nucleotide exchange. The conformational changes induced during the GDP+GTP transition triggers the exposure of the amphipatic amino- terminal helix (hexagons) of ARFl *GTP which becomes stably

associated with the lipid bilayer. Coatomer is recruited and assembled

through a bivalent interaction with ARFI .GTP and the cytoplasmic tail of membrane cargo receptors and the membrane deforms into a coated

bud. An ARF.GAP protein is recruited during this process by its interaction with a membrane cargo protein. ARF*GAP, together with the

coatomer, stimulates hydrolysis of GTP to release ARFl -GDP which can recycle for another round. Pi, inorganic phosphate.

ARF, coat and beyond The entire process of Golgi vesicle budding can be repro- duced with pure components in vitro [l]. The simplest system consists of coatomer and GTP-bound ARFl added to liposomes of defined composition that support the formation of small coated vesicles (40-70 nm diameter) [ 19”]. These data provide direct confirmation that ARFl.GTP and coatomer are sufficient to pinch off vesicles [ 11. In addition, acidic phospholipids, such as phosphatidic acid (PA),

are not sufficient to trigger coat protein recruitment but might promote coatomer binding in the presence of ARFl.GTP [19”]. These results demonstrate a direct role for ARFl in coatomer recruitment to membranes and refute the hypothesis that ARFl acts as an activator of phospholipase D (PLD)-mediated PA production, which was proposed to initiate coat protein binding and vesicle formation [1,2]. Using a photo-cross-linking approach, Wieland and coworkers [ZO] have demonstrated that membrane bound ARFl.GTP serves as an adaptor for coatomer through an interaction with coatomer--subunit. When using liposomes of composition mimicking mammalian Golgi membranes, the cytoplasmic tail of a putative cargo receptor (containing the dibasic motif KKXX [Lys-Lys-X-X]) is required, in addition to ARFl.GTP, for coatomer binding and vesicle budding [Zl”]. These observations confirm that vesicle formation and cargo selection are coupled through a bivalent interaction of coatomer with ARFl.GTP and cytoplasmic tails of putative cargo receptor or membrane cargo proteins (Figure 2). Similarly, ARFl.GTP might be involved in interactions with transmembrane molecules required at later stages of transport, including vesicle-associated V-SNARES that participate in vesicle targeting. Experiments in Sheckman’s laboratory [ZZ] demonstrate that this indeed the case during the formation of endoplasmic reticulum (ER)-to-Golgi COP11 transport vesicles. The coat subunit Sec23p-Sec24p complex was shown to interact simultaneously with Sarlp.GTP (a close relative of ARFl) and Betlp (a v-SNARE) in a co- operative manner [ZZ].

Finally, either during or after completion of budding, GTP should be hydrolyzed in order to release ARFl.GTP (which can be recycled for another round) and to allow dissociation of the coat and vesicle fusion [Z]. As nucleotide exchange is catalyzed by GEFs, GTP hydrolysis is an enzyme-stimulated reaction requiring the activity of a Golgi-associated GAP specific for ARFl ( Figure 2; for review see [23]). Recent findings provide some new insights into how structural components of the vesicles -

that is the coatomer and a transmembrane cargo receptor - might control GTP hydrolysis by regulating ARFl-GAP activity. Aoe et nl’. [24] have shown that the KDEL receptor, ERDZ, which recognizes a Lys-Asp-Glu-Leu carboxy-terminal motif on certain soluble proteins destined for Golgi-to-ER retrieval, is able to recruit ARFl GAP to Golgi membranes and thereby to regulate ARFl inactivation. Moreover, Goldberg [25’*] has determined the crystal structure of GDP-bound ARFl complexed with the minimal GAP domain of ARFl GAP [ZS”]. Strikingly, unlike most GTP-binding proteins, the GAP binding site on ARFl does not overlap with its effector binding site, the region of the molecule that interacts with coatomer in the GTP-bound state (see [ZS**]). In addition, in contrast to previously characterized GAPS, ARFl GAP does not supply any catalytic residues into the active site. Thus, ARFl might be simultaneously engaged in a tripartite interaction with ARFl GAP and coatomer,


raising the possibility that coatomer might itself participate in the hydrolysis reaction by providing catalytic residues [ZS”]. Indeed, when coatomer is added to ARFl*GTP and ARFl GAP, GTP hydrolysis is increased by three orders of magnitude [Z”].

ARFG: a special case ARF6, the least conserved ARF protein, controls the integrity of peripheral membranes and appears to cycle between the plasma membrane and recycling endosomes depending on its nucleotide status ([26’] and references therein). Immuno-electron microscopic studies have shown that a mutant of ARF6 (ARF6Gln67+Leu) that is constitutively bound to GTP accumulates at the plasma membrane. In contrast, a mutant defective for GTP-binding (ARFbThrZ7+Asn), colocalizes with the transferrin receptor and cellubrevin on perinuclear recycling endosomes [26’] and inhibits recycling to the plasma membrane [27]. In addition to its effect on endosomal membrane recycling, ARF6 is unique among the six mammalian ARFs in its ability to stimulate cortical actin rearrangements [27,28]. Therefore, it has been postulated that ARF6 might link membrane traffic with the organization of the actin cytoskeleton [27,28]. This possibility is support- ed by the recent observation that ARF6 activation and subsequent morphological changes are required for cell spreading [29]. In adrenal chromaffin cells, ARF6 localizes to chromaffin granules and is implicated in regulated exocytosis, a process that also requires cortical actin reorganization [30]. Interestingly, ARF6 appears to be con- nected to Racl, a member of the Rho family of small Ras-related GTP-binding proteins that regulates membrane ruffling and lamellipodia formation [31]. ARF6 and Racl colocalize partially in the perinuclear endosomal compartment and the recycling of Racl to the plasma membrane requires ARF6 activity ([32’]; C D’Souza- Schorey, personal communication). In addition, GTP-bound ARF6 interacts with PORl, a protein identified initially as a Racl downstream effector of membrane ruffling [28]. Therefore, it appears that the definition of a balance of ARF6 and Racl activities is certainly an important parameter in adapting cell surface morphology to various external stimuli.

Frank et al. [33] have published recent observations indicating that in vitro, ARNO activates not only ARFl but also ARF6, implying that ARNO is promiscuous in its substrate specificity. In addition, ARNO and ARF6 have been shown to colocalize in actin-rich lamellipodia at the cell periphery but only upon treatment of cells with phorbol ester [34]. These observations have been taken as a sug- gestion that ARF6 activation depends on ARNO activity at the plasma membrane [33]. In support of this conclusion is the finding that, upon activation of cells with certain ago- nists, GEFs of the ARNO family are targeted to the plasma membrane via binding of the their PH domain to PIP, that is generated by activation of phosphoinositide 3’-kinase (PI3-kinase) [35,36]. Other in vitro studies have shown,

however, that ARNO and Cytohesin-1 activate ARFl but not ARF6 [10,37]. A novel GEF, termed EFA6 (exchange factor for ARF6), has been recently identified that contains Sec7 and PH domains (see Figure 1) [38’]. EFA6 stimulates GTP/GDP exchange on ARF6 in vitro, but not on ARFl. Overexpression studies have shown that EFA6 is associated with the plasma membrane and regulates endosomal recycling. In addition, EFA6 triggers the formation of actin-based membrane ruffles similar to the structures induced by ARFbGln67+Leu, the active form of ARF6. EFA6-mediated actin reorganization requires the activity of Racl. All these observations support a role for EFA6 in the activation of ARF6 at the plasma membrane.

Rab proteins Rab/Ypt proteins exist in all eukaryotic cells and form the largest branch of the Ras superfamily of GTPases. In Sac- charomyces cerevisiae, the genome of which has been fully sequenced, a total of 11 Ypt proteins has been identified [39]. In mammalian cells, over 30 Rabs (including isoforms) are known - Rab33 and Rab36 being the two most recent [40,41]. It is likely that more remain to be discovered, especially tissue or cell-type-specific proteins, as well as novel Rab isoforms.

Rab proteins are localized to the cytoplasmic face of all organelles involved in intracellular transport. It is now widely accepted that Rabs regulate discrete transport steps along the biosynthetic/secretory and endocytic pathways [42-44]. For example, Rabl, Rab2 and Rab6 act at the level of ER and Golgi along the biosynthetic/secretory pathway. Rab4 and RabS regulate early steps of the endocytic process and endosome-endosome fusion [42114]. Within the past two years, the subcellular localization and the function of several Rab proteins - such as Rab3, Rab5, Rab7, Rabll, Rab17 and Rab25 - have been stud- ied in further detail [45-511. Nevertheless, more work needs to be done to determine the site of action of the majority of Rabs. In addition, it seems more and more likely that some functional specialization of Rabs takes place, both in different cell types and in different organisms. One example is Rabll (A and B) and its two yeast homologues Ypt3lp and Ypt32p. A role for Rabll has been documented in late recycling of transferrin receptors [47], in tram-Golgi network (TGN) to plasma membrane transport [48] and in endosome-to-TGN transport (E Wilcke, L Johannes, B Goud, J Salamero, unpublished data). On the other hand, yeast YpQlp is involved in intra-Golgi transport [39].

The Rab cycle Like ARF and other Ras-like proteins, Rabs cycle between a GDP-bound (‘off’) and a GTP-bound (‘on’) conformation. The GDP/GTP exchange reaction is catalyzed by GEFs. Although less well characterized than Ras or Rho/Rat GEFs, some Rab exchange factors have now been identified. They include Sec2p - an essential 178 kDa cytosolic protein specific for prenylated Rab3A,


Figure 3

Vectorial transport

-t GDP

Current Op~nm in Cell Blologj

Model of the functional cycle of Rab proteins. Rab proteins are cytoskeleton, and docking complexes, which are recruited from the

incorporated into a transport vesicle, either during or after its formation. cytosol. During or after docking of the vesicle with the acceptor

The attachment process of Rab.GDP-GDI complexes involves several membrane, other sets of Rab effecters activate SNARES through

proteins including GDF and is coupled to GDP/GTP exchange catalyzed interactions with SNARE regulatory proteins, such as Secl p (or its

by GEF proteins. Rab.GTP interacts with a select set of effecters to mammalian homolog Munclg). After fusion of the vesicle, GTP hydrolysis

target the vesicle to appropriate sites on the acceptor membrane. The is mediated by GAP..GDI releases Rab.GDP from membranes and

effecters include motor proteins (motor), which link the vesicle to the RablGDl complexes are reused for another round of transport.

which is required for Golgi to plasma membrane traffic and mediates nucleotide exchange on Sec4p - and Rabex-5, which catalyzes GDP/GTP exchange upon delivery of Rab5 to membranes [42-44]. A GEF activity that stimulates GDP release and GTP uptake by Yptlp has recently been detected in yeast extracts [52]. Signifi- cant progress has also been made in the identification of GAPS that stimulate the very low intrinsic GTPase activities of Rabs. In S. cermisiae, three proteins termed Gyp have been characterized - Gyplp, Gyp6p and Gyp7p acting on Sec4p, Ypt6p and Ypt7p, respectively

J53,54]. In mammalian cells, a 130 kDa protein that acts as a GAP for the Rab3 subfamily has been isolated. Rab3A GAP is associated with a noncatalytic subunit [55]. Recently, a protein termed GAPCenA active on Rab6 has been identified [56’]. Interestingly, Gyp proteins and GAPCenA (but not Rab3GAP) share significant homology with yeast cell cycle checkpoint proteins BubZp and Cdcl6p. This raises the intriguing possibility that Rab- GAPS serve to co-ordinate membrane traffic with other events taking place during mitosis, such as the control of microtubule nucleation.

A characteristic of Rab proteins is that a cycle of association with and dissociation from membranes is superimposed onto their GDP/GTP cycle (Figure 3). At steady state, lo-50% of a given Rab is present in the cytosol. This pool is maintained in the GDP-bound conformation through an interaction with GDP-dissociation inhibitor (GDI) proteins. To date, three isoforms of GDI have been characterized, and possibly two other isoforms exist. The amino-acid residues important for the binding of Rab3A GDI/GDIa to RablA have been determined [57].

Prenylation of Rab proteins (stable covalent addition of one or two C’ZO geranylgeranyl groups via thioester bonds to carboxy-terminal cysteine residues) serves as a membrane-anchoring device. The enzymology of Rab prenylation, a reaction catalyzed by Rab geranylgeranyl transferase (GGTase), has been characterized [58]; however, the process of attachment of Rab proteins to specific membranes is still poorly understood. As all Rabs are prenylated, prenylation cannot constitute the sole membrane targeting signal. GDP/GTP exchange does not seem to be a prerequisite for Rab binding to membranes, as


GDP-bound Rab can be transiently detected in membranes after delivery of Rab-GDI complexes. The binding of Rab.GDP-GDI complexes onto membranes is likely to be followed by GDP/GTP exchange and the release of GDI. A GDI-displacement factor (GDF) that is active on a subset of Rab proteins and devoid of exchange activity has been identified but not yet fully characterized. Whether or not saturable ‘receptors’ for Rabs are present on membranes is still a matter of debate. Nevertheless, a protein (Yipl) capable of recruiting Yptlp and Ypt3lp on Golgi membranes has been described [59’].

After Rabs become membrane-bound, they interact with their specific effecters (see below). Following Rab.GTP interaction with effector( hydrolysis of GTP is catalyzed by GAPS. It is not yet clear whether GTP hydrolysis only serves to switch off the GTPase or whether it has a direct role in the function of Rab effecters. In the case of Rab5, Zerial and coworkers [60] have convincingly shown that the cycle of GTP hydrolysis is not coupled to endosome-endosome fusion, a process regulated by this GTPase. Similarly, GTP hydrolysis seems dispensable for Yptlp mediated fusion events [61]. The Rab cycle is com- pleted by the GDI-mediated release of membrane-bound RabeGDP Other proteins unrelated to GDIs might also serve to remove Rabs from membranes [62].

Rabs function in targeting and docking On the basis of the initial studies on the role of Sedp and Yptlp in transport, Henri Bourne proposed ten years ago [63] that RabGTPases could specify the direction traveled by transport vesicles between intracellular compartments. By analogy to ribosomal elongation factors, Bourne’s model predicted that a GTPase specific for each transport step would ensure that the labeled vesicle is deposited at the appropriate address. A few years later, the identification of SNARES, a family of proteins present on both vesicle (v-SNARE) and target (t-SNARE) membranes led Roth- man and colleagues to propose that the specificity of interactions between the SNARES accounted for the specificity of membrane trafficking. Recent results suggest, however, that SNARE pairing cannot be solely responsible for the appropriate targeting/docking of transport vesicles. For example, the docking of ER-derived transport vesicles with their target Golgi membranes can occur in the absence of SNARES [64’]. Similarly, initial events leading to the homotypic fusion of vacuoles are SNARE-independent [65,66-l. These data, together with studies showing that SNARES can form promiscuous complexes in vitro, suggests that SNARES are not primarily involved in the specific targeting/docking of vesicles. In addition, a grow- ing number of proteins that are not related to SNARES and that play key roles in vesicle targeting have been now identified. A yeast complex termed TRAPP [67] is implicated in ER-to-Golgi transport, and the exocyst - a complex con- sisting of one copy each of Sec3p, Se&p, Sec6p, SecBp, SeclOp, Secl5p and Exo70p [68”] -targets post-Golgi vesicles to the plasma membrane. Mammalian homologs of

some components of the exocyst have been characterized and are involved in targeting of vesicles to the basolateral membrane of epithelial cells [69]. In animal cells, Golgi- derived vesicles interact with Golgi membranes through a complex of ~115, GM130, and giantin.

Together, the above data suggest that the events that pre- cede stable, SNARE-dependent, docking of vesicles or membranes are the result of a network of interactions between many proteins. Rabs again appear to be excellent candidates for orchestrating these events. They could per- form this task sequentially one of in three ways (Figure 3). First, Rabs might directly link transport vesicles or membranes to the cytoskeleton. Rab6, a Golgi-associated Rab, has been shown to interact with a kinesin-like protein termed Rabkinesin-6 [70”]. Rab6 labels highly dynamic tubular structures moving along microtubules from the Golgi to the cell periphery (J White, personal communication). Rab6 could facilitate the transport of these structures to their acceptor compartment, probably the ER , either by recruiting Rabkinesin-6 onto membranes or by changing the biochemical properties of the motor. Rab4 interacts with a dynein intermediate chain (M McCaffrey, personal communication). Similarly, Sec4p could promote polarized transport of post-Golgi vesicles along the actin cytoskeleton through an interaction with its exchange factor Se&p. Another possibility is that Rabs serve to link vesicles and target membranes to the cytoskeleton at docking sites. For example, the Rab3A effector Rabphilin-A has been shown to interact with the actin-bundling protein cx actinin. Such an interaction could favor the local remodeling of the actin cytoskeleton prior to the fusion of synaptic vesicles with the plasma membrane [42-44].

Second, Rabs might trigger the recruitment of docking complexes. Such a role has been documented both in heterotypic (Sec4p) and homotypic (RabS and Ypt7p) fusion events. In its GTP-bound form, Sec4p interacts with Secl5p, a component of the exocyst [68”]. Then, a chain of protein-protein interactions leads to the assembly of the exocyst and its binding to Sec3p, which marks the specific sites of exocytosis at the plasma membrane (bud region). After activation through binding of RabaptinS-Rabex5 complex, Rab5, recruits EEA1 -a protein that interacts with the PI3-kinase product phosphatidylinositol-3-phosphate (PIP,) - to early endosomes [71”,72”]. PIP, might serve to stabilize EEA1 on membrane through interaction with its phospholipid binding domain (FYVE domain) [71”,72”]. Ypt7 has been shown to promote the reversible docking of vacuoles, a step that pre- cedes SNARE pairing [65,66-l. It is not clear, however, whether other proteins are also involved in this process. Other docking complexes regulated by Rabs could be soon characterized. For example, Rabphilin-A and Rim, two effec- tors of Rab3A, could be part of a complex that links synaptic vesicles to the presynaptic membrane in neurons.

Finally, Rabs could regulate or facilitate the assembly of SNARE complexes. It seems clear now that Rabs are not


core components of SNARE complexes. Nevertheless, experiments in yeast have clearly shown that Rab and SNARE functions are genetically linked. For example, the effects of a mutation in the effector domain of Sec4p can be suppressed by the overexpression of the SNAPZ5-like protein Sec9p. It is a possibility that Rab proteins are involved in the priming of t-SNARES. Such a role has been suggested for Yptl. Rabs could, for example, favor the dissociation of the members of the Secl/munclB family, which impairs the association of t-SNARES with v- SNARES. In further support of this hypothesis, it has been recently shown that Vaclp, an effector of the Rab5-like GTPase VpsZlp, directly interacts with Vps45p, a Seclp homologue [73’].

Although many arguments now support the model presented in Figure 2, more work is needed before one can have a precise picture of how Rabs function. In particular, many other Rab effecters remain to be discovered. Within the past year, three novel proteins interacting specifically with Rab4 and Rabll have been characterized, and a total of 22 proteins are specifically retained on GST-Rab5eGTP columns ([71”,74]; Y Lemarchand-Brustel, personal communication). In addition, the recent determination of the X-ray crystal structure of Rab3A complexed to the Rab-binding domain of Rabphilin-A has allowed the identification of complementarity-determining regions, which are potential- ly involved in the interactions of Rabs with their effecters [75”]. These regions exhibit a high degree of sequence vari- ability among the Rab family and might enable them to interact with a wide variety of effecters.

Conclusions Our knowledge of ARF and Rab GTP-binding proteins has made tremendous progress over the past few years. Rabs and ARFs function through the common principle that a cycle of nucleotide exchange and hydrolysis coupled to a cycle of association to and dissociation from membranes ensures the vectorial nature of vesicular transport. In the case of ARFl, this allows vesicles to bud off the donor membrane with its specific set of cargo molecules, whereas in the case of Rabs, it ensures that the vesicle is targeted to the appropriate acceptor membrane.

The reality might, however, be more complex. Indeed, evidence exists that Rabs are also involved in vesicle formation. For example, Rab5-GDI complexes participate in the sequestration of transferrin receptors into clathrin coated pits [76]. In addition, even though the function of ARF6 in membrane recycling seems well established, the question remains whether ARF6 controls the formation of recycling vesicles/membranes from the perinuclear endosomes or whether it regulates their transport and/or delivery to the plasma membrane. It is also important to consider that Rabs and ARFs might work in concert with other members of the Ras superfamily. Such a connection has been demonstrated in the case of Racl, which appears to be transported to the plasma membrane under the control of

ARF6 [32’]. Rabs might also work in concert with members of the Rho/Rat family to reorganize stress fibers and focal adhesions in response to the protein kinase C activator, TPA [77]. Similarly, it remains to be seen whether Rab and ARF functions are coupled. A tentative hypothesis is that accessory proteins such as GEFs might link these two families and co-ordinate their different functions in an integrated machinery of intracellular transport.

Acknowledgements The authors thank Arnaud Echard, Michel France, Ludger Johannes, Gordon Langsley, and Solange Monier for helpful comments on this manuscript.

Note Due to space limits, only the references from 1998 are listed for Rab proteins. The other references can be found in recent reviews [39,42-441.

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

l of special interest **of outstanding interest

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2. Rothman JE: The protein machinery of vesicle budding and fusion. Protein Sci 1996, 5:185-l 94.

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4. Paris S. Beraud-Dufour S. Robineau S. Biaav J. Antonnv B. Chabre M. Chardin P: Role of protein-phosphotipidinteractions in the activation of ARFI by the guanine nucleotide exchange factor Arno. J Viol Chem 1997, 272:22221-22226.

5. Peyroche A, Paris S, Jackson CL: Nucleotide exchange on ARF mediated by yeast Geal protein. Nature 1996, 384:479-481.

6. Chardin P, Paris S, Antonny B, Robineau S, Beraud-Dufour S, Jackson CL, Chabre M: A human exchange factor for ARF contains Sec7- and pleckstrin-homology domains. Nature 1996,384:481-484.

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Sata M, Donaldson JG, Moss J, Vaughan M: Brefeldin A-inhibited guanine nucleotide-exchange activity of Sec7 domain from yeast Sec7 with yeast and mammalian ADP ribosylation factors. Proc Nafl Acad Sci USA 1998, 95:4204-4208.

Meacci E, Tsai SC, Adamik R, Moss J, Vaughan M: Cytohesin-I, a cytosolic guanine nucleotide-exchange protein for ADP- ribosylation factor. Proc Nat/ Acad Sci USA 1997, 94:1745-l 748.

Klarlund JK, Guilherme A, Holik JJ, Virbasius JV, Chawla A, Czech MP: Sionalins bv ohosohoinositide-3.4.5~trisohosphate throuah proteins-containing pleckstrin and’Sec7’homblogy domayns. Science 1997, 27&i 927-1930.

Franc0 M, Boretto J, Robineau S, Monier S, Goud B, Chardin P, Chavrier P: ARN03, a Sec7-domain guanine nucleotide exchange factor for ADP ribosylation factor 1, is involved in the control of Golgi structure and function. Proc Nat/ Acad Sci USA 1998, 95:9926-9931.

Mossessova E, Gulbis JM, Goldberg J: Structure of the guanine nucleotide exchange factor Sec7 domain of human Arno and analysis of the interaction with ARF GTPase. Cell 1998, 92:415-423.

The authors have determmed the crystal structure of the Sec7 domain of human ARNO (23 kDa). It shows a domain of 10 a-helices with a hydrophobic groove for interaction with ARFl. The edges of the groove are formed by two amino acid sequences, which are conserved in all Sec7 domains.

12. Cherfils J, Menetrey J, Mathieu M, Le Bras G, Robineau S, Beraud . Dufour S, Antonny B, Chardin P: Structure of the Sec7 domain of

the Arf exchange factor ARNO. Nature 1998, 392:101-l 05. See annotation [l 1’1.


13. BBraud-Dufour S, Robineau S, Chardin P, Paris S, Chabre M, . Cherfils J, Antonny B: A glutamic finger in the guanine nucleotide

exchange factor ARNO displaces Mg*+ and the beta-phosphate to destabilize GDP on ARFl. EMBO J 1998,17:3651-3659.

Site-directed mutaaenesis was used to identifv crucial residues at the interface between ARFY and the ARNO Sec7 dokain. Hydrophobic residues in the switch 1 region (lle46, lle49) and Lys73 in the switch 2 region of ARFl are critical for ihe interaction with the-Sec7 domain groove.-8 glutamate residue located at the edge of the groove and conserved in all Sec7 domains (Glul56 of ARNO) was identified as a residue crucial for the catalytic mechanism as substitution with a lysine decreases the nucleotide exchange reaction by three orders of magnitude without preventing interaction with ARFl (at 10~3 Mg*+ concentration).

14. Goldberg J: Structural basis for activation of ARF GTPase: . . mechanisms of guanine nucleotide exchange and GTP-myristoyl

switching. Cell 1998, 95237-248. The crystal structures of GTP.ARFl (with a deletion of the amino-terminal myristoylated helix and bound to a non nonhydrolyzable GTP analog) and of the complex between nucleotide-free ARFl bound to the Sec7 domain of yeast Gea2 have been determined. As for Ras, the GDP/GTP transition induces conformational changes in the switch 1 and 2 regions of ARFl that expose the effector binding site. Specific to ARFl, the GDP/GTP switch dri- ves the amino-terminal myristoylated helix from the protein core, allowing it to become available for membrane interaction. Nucleotide-free ARFl bound to the Sec7 domain is in the GTP conformation. The switch 1 and switch 2 regions of ARFl insert into the Sec7 domain hydrophobic groove. Interaction with the Sec7 domain destabilizes the Mg 2+ in the nucleotide binding pocket of ARFl and dissociation of bound GDP is further favored by the repulsive effect of the negatively charged sidechain of a conserved glutamate residue of the Sec7 domain (corresponding to Glu156 of human ARNO).

15. Morinaga N, Moss J, Vaughan M: Cloning and expression of a cDNA encoding a bovine brain brefeldin A- sensitive guanine nucleotide-exchange protein for ADP-ribosylation factor. Proc Nat/ Acad Sci USA 1997,94:12926-l 2931.

16. Sata M, Moss J, Vaughan M: Structural basis for the inhibitory . effect of brefeldin A on guanine nucleotide-exchange proteins for

ADP-ribosylation factors. Proc Nat/ Acad Sci USA 1999, 9612752-2757.

The authors have constructed chimeric proteins composed of segments of the Sec7 domains of yeast Sec7 (BFA-sensitive) and human Cytohesin-1 (BFA-resistant) to identify the structural basis of BFA-sensitivity. Substitution of two residues of Cytohesin-1 (Serl 99, Pro209 - present in all BFA-resistant GEFs) with the corresponding amino acids of yeast Sec7 (Asp965, Met975 - conserved in all BFA-sensitive GEFs) is sufficient to confer BFA- sensitivity to the Sec7 domain of Cytohesin-1

17. Peyroche A, Antonny B, Robineau S, Acker J, Cherfils J, Jackson CL: . . Brefeldin A acts to stabilize an abortive ARF-GDP-Sec7 domain

protein complex: involvement of specific residues of the SecT domain. MO/ Cell 1999, 3:275-285.

GEAI is an yeast gene encoding a BFA-sensitive Sec7 domain GEF (see [5]). The authors have used a genetic approach to identify mutations in GEAl that confers BFA resistance in viva (i.e. growth in the presence of BFA). Residues that are critical for BFA-sensitivitv are all located in the region of the Sec7 domain that interacts with ARFi and are not conserved in BFA-resistant ARNO-like GEFs (note that this region overlaps with the one identified in (I 6’1). Interestingly, biochemical procedures demonstrated that BFA exerts its inhibitory effect by binding to and stabilizing an abortive complex between ARFl .GDP and the Sec7 domain.

18. Monier S, Chardin P, Robineau S, Goud B: Overexpression of the ARFl exchange factor ARNO inhibits the early secretory pathway and causes the disassembly of the Golgi complex. J Cell Sci 1998, 111:3427-3436.

19. Spang A, Matsuoka K, Hamamoto S, Schekman R, Orci L: Coatomer, .a Arfl p, and nucleotide are required to bud coat protein complex I-

coated vesicles from large synthetic liposomes. froc Nat/ Acad Sci USA 1998, 95:11199-i 1204.

What are the minimal requirements for COPI-coated vesicle budding? The authors succeeded in reconstituting the budding of small coated vesicles (~70 nm) by incubating liposomes of chemically defined composition (similar to yeast ER membranes) with purified, myristoylated ARFl.GTP and coatomer. Furthermore, this paper shows that acidic phospholipids such as phosphatidic acid (PA) are not sufficient to trigger coat protein recruitment but might promote coatomer bindinq in the presence of ARFl .GTP. These results-demonstrate a direct role forARF1 in coatomer recruitment to membranes and refute the hypothesis that ARFl is acting as an activator of PLD- mediated PA production that was proposed to initiate coat protein binding and vesicle formation.

20. Zhao L, Helms JB, Brugger B, Harter C, Martoglio B, Graf R, Brunner J, Wieland FT: Direct and GTP-dependent interaction of

ADP ribosylation factor 1 with coatomer subunit beta. Proc Nat/ Acad Sci USA 1997,94:4418-4423.

21. Bremser M, Nickel W, Schweikert M, Ravazzola M, Amherdt M, l 0 Hughes CA, Sollner TH, Rothman JE, Wieland FT: Coupling of coat

assembly and vesicle budding to packaging of putative cargo receptors. Cell 1999, 96:495-506.

In an assay that reconstitutes COPI-coated vesicle formation, the authors have explored the function of transmembrane proteins in coatomer recruitment. A peptide corresponding to the cytoplasmic tail of p23 - a putative transmembrane cargo receptor - was coupled to a lipid and this lipopeptide included during the preparation of liposomes of composition mimicking mammalian Golgi membranes. Budding of COPI-coated vesicles required the presence of ARFl .GTP and coatomer added to the ~23 lipopeptide- containina lioosomes. Buddina necessitates the oresence of an intact KKXX (in the sin”glk letter code for a&no acids where i is any amino acid) dibasic motif in the p23 cytoplasmic peptide that is known to bind the y-COP subunit of coatomer. -These results. provide a strong indication that coatomer recruitment to Golgi membranes results from a bivalent interaction of coatomer with ARFl -GTP and the cytoplasmic tails of membrane cargo and putative cargo receptors.

22. Springer S, Schekman R: Nucleation of COPII vesicular coat complex by endoplasmic reticulum to Golgi vesicle SNARES. Science 1998, 281:698-700.

23. Moss J, Vaughan M: Molecules in the ARF orbit. J Biol Chem 1998, 273:21431-21434.

24. Aoe T, Cukierman E, Lee A, Cassel D, Peters PJ, Hsu VW: The KDEL receptor, ERD2, regulates intracellular traffic by recruiting a GTPase-activating protein for ARFl. EMBO J 1997,16:7305-7316.

25. Goldberg J: Structural and functional analysis of the ARFl l 0 ARFGAP complex reveals a role for coatomer in GTP hydrolysis.

Cell 1999, 96:893-902. This oaoer reoorts the crvstal structure (1.95 8, resolution) of ARFI.GDP bounb io the’catalytic do’main of ARF.dlAP (residues 6-i36). Like other GAPS for small GTP-binding proteins, ARF*GAP acts by stabilizing the switch 2 region of ARFl to bosition Glu71 to participate in the hydrolysis reaction. In contrast to other GAPS, however, the GAP binding site does not overlap with the effector binding site on ARFl, nor does ARF.GAP provide anv residues oarticioatino in hvdrolvsis. Furthermore. addina coatomer to AdFl .GTP an’d ARi.GAp accelerates hydrolysis by ihree orders of magnitude. The proposal of the author is that, in this tripartite complex, the coatomer supplies the residues that enter the active site and accelerate hydrolysis.

26. D’Souza-Schorey C, van Donselaar E, Hsu VW, Yang C, Stahl PD, . Peters PJ: ARF6 targets recycling vesicles to the plasma

membrane: insights from an ultrastructural investigation. J Cell Biol 1998, 140:603-616.

The authors have used cryo-immunogold electron microscopy to analyze the intracellular distribution of ARF6 and ARF6 mutant forms in transfected CHO cells. At levels of expression close to that of the endogenous protein, ARF6 and a GTP-binding-defective mutant colocalize with the transferrin receptor and cellubrevin to the pericentriolar recycling compartment. In contrast, a GTP- bound mutant accumulates at the plasma membrane where it induces exten- sive membrane invaginations. Therefore, depending on its nucleotide status, ARF6 appears to cycle between recycling endosomes and the plasma membrane and might serve to regulate the outward flow of recycling membrane.

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The

Radhakrishna H, Donaldson JG: ADP-ribosylation factor 6 regulates a novel plasma membrane recycling pathway. J Cell Bioll997, 139:49-61.

D’Souza-Schorey C, Boshans RL, McDonough M, Stahl PD, Van Aelst L: A role for PORI, a Racl-interacting protein, in ARFG- mediated cytoskeletal rearrangements. EMBO J 1997, 16:5445-5454.

Song J, Khachikian Z, Radhakrishna H, Donaldson JG: Localization of endogenous ARF6 to sites of cortical actin rearrangement and involvement of ARF6 in cell spreading. J Cell Sci 1998, 111:2257-2267.

Caumont AS, Galas MC, Vitale N, Aunis D, Bader MF: Regulated exocytosis in chromaffin cells. Translocation of ARF6 stimulates a plasma membrane-associated phospholipase D. J Biol Chem 1998, 273:1373-l 379.

Van Aelst L, D’Souza-Schorey C: Rho GTPases and signaling networks. Genes Dev 1997,11:2295-2322.

Radhakrishna H, Al-Awar 0, Khachikian Z, Donaldson JG: ARF6 requirement for Rat ruffling suggests a role for membrane trafficking in cortical actin rearrangements. J Cell Sci 1999, 112:855-866.

authors show that ARF6 and Racl (a member of the Rho subfamily of .

Ras-related tiTP-blndmg protein Involved In the regulation ot actln-based


membrane ruffling) colocalize in the perinuclear recycling endosomal compartment. Evidence is presented that Racl -induced membrane ruffling requires the activity of ARFG.

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Frank S, Upender S, Hansen SH, Casanova JE: ARNO is a guanine nucleotide exchange factor for ADP-ribosylation factor 6. J Biol Chem 1998, 273123-27.

Frank SR, Hatfield JC, Casanova JE: Remodeling of the actin cytoskeleton is coordinately regulated by protein kinase C and the ADP-ribosylation factor nucleotide exchange factor ARNO. MO/ Eiol Cell 1998, 9:3133-3146.

Klarlund JK, Rameh LE, Cantley LC, Buxton JM, Holik JJ, Sakelis C, Patki V, Corvera S, Czech MP: Regulation of GRPI-catalyzed ADP ribosylation factor guanine nucleotide exchange by phosphatidylinositol3,4,5+risphosphate. J Viol Chem 1998, i73:1859-1862.

Venkateswarlu K, Oatey PB, Tavare JM, Cullen PJ: Insulin-dependent translocation of ARNO to the plasma membrane of adipocytes requires phosphatidylinositol 3-kinase. Curr Biol 1998, 8:463-466.

Pacheco-Rodriguez G, Meacci E, Vitale N, Moss J, Vaughan M: Guanine nucleotide exchange on ADP-ribosylation factors catalyzed by cytohesin-1 and its Sec7 domain. J Biol Chem 1998, 273:26543-26548,

Franc0 M, Peters PJ, Boretto J, van Donselaar E, Neri A, D’Souza Schorey C, Chavrier P: EFAG, a Sec7 domain-containing exchange factor for ARFG, coordinates membrane recycling and actin cytoskeleton organization. EMBO J 1999, 18:1480-l 491.

The authors have identified the first Sec7- and PH-domain-containing GEF, termed EFAG, as a specific activator of ARFG. Overexpressed EFA6 localizes to the cytoplasmic face of actin-based plasma membrane ruffles, which are induced on its expression. As previously shown for ARFG, EFA6 regulates transferrin receptor trafficking. EFA6 might co-ordinate membrane recycling in the endocytic pathway with cytoskeletal rearrangements.

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Lazar T, Gotte M, Gallwitz D: Vesicular transport: how many YpVRab-GTPases make a eukaryotic cell? Trends Biochem Sci 1997, 22:468-472.

Mori T, Fukuda Y, Kuroda H, Matsumura T, Ota S, Sugimoto T, Nakamura Y, lnazawa J: Cloning and characterization of a novel Rab-family gene, Rab36, within the region at 22qll.2 that is homozygously deleted in malignant rhabdoid tumors. Biochem Biophys Res Commun 1999, 254:594-600.

Zheng JY, Koda T, Fujiwara T, Kishi M, lkehara Y, Kakinuma M: A novel Rab GTPase, Rab33B, is ubiquitously expressed and localized to the medial Golgi cisternae. J Cell Sci 1998, Ill :1061-l 069.

Schimmoller F, Simon I, Pfeffer SR: Rab GTPases, directors of vesicle docking. J Biol Chem 1998, 273:22161-22164.

Martinez 0, Goud B: Rab proteins. Biochim Siophys Acfa 1998, 1404:101-112.

Gonzalez L Jr, Scheller RH: Regulation of membrane trafficking: structural insights from a Rab/effector complex. Cell 1999, 96:755-758.

leui M, Escher G, Meda P, Charollais A, Baldini G, Darchen F, Wollheim CB. Reaaui R: Subcellular distribution and function of Rab3A, 6, C, and-D isoforms in insulin-secreting cells. MO/ Endocrinol 1999, 13:202-212.

Press B, Feng Y, Hoflack B, Wandinger-Ness A: Mutant Rab7 causes the accumulation of cathepsin D and cation- independent mannose B-phosphate receptor in an early endocytic compartment J Cell B/o/ 1998, 140:1075-l 089.

Ren M, Xu G, Zeng J, De Lemos-Chiarandini C, Adesnik M, Sabatini DD: Hydrolysis of GTP on Rabll is required for the direct delivery of transferrin from the pericentriolar recycling compartment to the cell surface but not from sorting endosomes. Proc Nat/ Acad Sci USA 1998, 95:6187-6192.

Chen W, Feng Y, Chen D, Wandinger-Ness A: Rabll is required for frans-Golgi network-to-plasma membrane transport and a preferential target for GDP dissociation inhibitor. MO/ Biol Cell 1998, 9:3241-3257.

Zacchi P, Stenmark H, Parton RG, Orioli D, Lim F, Giner A, Mellman I, Zerial M, Murphy C: Rab17 regulates membrane trafficking through apical recycling endosomes in polarized epithelial cells. J Cell Biol 1998, 140:1039-l 053.

Casanova JE, Wang X, Kumar R, Bhartur SG, Navarre J, Woodrum JE, Altschuler Y, Ray GS, Goldenring JR: Association of Rab25 and

51. Barbieri MA, Hoffenberg S, Roberts R, Mukhopadhyay A, Pomrehn A, Dickey BF, Stahl PD: Evidence for a symmetrical requirement for Rab5-GTP in in vitro endosome-endosome fusion. J Biol Chem 1998, 273:25850-25855,

52. Jones S, Richardson CJ, Litt RJ, Segev N: Identification of regulators for Yptl GTPase nucleotide cycling. MO/ Biol Cell 1998, 9:?!81 Q-2837.

53. Vollmer P, Will E, Scheglmann D, Strom M, Gallwitz D: Primary structure and biochemical characterization of yeast GTPase- activating proteins with substrate preference for the transport GTPase Ypt7p. fur J Biochem 1999, 260:284-290.

54. Du LL, Collins RN, Novick PJ: Identification of a Sec4p GTPase- activating protein (GAP) as a novel member of a Rab GAP family. J Biol Chem 1998, 273:3253-3256.

55. Nagano F, Sasaki T, Fukui K, Asakura T, lmazumi K, Takai Y: Molecular cloning and characterization of the noncatalytic subunit of the Rab3 subfamily-specific GTPase-activating protein. J Biol Chem 1998,273:24781-24785.

56. Cuif MH, Possmayer F, Zander H, Bordes N, Jollivet F, Couedel . Courteille A. Janoueix-Lerosev I. Lanaslev G. Bornens M. Goud B:

Characterization of GAPCe& a G?Pa>e activating protein for Rab6, part of which associates with the centrosome. EM60 J 1999, 18:i 772-i 782.

The authors have ldentifled a new protein termed tiAPC;enA that acts as a GAP (GTPase activatina orotein) for Rab6. A minor DOOI of GAPCenA is asso-

Rabl IA with the apical recycling system of polarized Madin- Darby canine kidney cells. MO/ Viol Cell 1999, 10:47-61.

-, ciated with the centrosome, and forms complexes’with cytosolic rtubulin, a protein involved in microtubule nucleation. This suggests that RabGAPs (which share homology domains with mitotic checkpoint proteins, see also [16’]) might be involved in the co-ordination of microtubule and membrane dynamics during mitosis.

57. Wu SK, Luan P, Matteson J, Zeng K, Nishimura N, Balch WE: Molecular role for the Rab binding platform of guanine nucleotide dissociation inhibitor in endoplasmic reticulum to Golgi transport J Biol Chem 1998, 273:26931-26938.

58. Anant JS, Desnoyers L, Machius M, Demeler B, Hansen JC, Westover KD, Deisenhofer J, Seabra MC: Mechanism of Rab geranylgeranylation: formation of the catalytic ternary complex. Biochemistry 1998, 37:12559-l 2568.

59. Yang X, Matern HT, Gallwitz D: Specific binding to a novel and . essential Golgi membrane protein (Yip1 p) functionally links the

transport GTPases Yptlp and Ypt31 p. EMBO J 1998,17:4954-4963. The authors have identified a new Golgi-associated integral membrane protein termed Yip1 p, which binds to Yptl p and Ypt31 p. They provide evidence that Yip1 p might serve to recruit Yptl p and Ypt31 p onto Golgi membranes.

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Cao X, Ballew N, Barlowe C: Initial docking of ER-derived vesicles requires Us01 p and Yptl p but is independent of SNARE proteins. EMBO J 1998, 17:2156-2165.

--. -I. In an assay that reconstitutes tK-to-Uolgl transport In yeast, the authors have investigated the role of Yptl p. GDI inhibits vesicle docking but not fusion of ER-derived vesicles with Golgi membranes. They also provide evidence that the SNARE proteins Sed5p. Bet1 p and Bosl p are not required for the initial docking of vesicles. This study points to a role for Rab proteins in SNARE-independent docking/tethering of vesicles with target membranes (see also Ungermann et a/. [66-l).

Rybin V, Ullrich 0, Rubino M, Alexandrov K, Simon I, Seabra C, Goody R, Zerial M: GTPase activity of Rab5 acts as a timer for endocytic membrane fusion. Nature 1996, 383:266-269.

Richardson CJ, Jones S, Litt RJ, Segev N: GTP hydrolysis is not important for Yptl GTPase function in vesicular transport. MO/ Cell Biol 1998, l&827-838.

Marzesco AM, Galli T, Louvard D, Zahraoui A: The rod cGMP phosphodiesterase delta subunit dissociates the small GTPase Rab13 from membranes. J Biol Chem 1998, 273:22340-22345.

Bourne HR: Do GTPases direct membrane traffic in secretion? Cell 1988, 53:669-671.

65. Sato K, Wickner W: Functional reconstitution of Ypt7p GTPase and a purified vacuole SNARE complex. Science 1998, 281:700-702.

66. Ungermann C, Sato K, Wickner W: Defining the functions of frans SNARE pairs. Nature 1998, 396:543-548.

The authors have dissected the steps that lead to vacuole-vacuole fusion in vitro. They define a reversible Ypt7p-dependent ‘tethering’ step that pre- cedes irreversible SNARE pairing.


67. Sacher M, Jiang Y, Barrowman J, Scarpa A, Burston J, Zhang L, Schieltz D, Yates JR III, Abeliovich H, Ferro-Novick S: TRAPP, a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion. EM60 J 1998, 17:2494-2503.

68. Guo W, Roth D, Walch-Solimena C, Novick P: The exocyst is an . . effector for Sec4p, targeting secretory vesicles to sites of

exocytosis. EM60 J 1999,18:1071-1080. The authors show that SeclSp, a component of the exocyst, associates with post-Golgi vesicles and binds to GTP-bound Sec4p. They also provide evidence that Sec4p is directly involved in the assembly of the exocyst proteins and in its binding to Sec3p, which marks specific sites of exocytosis at the plasma membrane.

69. Hsu SC, Hazuka CD, Foletti DL, Scheller RH: Targeting vesicles to specific sites on the plasma membrane: the role of the Sec6/8 complex. Trends Biochem Sci 1999, 9: 150-l 53.

70. Echard A, Jollivet F, Martinez 0, Lacapere JJ, Rousselet A, Janoueix l * Lerosey I, Goud B: Interaction of a Golgi-associated kinesin-like

protein with Rab6. Science 1998, 279:580-585. The authors have identified a new Golgi-associated kinesin-like protein termed Rabkinesin-6 as an effector of Rab6. This result provides the first direct evidence that one Rab function might be to regulate the movement of transport vesicles along the cytoskeleton.

71. Christoforidis S, McBride HM, Burgoyne RD, Zerial M: The Rab5 . . effector EEA1 is a core component of endosome docking. Nature

1999, 397:621-625. The authors have determined the minimal machinery needed for in vitro endosome-endosome fusion. EEA1 alone (or at least a fractionated cytosolic frac- tion highly enriched in this protein) can drive the fusion reaction, suggesting that EEA1 is an integral component of the docking and fusion machinery. The other putative Rab5 effecters might serve to regulate EEA1 activity.

72. Simonsen A, Lippe R, Christoforidis S, Gaullier JM, Brech A, l * Callaghan J, Toh BH, Murphy C, Zerial M, Stenmark H: EEA1 links

Pl(3)K function to Rab5 regulation of endosome fusion. Nature 1998, 394:494-498.

The authors identify the early-endosomal autoantigen EEA1 as an effector of Rab5. EEA1 binds to PIP, on endosomal membranes. As PI3-kinase

activity is required for endosome fusion, this result provides the first molecular link between PI3-kinase and Rab5 in the regulation of endocytic membrane traffic.

73. Peterson MR, Burd CG, Emr SD: Vacl p coordinates Rab and . phosphatidylinositol3-kinase signaling in Vps45p-dependent

vesicle docking/fusion at the endosome. Curr Biol 1999, 9:159-l 62.

The authors show that Vacl p, a yeast endosomal protein closely related to EEAl, might function as a downstream effector of Vpsll p, a RabGTPase that functions in Golgi-to-endosome transport. Vacl p directly interacts with Vps45p, a Secl p homologue.

74. Zeng J, Ren M, Gravotta D, De Lemos-Chiarandini C, Lui M, Erdjument-Bromage H, Tempst P, Xu G, Shen TH, Morimoto T et a/.: Identification of a putative effector protein for Rabll that participates in transferrin recycling. Proc Nat/ Acad Sci USA 1999, 96:2640-2845.

75. . .

Ostermeier C, Brunger AT: Structural basis of Rab effector specificity: crystal structure of the small G protein Rab3A complexed with the effector domain of rabphilin-3A. Cell 1999, 96:363-374.

The authors present the first crystal structure (2.6 A resolution) of a Rab (Rab3A) in complex with an effector (Rabphilin-A). The crystal has been obtained with the GTPase mutant of Rab3A (Rab3 Glu81 -+Leu ), with a few amino acids at the amino and carboxyl terminus deleted (leaving amino acids 1 Q-217), and residues 40-l 70 of Rabphilin-A. The Rab3A interface with Rabphilin-A involves the switch I and switch II regions of Rab3A and three domains termed RabCDR. These domains are hypetvariable among the Rab family and could enable Rab proteins to interact with a wide variety of effecters.

76. McLauchlan H, Newell J, Morrice N, Osborne A, West M, Smythe E: A novel role for Rab(i-GDI in ligand sequestration into clathrin- coated pits. Curr Bioll998,8:34-45.

77. lmamura H, Takaishi K, Nakano K, Kodama A, Oishi H, Shiozaki H, Monden M, Sasaki T, Takai Y: Rho and Rab small G proteins coordinately reorganize stress fibers and focal adhesions in MDCK cells. MO/ Biol Cell 1998, 9:2561-2575.

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The role of ARF and Rab GTPases in membrane transport ...didattica.uniroma2.it/assets/uploads/corsi/145667/rab_e_arf_nel_t... · yeast secretory pathway. ... idea that member(s) of