Rab1 interaction with a GM130 effector complex regulates COPII vesicle cis--Golgi tethering

Members of the Rab family of small molecular weight GTPases regulate the fusion of transport intermediates to target membranes along the biosynthetic and endocytic pathways. We recently demonstrated that Rab1 recruitment of the tethering factor p115 into a cis -SNARE complex programs coat protein II vesicles budding from the endoplasmic reticulum (donor compartment) for fusion with the Golgi apparatus (acceptor compartment) (Allan BB, Moyer BD, Balch WE. Science 2000; 289: 444–448). However, the molecular mechanism(s) of Rab regulation of Golgi acceptor compartment function in endoplasmic reticulum to Golgi transport are unknown. Here, we demonstrate that the cis -Golgi tethering protein GM130, complexed with GRASP65 and other proteins, forms a novel Rab1 effector complex that interacts with activated Rab1-GTP in a p115-independent manner and is required for coat protein II vesicle targeting/fusion with the cis -Golgi. We propose a 'homing hypothesis' in which the same Rab interacts with distinct tethering factors at donor and acceptor membranes to program heterotypic membrane fusion events between transport intermediates and their target compartments.     

Multiple aspects of Rab protein action in the secretory pathway: focus on Rab3 and Rab6

Rab proteins form the largest branch of the Ras superfamily of GTPases. They are localized to the cytoplasmic face of organelles and vesicles involved in the biosynthetic/secretory and endocytic pathways in eukaryotic cells. It is now well established that Rab proteins play an essential role in the processes that underlie the targeting and fusion of transport vesicles with their appropriate acceptor membranes. They perform this task through interactions with a wide variety of effector molecules. In this review, we illustrate recent advances in the field of Rab GTPases, taking as examples two proteins involved in the biosynthetic pathway, Rab3 and Rab6.     

Small, monomeric G proteins in plants

The superfamily of small, monomeric GTP-binding proteins, in Arabidopsis thaliana comprising 93 members, is classified into four families: Arf/Sar, Rab, Rop/Rac, and Ran families. All monomeric G proteins function as molecular switches that are activated by GTP and inactivated by the hydrolysis of GTP to GDP. GTP/GDP cycling is controlled by three classes of regulatory protein: guanine-nucleotide-exchange factors (GEFs), GTPase-activating proteins (GAPs), and guanine-nucleotide-dissociation inhibitors (GDIs). Proteins of Arf family are primarily involved in regulation of membrane traffic and organization of the cytoskeleton. Arf1/Sar1 proteins regulate the formation of vesicle coat at different steps in the exocytic and endocytic pathways. Rab GTPases are regulators of vesicular transport. They are involved in vesicle formation, recruitment of cytoskeletal motor proteins, and in vesicle tethering and fusion. Rop proteins serve as key regulators of cytoskeletal reorganization in response to extracellular signals. Several data have also shown that Rop proteins play additional roles in membrane trafficking and regulation of enzymes activity. Ran proteins are involved in nucleocytoplasmic transport.     

Mapping the interactions between a RUN domain from DENND5/Rab6IP1 and sorting nexin 1

Eukaryotic cells have developed a diverse repertoire of Rab GTPases to regulate vesicle trafficking pathways. Together with their effector proteins, Rabs mediate various aspects of vesicle formation, tethering, docking and fusion, but details of the biological roles elicited by effectors are largely unknown. Human Rab6 is involved in the trafficking of vesicles at the level of Golgi via interactions with numerous effector proteins. We have previously determined the crystal structure of Rab6 in complex with DENND5, alternatively called Rab6IP1, which comprises two RUN domains (RUN1 and RUN2) separated by a PLAT domain. The structure of Rab6/RUN1-PLAT (Rab6/R1P) revealed the molecular basis for Golgi recruitment of DENND5 via the RUN1 domain, but the functional role of the RUN2 domain has not been well characterized. Here we show that a soluble DENND5 construct encompassing the RUN2 domain binds to the N-terminal region of sorting nexin 1 by surface plasmon resonance analyses.     

The Legionella pneumophila effector DrrA is sufficient to stimulate SNARE-dependent membrane fusion

The intracellular bacterial pathogen Legionella pneumophila subverts host membrane transport pathways to promote fusion of vesicles exiting the endoplasmic reticulum (ER) with the pathogen-containing vacuole. During infection there is noncanonical pairing of the SNARE protein Sec22b on ER-derived vesicles with plasma membrane (PM)-localized syntaxin proteins on the vacuole. We show that the L.?pneumophila Rab1-targeting effector DrrA is sufficient to stimulate this noncanonical SNARE association and promote membrane fusion. DrrA activation of the Rab1 GTPase on PM-derived organelles stimulated the tethering of ER-derived vesicles with the PM-derived organelle, resulting in vesicle fusion through the pairing of Sec22b with the PM syntaxin proteins. Thus, the effector protein DrrA stimulates a host membrane transport pathway that enables ER-derived vesicles to remodel a PM-derived organelle, suggesting that Rab1 activation at the PM is sufficient to promote the recruitment and fusion of ER-derived vesicles.     

Rab and Arf proteins at the crossroad between membrane transport and cytoskeleton dynamics

The intracellular movement and positioning of organelles and vesicles is mediated by the cytoskeleton and molecular motors. Small GTPases like Rab and Arf proteins are main regulators of intracellular transport by connecting membranes to cytoskeleton motors or adaptors. However, it is becoming clear that interactions between these small GTPases and the cytoskeleton are important not only for the regulation of membrane transport. In this review, we will cover our current understanding of the mechanisms underlying the connection between Rab and Arf GTPases and the cytoskeleton, with special emphasis on the double role of these interactions, not only in membrane trafficking but also in membrane and cytoskeleton remodeling. Furthermore, we will highlight the most recent findings about the fine control mechanisms of crosstalk between different members of Rab, Arf, and Rho families of small GTPases in the regulation of cytoskeleton organization.     

Structure of membrane tethers and their role in fusion

Vesicular transport between different membrane compartments is a key process in cell biology required for the exchange of material and information. The complex machinery that executes the formation and delivery of transport vesicles has been intensively studied and yielded a comprehensive view of the molecular principles that underlie the budding and fusion process. Tethering also represents an essential step in each trafficking pathway. It is mediated by Rab GTPases in concert with so‐called tethering factors, which constitute a structurally diverse family of proteins that share a similar role in promoting vesicular transport. By simultaneously binding to proteins and/or lipids on incoming vesicles and the target compartment, tethers are thought to bridge donor and acceptor membrane. They thus provide specificity while also promoting fusion. However, how tethering works at a mechanistic level is still elusive. We here discuss the recent advances in the structural and biochemical characterization of tethering complexes that provide novel insight on how these factors might contribute the efficiency of fusion.     

The Rab5 effector EEA1 interacts directly with syntaxin-6.

The fusion of transport vesicles with their cognate target membranes, an essential event in intracellular membrane trafficking, is regulated by SNARE proteins and Rab GTPases. Rab GTPases are thought to act prior to SNAREs in vesicle docking, but the exact biochemical relationship between the two classes of molecules is not known. We recently identified the early endosomal autoantigen EEA1 as an effector of Rab5 in endocytic membrane fusion. Here we demonstrate that EEA1 interacts directly and specifically with syntaxin-6, a SNARE implicated in trans-Golgi network to early endosome trafficking. The binding site for syntaxin-6 overlaps with that of Rab5-GTP at the C terminus of EEA1. Syntaxin-6 and EEA1 were found to colocalize extensively on early endosomes, although syntaxin-6 is present in the trans-Golgi network as well. Our results indicate that SNAREs can interact directly with Rab effectors, and suggest that EEA1 may participate in trans-Golgi network to endosome as well as in endocytic membrane traffic.     

SNARE status regulates tether recruitment and function in homotypic COPII vesicle fusion

In mammals, coat complex II (COPII)-coated transport vesicles deliver secretory cargo to vesicular tubular clusters (VTCs) that facilitate cargo sorting and transport to the Golgi. We documented in vitro tethering and SNARE-dependent homotypic fusion of endoplasmic reticulum-derived COPII transport vesicles to form larger cargo containers characteristic of VTCs ( Xu, D., and Hay, J. C. (2004) J. Cell Biol. 167, 997-1003). COPII vesicles thus appear to contain all necessary components for homotypic tethering and fusion, providing a pathway for de novo VTC biogenesis. Here we demonstrate that antibodies against the endoplasmic reticulum/Golgi SNARE Syntaxin 5 inhibit COPII vesicle homotypic tethering as well as fusion, implying an unanticipated role for SNAREs upstream of fusion. Inhibition of SNARE complex access and/or disassembly with dominant-negative alpha-soluble NSF attachment protein (SNAP) also inhibited tethering, implicating SNARE status as a critical determinant in COPII vesicle tethering. The tethering-defective vesicles generated in the presence of dominant-negative alpha-SNAP specifically lacked the Rab1 effectors p115 and GM130 but not other peripheral membrane proteins. Furthermore, Rab effectors, including p115, were shown to be required for homotypic COPII vesicle tethering. Thus, our results demonstrate a requirement for SNARE-dependent tether recruitment and function in COPII vesicle fusion. We anticipate that recruitment of tether molecules by an upstream SNARE signal ensures that tethering events are initiated only at focal sites containing appropriately poised fusion machinery.     

Myosin V transports secretory vesicles via a Rab GTPase cascade and interaction with the exocyst complex

Vesicle transport requires four steps: vesicle formation, movement, tethering, and fusion. In yeast, two Rab GTPases, Ypt31/32, are required for post-Golgi vesicle formation. A third Rab GTPase, Sec4, and the exocyst act in tethering and fusion of these vesicles. Vesicle production is coupled to transport via direct interaction between Ypt31/32 and the yeast myosin V, Myo2. Here we show that Myo2 interacts directly with Sec4 and the exocyst subunit Sec15. Disruption of these interactions results in compromised growth and the accumulation of secretory vesicles. We identified the Sec15-binding region on Myo2 and also identified residues on Sec15 required for interaction with Myo2. That Myo2 interacts with Sec15 uncovers additional roles for the exocyst as an adaptor for molecular motors and implies similar roles for structurally related tethering complexes. Moreover, these studies predict that for many pathways, molecular motors attach to vesicles prior to their formation and remain attached until fusion.     

The Rab6 GTPase regulates recruitment of the dynactin complex to Golgi membranes

Dynactin is a multisubunit protein complex required for the activity of dynein in diverse intracellular motility processes, including membrane transport. Dynactin can bind to vesicles and liposomes containing acidic phospholipids, but general properties such as this are unlikely to explain the regulated recruitment of dynactin to specific sites on organelle membranes. Additional factors must therefore exist to control this process. Candidates for these factors are the Rab GTPases, which function in the tethering of vesicles to their target organelle prior to membrane fusion. In particular, Rab27a tethers melanosomes to the actin cytoskeleton. Other Rabs have been implicated in microtubule-dependent organelle motility; Rab7 controls lysosomal transport, and Rab6 is involved in microtubule-dependent transport pathways through the Golgi and from endosomes to the Golgi. We demonstrate that dynactin binds to Rab6 and shows a Rab6-dependent recruitment to Golgi membranes. Other Golgi Rabs do not bind to dynactin and are unable to support its recruitment to membranes. Rab6 therefore functions as a specificity or tethering factor controlling the recruitment of dynactin to membranes.     

Small GTPases in vesicle trafficking

Plant small GTPases belonging to the Rop, Arf, and Rab families are regulators of vesicle trafficking. Rop GTPases regulate actin dynamics and modulate H(2)O(2) production in polar cell growth and pathogen defence. A candidate Rop GDP to Rop GTP exchange factor (RopGEF) SPIKE1 is involved in the morphogenesis of leaf epidermal cells. The ArfGEF GNOM regulates the endosomal recycling of the PIN proteins, which are involved in polar auxin transport. Intracellular localisation of small GTPases and functional studies using dominant mutant versions of Arf and Rab GTPases are defining novel plant-specific membrane compartments, especially those that participate in endosomal vesicle trafficking.     

Functional Synergy between Rab5 Effector Rabaptin-5 and Exchange Factor Rabex-5 When Physically Associated in a Complex

Rab GTPases are central elements of the vesicular transport machinery. An emerging view is that downstream effectors of these GTPases are multiprotein complexes that include nucleotide exchange factors to ensure coupling between GTPase activation and effector function. We have previously shown that Rab5, which regulates various steps of transport along the early endocytic pathway, is activated by a complex consisting of Rabex-5, a Rab5 nucleotide exchange factor, and the effector Rabaptin-5. We postulated that the physical association of these two proteins is necessary for their activity in Rab5-dependent endocytic membrane transport. To evaluate the functional implications of such complex formation, we have reconstituted it with the use of recombinant proteins and characterized its properties. First, we show that Rabaptin-5 increases the exchange activity of Rabex-5 on Rab5. Second, Rab5-dependent recruitment of Rabaptin-5 to early endosomes is completely dependent on its physical association with Rabex-5. Third, complex formation between Rabaptin-5 and Rabex-5 is essential for early endosome homotypic fusion. These results reveal a functional synergy between Rabaptin-5 and Rabex-5 in the complex and have implications for the function of analogous complexes for Rab and Rho GTPases.     

Arf-like GTPases: not so Arf-like after all

ADP-ribosylation factor (Arf) GTP-binding proteins are among the best-characterized members of the Ras superfamily of GTPases, with well-established functions in membrane-trafficking pathways. A recent watershed of genomic and structural information has identified a family of conserved related proteins: the Arf-like (Arl) GTPases. The best-characterized Arl protein, Arl2, regulates the folding of beta tubulin, and recent data suggest that Arl1 and Arf-related protein 1 (ARFRP1) are localized to the trans-Golgi network (TGN), where they function, in part, to regulate the tethering of endosome-derived transport vesicles. Other Arl proteins are localized to the cytosol, nucleus, cytoskeleton and mitochondria, which indicates that Arl proteins have diverse roles that are distinct from the known functions of traditional Arf GTPases.     

Ciliary targeting motif VxPx directs assembly of a trafficking module through Arf4

Dysfunctions of primary cilia and cilia‐derived sensory organelles underlie a multitude of human disorders, including retinal degeneration, yet membrane targeting to the cilium remains poorly understood. Here, we show that the newly identified ciliary targeting VxPx motif present in rhodopsin binds the small GTPase Arf4 and regulates its association with the trans‐Golgi network (TGN), which is the site of assembly and function of a ciliary targeting complex. This complex is comprised of two small GTPases, Arf4 and Rab11, the Rab11/Arf effector FIP3, and the Arf GTPase‐activating protein ASAP1. ASAP1 mediates GTP hydrolysis on Arf4 and functions as an Arf4 effector that regulates budding of post‐TGN carriers, along with FIP3 and Rab11. The Arf4 mutant I46D, impaired in ASAP1‐mediated GTP hydrolysis, causes aberrant rhodopsin trafficking and cytoskeletal and morphological defects resulting in retinal degeneration in transgenic animals. As the VxPx motif is present in other ciliary membrane proteins, the Arf4‐based targeting complex is most likely a part of conserved machinery involved in the selection and packaging of the cargo destined for delivery to the cilium.     

beta 2-adrenergic receptor internalization, endosomal sorting, and plasma membrane recycling are regulated by rab GTPases

Rab GTPases are recognized as critical regulatory factors involved in vesicular membrane transport and endosomal fusion. For example, Rab5 directs the transport and fusion of endocytic vesicles to and with early endosomes, whereas Rab4 is thought to control protein trafficking from early endosomes back to the plasma membrane. In the present study, we investigated the role of Rab5 and Rab4 GTPases in regulating the endocytosis, intracellular sorting, and the plasma membrane recycling of the beta(2)AR. In cells expressing the dominant-negative Rab5-S34N mutant, beta(2)AR internalization was impaired, and beta(2)AR-bearing endocytic vesicles remained in either close juxtaposition or physically attached to the plasma membrane. In contrast, a constitutively active Rab5-Q79L mutant redirected internalized beta(2)AR to enlarged endosomes but did not prevent beta(2)AR dephosphorylation and recycling. The expression of either wild-type Rab4 or a Rab4-N121I mutant did not prevent beta(2)AR dephosphorylation. However, the dominant-negative Rab4-N121I mutant blocked beta(2)AR resensitization by blocking receptor recycling from endosomes back to the cell surface. Our data indicate that, in addition to regulating the intracellular trafficking and fusion of beta(2)AR-bearing endocytic vesicles, Rab5 also contributes to the formation and/or budding of clathrin-coated vesicles. Furthermore, beta(2)AR dephosphorylation occurs as the receptor transits between Rab5- and Rab4-positive compartments.     

Golgins in the structure and dynamics of the Golgi apparatus

Golgins are a family of coiled-coil proteins associated with the Golgi apparatus necessary for tethering events in membrane fusion and as structural supports for Golgi cisternae. Recent work has shown that golgins such as GM130, golgin-45 and p115 bind to Rab GTPases via their coiled-coil domains, and that GM130, rather than being part of a static structural matrix, is in dynamic exchange between the membrane surface and the cytoplasm. Golgins such as bicaudal-D1 and -D2 bind to Rab6, but, rather than tethering membranes together, link vesicles to the cytoskeleton, thus adding a new function for this class of proteins. Other golgins containing the Golgi targeting GRIP domain, rather than binding Rabs, interact with and are recruited to membranes by another class of GTPase, the Arls. Current evidence therefore suggests that golgins function in a variety of membrane-membrane and membrane-cytoskeleton tethering events at the Golgi apparatus, and that all these are regulated by small GTPases of the Rab and Arl families.     

Rab cascades and tethering factors in the endomembrane system

Rab GTPases are key proteins that determine organelle identity and operate at the center of fusion reactions. Like Ras, they act as switches that are connected to a diverse network of tethering factors, exchange factors and GTPase activating proteins. Recent studies suggest that Rabs are linked to each other via their effectors, thus coordinating protein transport in the endomembrane system. Within this review, we will focus on selected examples that highlight these issues.     

Crystal structure of inactive form of Rab3B

Rab proteins are the largest family of ras-related GTPases in eukaryotic cells. They act as directional molecular switches at membrane trafficking, including vesicle budding, cargo sorting, transport, tethering, and fusion. Here, we generated and crystallized the Rab3B:GDP complex. The structure of the complex was solved to 1.9 Å resolution and the structural base comparison with other Rab3 members provides a structural basis for the GDP/GTP switch in controlling the activity of small GTPase. The comparison of charge distribution among the members of Rab3 also indicates their different roles in vesicular trafficking.     

Purification and identification of novel Rab effectors using affinity chromatography

Rab GTPases are central regulatory elements of the intracellular transport machinery of eukaryotic cells. To regulate vesicle docking and fusion as well as organelle dynamics Rab proteins interact with effector molecules in the GTP-bound active state. The identification of Rab effectors is, therefore, of primary importance for the mechanistic understanding of intracellular transport. Here we describe the experimental system we have developed to biochemically purify and identify effectors of the small GTPase Rab5. The method, which is based on an affinity chromatography procedure, results in the large-scale purification of Rab effectors in amounts sufficient for both their identification by microsequencing techniques and their functional characterization. In the case of Rab5, the procedure allows a comprehensive analysis of the downstream effectors and regulators of this GTPase. We expect this strategy to provide fundamental insights into the molecular mechanism of membrane transport but also to be applicable to several other GTPase-dependent biological functions.