Rab11BP/Rabphilin-11, a downstream target of rab11 small G protein implicated in vesicle recycling.

Rab11 small G protein has been implicated in vesicle recycling, but its upstream regulators or downstream targets have not yet been identified. We isolated here a downstream target of Rab11, named rabphilin-11, from bovine brain. Moreover, we isolated from a rat brain cDNA library its cDNA, which encoded a protein with a M(r) of 100,946 and 908 amino acids (aa). Rabphilin-11 bound GTP-Rab11 more preferentially than GDP-Rab11 at the N-terminal region and was specific for Rab11 and inactive for other Rab and Rho small G proteins. Both GTP-Rab11 and rabphilin-11 were colocalized at perinuclear regions, presumably the Golgi complex and recycling endosomes, in Madin-Darby canine kidney cells. In HeLa cells cultured on fibronectin, both the proteins were localized not only at perinuclear regions but also along microtubules, which were oriented toward membrane lamellipodia. Treatment of HeLa cells with nocodazole caused disruption of microtubules and dispersion of GTP-Rab11 and rabphilin-11. Overexpression of the C-terminal fragment of rabphilin-11 (aa 607-730), lacking the GTP-Rab11 binding domain, in HeLa cells reduced accumulation of transferrin at perinuclear regions and cell migration. Rabphilin-11 turned out to be a rat counterpart of recently reported bovine Rab11BP. These results indicate that rabphilin-11 is a downstream target of Rab11 which is involved in vesicle recycling.     

Structural basis of Rab effector specificity: crystal structure of the small G protein Rab3A complexed with the effector domain of rabphilin-3A

The small G protein Rab3A plays an important role in the regulation of neurotransmitter release. The crystal structure of activated Rab3A/GTP/Mg2+ bound to the effector domain of rabphilin-3A was solved to 2.6 A resolution. Rabphilin-3A contacts Rab3A in two distinct areas. The first interface involves the Rab3A switch I and switch II regions, which are sensitive to the nucleotide-binding state of Rab3A. The second interface consists of a deep pocket in Rab3A that interacts with a SGAWFF structural element of rabphilin-3A. Sequence and structure analysis, and biochemical data suggest that this pocket, or Rab complementarity-determining region (RabCDR), establishes a specific interaction between each Rab protein and its effectors. RabCDRs could be major determinants of effector specificity during vesicle trafficking and fusion.     

Sec15 is an effector for the Rab11 GTPase in mammalian cells

Rab/Ypt GTPases play key roles in the regulation of vesicular trafficking. They perform most of their functions in a GTP-bound form by interacting with specific downstream effectors. The exocyst is a complex of eight polypeptides involved in constitutive secretion and functions as an effector for multiple Ras-related small GTPases, including the Rab protein Sec4p in yeast. In this study, we have examined the localization and function of the Sec15 exocyst subunit in mammalian cells. Overexpressed Sec15 associated with clusters of tubular/vesicular elements that were concentrated in the perinuclear region. The tubular/vesicular clusters were dispersed throughout the cytoplasm upon treatment with the microtubule-depolymerizing agent nocodazole and were accessible to endocytosed transferrin, but not exocytic cargo (vesicular stomatitis virus glycoprotein). Consistent with these observations, Sec15 colocalized selectively with the recycling endosome marker Rab11 and exhibited a GTP-dependent interaction with the Rab11 GTPase, but not with Rab4, Rab6, or Rab7. These findings provide the first evidence that the exocyst functions as a Rab effector complex in mammalian cells.     

A catalytic coiled coil: structural insights into the activation of the Rab GTPase Sec4p by Sec2p

Rab GTPases, the largest subgroup in the superfamily of Ras-like GTPases, play regulatory roles in multiple steps of intracellular vesicle trafficking. They are activated by guanine nucleotide exchange factors (GEFs), which catalyze the interconversion of the GDP-bound, or inactive, form of Rab to the GTP-bound, or active, form. Relatively little is known of the mechanisms by which GEFs activate Rabs. Here, we present the crystal structure of the GEF domain of Sec2p in complex with its Rab partner Sec4p. The Sec2p GEF domain is a 220 Angstroms long coiled coil, striking in its simplicity and in the use of the coiled-coil motif for catalysis. The structure suggests a mechanism whereby Sec2p induces extensive structural rearrangements in the Sec4p switch regions and phosphate-binding loop that are incompatible with nucleotide binding. We show that Sec2p is specific for Sec4p and that specificity determinants reside in the two switch regions of Sec4p.     

Bchs, a BEACH domain protein, antagonizes Rab11 in synapse morphogenesis and other developmental events

BEACH proteins, an evolutionarily conserved family characterized by the presence of a BEACH (Beige and Chédiak-Higashi) domain, have been implicated in membrane trafficking, but how they interact with the membrane trafficking machinery is unknown. Here we show that the Drosophila BEACH protein Bchs (Blue cheese) acts during development as an antagonist of Rab11, a small GTPase involved in vesicle trafficking. We find that reduction in, or loss of, bchs function restores viability and normal bristle development in animals with reduced rab11 function, while reductions in rab11 function exacerbate defects caused by bchs overexpression in the eye. Consistent with a role for Bchs in modulating Rab11-dependent trafficking, Bchs protein is associated with vesicles and extensively colocalized with Rab11 at the neuromuscular junction (NMJ). At the NMJ, we find that rab11 is important for synaptic morphogenesis, as reductions in rab11 function cause increases in bouton density and branching. These defects are also suppressed by loss of bchs. Taken together, these data identify Bchs as an antagonist of Rab11 during development and uncover a role for these regulators of vesicle trafficking in synaptic morphogenesis. This raises the interesting possibility that Bchs and other BEACH proteins may regulate vesicle traffic via interactions with Rab GTPases.     

Sec24p and Sec16p cooperate to regulate the GTP cycle of the COPII coat

Vesicle budding from the endoplasmic reticulum (ER) employs a cycle of GTP binding and hydrolysis to regulate assembly of the COPII coat. We have identified a novel mutation (sec24-m11) in the cargo-binding subunit, Sec24p, that specifically impacts the GTP-dependent generation of vesicles in vitro. Using a high-throughput approach, we defined genetic interactions between sec24-m11 and a variety of trafficking components of the early secretory pathway, including the candidate COPII regulators, Sed4p and Sec16p. We defined a fragment of Sec16p that markedly inhibits the Sec23p- and Sec31p-stimulated GTPase activity of Sar1p, and demonstrated that the Sec24p-m11 mutation diminished this inhibitory activity, likely by perturbing the interaction of Sec24p with Sec16p. The consequence of the heightened GTPase activity when Sec24p-m11 is present is the generation of smaller vesicles, leading to accumulation of ER membranes and more stable ER exit sites. We propose that association of Sec24p with Sec16p creates a novel regulatory complex that retards the GTPase activity of the COPII coat to prevent premature vesicle scission, pointing to a fundamental role for GTP hydrolysis in vesicle release rather than in coat assembly/disassembly.     

GTP drives myosin light chain 1 interaction with the class V myosin Myo2 IQ motifs via a Sec2 RabGEF-mediated pathway

The yeast myosin light chain 1 (Mlc1p) belongs to a branch of the calmodulin superfamily and is essential for vesicle delivery at the mother-bud neck during cytokinesis due to is ability to bind to the IQ motifs of the class V myosin Myo2p. While calcium binding to calmodulin promotes binding/release from the MyoV IQ motifs, Mlc1p is unable to bind calcium and the mechanism of its interaction with target motifs has not been clarified. The presence of Mlc1p in a complex with the Rab/Ypt Sec4p and with Myo2p suggests a role for Mlc1p in regulating Myo2p cargo binding/release by responding to the activation of Rab/Ypt proteins. Here we show that GTP or GTPgammaS potently stimulate Mlc1p interaction with Myo2p IQ motifs. The C-terminus of the Rab/Ypt GEF Sec2p, but not Sec4p activation, is essential for this interaction. Interestingly, overexpression of constitutively activated Ypt32p, a Rab/Ypt protein that acts upstream of Sec4p, stimulates Mlc1p/Myo2p interaction similarly to GTP although a block of Ypt32 GTP binding does not completely abolish the GTP-mediated Mlc1p/Myo2p interaction. We propose that Mlc1p/Myo2p interaction is stimulated by a signal that requires Sec2p and activation of Ypt32p.     

A Rab11/Rip11 protein complex regulates apical membrane trafficking via recycling endosomes

Rab11 is a GTPase that regulates endosomal trafficking to apical plasma membrane domains in polarized epithelial cells. We report the identification of a novel Rab11 effector, Rip11. Rip11 is enriched in polarized epithelial cells where, like Rab11, it is localized to subapical recycling endosomes (ARE) and the apical plasma membrane. Using various transport assays, we demonstrate that Rip11 is important for protein trafficking from ARE to the apical plasma membrane. Rip11 is recruited to ARE by binding to Rab11 as well as through a Mg(2+)-dependent interaction of its C2 domain with neutral phospholipids. The association of Rip11 with membranes is regulated by a phosphorylation and dephosphorylation cycle. We propose a model whereby the Rab11/Rip 11 complex regulates vesicle targeting from the ARE.     

Sec15, a component of the exocyst, promotes notch signaling during the asymmetric division of Drosophila sensory organ precursors

Asymmetric division of sensory organ precursors (SOPs) in Drosophila generates different cell types of the mature sensory organ. In a genetic screen designed to identify novel players in this process, we have isolated a mutation in Drosophila sec15, which encodes a component of the exocyst, an evolutionarily conserved complex implicated in intracellular vesicle transport. sec15(-) sensory organs contain extra neurons at the expense of support cells, a phenotype consistent with loss of Notch signaling. A vesicular compartment containing Notch, Sanpodo, and endocytosed Delta accumulates in basal areas of mutant SOPs. Based on the dynamic traffic of Sec15, its colocalization with the recycling endosomal marker Rab11, and the aberrant distribution of Rab11 in sec15 clones, we propose that a defect in Delta recycling causes cell fate transformation in sec15(-) sensory lineages. Our data indicate that Sec15 mediates a specific vesicle trafficking event to ensure proper neuronal fate specification in Drosophila.     

Rab11 function in Trypanosoma brucei: identification of conserved and novel interaction partners

The Ras-like GTPase Rab11 is implicated in multiple aspects of intracellular transport, including maintenance of plasma membrane composition and cytokinesis. In metazoans, these functions are mediated in part via coiled-coil Rab11-interacting proteins (FIPs) acting as Rab11 effectors. Additional interaction between Rab11 and the exocyst subunit Sec15 connects Rab11 with exocytosis. We find that FIPs are metazoan specific, suggesting that other factors mediate Rab11 functions in nonmetazoans. We examined Rab11 interactions in Trypanosoma brucei, where endocytosis is well studied and the role of Rab11 in recycling well documented. TbSec15 and TbRab11 interact, demonstrating evolutionary conservation. By yeast two-hybrid screening, we identified additional Rab11 interaction partners. Tb927.5.1640 (designated RBP74) interacted with both Rab11 and Rab5. RBP74 shares a coiled-coil architecture with metazoan FIPs but is unrelated by sequence and appears to play a role in coordinating endocytosis and recycling. A second coiled-coil protein, Tb09.211.4830 (TbAZI1), orthologous to AZI1 in Homo sapiens, interacts exclusively with Rab11. AZI1 is restricted to taxa with motile cilia/flagella. These data suggest that Rab11 functions are mediated by evolutionarily conserved (i.e., AZI1 and Sec15) and potentially lineage-specific (RBP74) interactions essential for the integration of the endomembrane system.     

Sec14 homology domain targets p50RhoGAP to endosomes and provides a link between Rab and Rho GTPases

Sec14 protein was first identified in Saccharomyces cerevisiae, where it serves as a phosphatidylinositol transfer protein that is essential for the transport of secretory proteins from the Golgi complex. A protein domain homologous to Sec14 was identified in several mammalian proteins that regulates Rho GTPases, including exchange factors and GTPase activating proteins. P50RhoGAP, the first identified GTPase activating protein for Rho GTPases, is composed of a Sec14-like domain and a Rho-GTPase activating protein (GAP) domain. The biological function of its Sec14-like domain is still unknown. Here we show that p50RhoGAP is present on endosomal membranes, where it colocalizes with internalized transferrin receptor. We demonstrate that the Sec14-like domain of P50RhoGAP is responsible for the endosomal targeting of the protein. We also show that overexpression of p50RhoGAP or its Sec14-like domain inhibits transferrin uptake. Furthermore, both P50RhoGAP and its Sec14-like domain show colocalization with small GTPases Rab11 and Rab5. We measured bioluminescence resonance energy transfer between p50RhoGAP and Rab11, indicating that these proteins form molecular complex in vivo on endosomal membranes. The interaction was mediated by the Sec 14-like domain of p50RhoGAP. Our results indicate that Sec14-like domain, which was previously considered as a phospholipid binding module, may have a role in the mediation of protein-protein interactions. We suggest that p50RhoGAP provides a link between Rab and Rho GTPases in the regulation of receptor-mediated endocytosis.     

Rim1 and rabphilin-3 bind Rab3-GTP by composite determinants partially related through N-terminal alpha -helix motifs.

Rim1 is a protein of the presynaptic active zone, the area of the plasma membrane specialized for neurotransmitter exocytosis, and interacts with Rab3, a small GTPase implicated in neurotransmitter vesicle dynamics. Here, we have studied the molecular determinants of Rim1 that are responsible for Rab3 binding, employing surface plasmon resonance and recombinant, bacterially expressed Rab3 and Rim1 proteins. A site that binds GTP- but not GDP-saturated Rab3 was localized to a short alpha-helical sequence near the Rim1 N terminus (amino acids 19-55). Rab3 isoforms A, C, and D were bound with similar affinities (K(d) = 1-2 microm). Low affinity binding of Rab6A-GTP was also observed (K(d) = 16 microm), whereas Rab1B, -5, -7, -8, or -11A did not bind. Adjacent sequences up to amino acid 387, encompassing differentially spliced sequences, the zinc finger module, and the SGAWFF motif of Rim1, did not significantly contribute to the strength or the specificity of Rab3 binding, whereas a point mutation within the helix (R33G) abolished binding. This Rab3 binding site of Rim1 is reminiscent of the N-terminal alpha-helix that is part of the Rab3-binding region of rabphilin-3, and indeed we observed low affinity, specific binding of Rab3A (K(d) on the order of magnitude of 10-100 microm) to this region of rabphilin-3 alone (amino acids 40-88), whereas additional sequences up to amino acid 178 are needed for high affinity Rab3A binding to rabphilin-3 (K(d) = 10-20 nm). In contrast, an N-terminal alpha-helix motif in aczonin, with sequence similarity to the Rab3-binding site of Rim1, did not bind Rab3A, -C, or -D or several other Rab proteins. These results were qualitatively confirmed in pull-down experiments with native, prenylated Rab3 from brain lysate in Triton X-100. Munc13 bound to the zinc finger domain of Rim1 but not to the rabphilin-3 or aczonin zinc fingers. Pull-down experiments from brain lysate in the presence of cholate as detergent detected binding to downstream Rim1 sequences, between amino acids 56 and 387, of syntaxin and of Rab3. The latter, however, was inhibited rather than stimulated by GTP.     

Tumor susceptibility gene 101 (TSG101) is a novel binding-partner for the class II Rab11-FIPs

The Rab11-FIPs (Rab11-family interacting proteins; henceforth, FIPs) are a family of Rab11a/Rab11b/Rab25 GTPase effector proteins implicated in an assortment of intracellular trafficking processes. Through proteomic screening, we have identified TSG101 (tumor susceptibility gene 101), a component of the ESCRT-I (endosomal sorting complex required for transport) complex, as a novel FIP4-binding protein, which we find can also bind FIP3. We show that α-helical coiled-coil regions of both TSG101 and FIP4 mediate the interaction with the cognate protein, and that point mutations in the coiled-coil regions of both TSG101 and FIP4 abrogate the interaction. We find that expression of TSG101 and FIP4 mutants cause cytokinesis defects, but that the TSG101-FIP4 interaction is not required for localisation of TSG101 to the midbody/Flemming body during abscission. Together, these data suggest functional overlap between Rab11-controlled processes and components of the ESCRT pathway.     

Interaction cloning of Rabin3, a novel protein that associates with the Ras-like GTPase Rab3A.

Rab3A is a small, Ras-like GTPase expressed in neuroendocrine cells, in which it is associated with secretory vesicle membranes and regulates exocytosis. Using the yeast two-hybrid system, we have identified a rat brain cDNA encoding a novel 50-kDa protein, which we have named Rabin3, that interacts with Rab3A and Rab3D but not with other small GTPases (Rab3C, Rab2, Ran, or Ras). Several independent point mutations in the effector domain of Rab3A (F51L, V55E, and G56D) which do not alter nucleotide binding by the GTPase abolish the interaction with Rabin3, while another mutation (V52A) appears to increase the interaction. These results demonstrate that the interaction is highly specific. However, a glutathione S-transferase-Rabin3 fusion protein associates only weakly in vitro with recombinant Rab3A and possesses no detectable GTPase-activating protein or nucleotide exchange activity, and Rabin3 overexpressed in adrenal chromaffin cells has no observable effect on secretion. The protein possess a sequence characteristic of coiled-coil domains and a second small region with sequence similarity to a Saccharomyces cerevisiae protein, Sec2p, Sec2p is essential for constitutive secretion in yeast cells and interacts with Sec4p, a close relative of the Rab3A GTPase. Rabin3 mRNA and protein are widely expressed but are particularly abundant in testes.     

A novel snare N-terminal domain revealed by the crystal structure of Sec22b

Intra-cellular membrane fusion is facilitated by the association of SNAREs from opposite membranes into stable alpha-helical bundles. Many SNAREs, in addition to their alpha-helical regions, contain N-terminal domains that likely have essential regulatory functions. To better understand this regulation, we have determined the 2.4-A crystal structure of the 130-amino acid N-terminal domain of mouse Sec22b (mSec22b), a SNARE involved in endoplasmic reticulum/Golgi membrane trafficking. The domain consists of a mixed alpha-helical/beta-sheet fold that resembles a circular permutation of the actin/poly-proline binding protein, profilin, and the GAF/PAS family of regulatory modules. The structure is distinct from the previously characterized N-terminal domain of syntaxin 1A, and, unlike syntaxin 1A, the N-terminal domain of mSec22b has no effect on the rate of SNARE assembly in vitro. An analysis of surface conserved residues reveals a potential protein interaction site. Key residues in this site are distinct in two mammalian Sec22 variants that lack SNARE domains. Finally, sequence analysis indicates that a similar domain is likely present in the endosomal/lysosomal SNARE VAMP7.     

Munc13-4 Is a Rab11-binding Protein That Regulates Rab11-positive Vesicle Trafficking and Docking at the Plasma Membrane

The small GTPase Rab11 and its effectors control trafficking of recycling endosomes, receptor replenishment and the up-regulation of adhesion and adaptor molecules at the plasma membrane. Despite recent advances in the understanding of Rab11-regulated mechanisms, the final steps mediating docking and fusion of Rab11-positive vesicles at the plasma membrane are not fully understood. Munc13-4 is a docking factor proposed to regulate fusion through interactions with SNAREs. In hematopoietic cells, including neutrophils, Munc13-4 regulates exocytosis in a Rab27a-dependent manner, but its possible regulation of other GTPases has not been explored in detail. Here, we show that Munc13-4 binds to Rab11 and regulates the trafficking of Rab11-containing vesicles. Using a novel Time-resolved Fluorescence Resonance Energy Transfer (TR-FRET) assay, we demonstrate that Munc13-4 binds to Rab11a but not to dominant negative Rab11a. Immunoprecipitation analysis confirmed the specificity of the interaction between Munc13-4 and Rab11, and super-resolution microscopy studies support the interaction of endogenous Munc13-4 with Rab11 at the single molecule level in neutrophils. Vesicular dynamic analysis shows the common spatio-temporal distribution of Munc13-4 and Rab11, while expression of a calcium binding-deficient mutant of Munc13-4 significantly affected Rab11 trafficking. Munc13-4-deficient neutrophils showed normal endocytosis, but the trafficking, up-regulation, and retention of Rab11-positive vesicles at the plasma membrane was significantly impaired. This correlated with deficient NADPH oxidase activation at the plasma membrane in response to Rab11 interference. Our data demonstrate that Munc13-4 is a Rab11-binding partner that regulates the final steps of Rab11-positive vesicle docking at the plasma membrane.     

Rab11 in dysplasia of Barrett's epithelia

Barrett's esophagus predisposes affected patients to the development of esophageal adenocarcinoma. The development of adenocarcinoma proceeds along a progression through low- and high-grade dysplasia. Surveillance of Barrett's patients requires serial endoscopic investigations and grading mucosal biopsies. Unfortunately, grading of biopsies by conventional hematoxylin and eosin staining is fraught with significant interobserver variations. We have found in both biopsy and resection specimens that immunostaining for the small GTP binding protein Rab11 is increased in low-grade dysplastic cells. This staining is lost in high-grade dysplastic cells. These results suggest that low-grade dysplastic cells undergo an apical trafficking blockade, which is released as cells progress to the less differentiated phenotype of high-grade dysplasia and adenocarcinoma. Examination of the SKGT-4 esophageal adenocarcinoma cell line demonstrated prominent mRNA and protein expression for Rab11. Rab11 immunostaining was present in SKGT-4 cells as a perinuclear nidus of punctate staining along with a more diffuse punctate pattern. Thus, Rab11 expression was present in a esophageal adenocarcinoma cells in culture. Markers of vesicle trafficking may be critical factors for grading of mucosal dysplastic transitions leading to adenocarcinoma.     

Myosin Va mediates Rab8A-regulated GLUT4 vesicle exocytosis in insulin-stimulated muscle cells

Rab-GTPases are important molecular switches regulating intracellular vesicle traffic, and we recently showed that Rab8A and Rab13 are activated by insulin in muscle to mobilize GLUT4-containing vesicles to the muscle cell surface. Here we show that the unconventional motor protein myosin Va (MyoVa) is an effector of Rab8A in this process. In CHO-IR cell lysates, a glutathione S-transferase chimera of the cargo-binding COOH tail (CT) of MyoVa binds Rab8A and the related Rab10, but not Rab13. Binding to Rab8A is stimulated by insulin in a phosphatidylinositol 3-kinase–dependent manner, whereas Rab10 binding is insulin insensitive. MyoVa-CT preferentially binds GTP-locked Rab8A. Full-length green fluorescent protein (GFP)–MyoVa colocalizes with mCherry-Rab8A in perinuclear small puncta, whereas GFP–MyoVa-CT collapses the GTPase into enlarged perinuclear depots. Further, GFP–MyoVa-CT blocks insulin-stimulated translocation of exofacially myc-tagged GLUT4 to the surface of muscle cells. Mutation of amino acids in MyoVa-CT predicted to bind Rab8A abrogates both interaction with Rab8A (not Rab10) and inhibition of insulin-stimulated GLUT4myc translocation. Of importance, small interfering RNA–mediated MyoVa silencing reduces insulin-stimulated GLUT4myc translocation. Rab8A colocalizes with GLUT4 in perinuclear but not submembrane regions visualized by confocal total internal reflection fluorescence microscopy. Hence insulin signaling to the molecular switch Rab8A connects with the motor protein MyoVa to mobilize GLUT4 vesicles toward the muscle cell plasma membrane.