The small GTPase Rab29 is a common regulator of immune synapse assembly and ciliogenesis

Accumulating evidence underscores the T-cell immune synapse (IS) as a site of intense vesicular trafficking, on which productive signaling and cell activation crucially depend. Although the T-cell antigen receptor (TCR) is known to exploit recycling to accumulate to the IS, the specific pathway that controls this process remains to be elucidated. Here we demonstrate that the small GTPase Rab29 is centrally implicated in TCR trafficking and IS assembly. Rab29 colocalized and interacted with Rab8, Rab11 and IFT20, a component of the intraflagellar transport system that regulates ciliogenesis and participates in TCR recycling in the non-ciliated T cell, as assessed by co-immunoprecipitation and immunofluorescence analysis. Rab29 depletion resulted in the inability of TCRs to undergo recycling to the IS, thereby compromizing IS assembly. Under these conditions, recycling TCRs accumulated in Rab11⁺ endosomes that failed to polarize to the IS due to defective Rab29-dependent recruitment of the dynein microtubule motor. Remarkably, Rab29 participates in a similar pathway in ciliated cells to promote primary cilium growth and ciliary localization of Smoothened. These results provide a function for Rab29 as a regulator of receptor recycling and identify this GTPase as a shared participant in IS and primary cilium assembly.T-cell activation is triggered by T-cell antigen receptor (TCR) engagement by MHC-bound peptide dispayed by an antigen presenting cell (APC). While a substantial proportion of the TCR has long been known to be associated with recycling endosomes,¹ it is only recently that the central function of this pool has emerged with the finding that, on assembly of the specialized interface with the APC, known as the immune synapse (IS), intracellular TCRs are delivered to this location by polarized recycling.² This process ensures a steady supply of TCRs at the IS to sustain signaling for T-cell activation³ and has been co-opted by other receptors, such as the transferrin receptor (TfR) and the chemokine receptor CXCR4,^{4, 5} as well as membrane-bound signaling mediators, such as the kinase Lck and the adaptor LAT.^{6, 7}Receptor recycling is orchestrated by the small GTPases Rab4 and Rab11.⁸ Cargo specificity is achieved with the assistance of specific regulators and effectors. The TCR recycling pathway has only started to be delineated with the identification of specific mediators, which include Rab35 and its GAP EPI64C⁹ and the actin adaptor WASH¹⁰. Specific combinations of Rabs and SNAREs have been recently associated with recycling endosomes carrying either Lck, or TCRζ and LAT.¹¹ We have moreover identified IFT20¹² and other components of the intraflagellar transport (IFT) system, which regulates ciliary assembly and function,¹³ as unexpected regulators of TCR recycling in the non-ciliated T cell.¹⁴Recently a Rab GTPase subfamily, which includes Rab29, Rab32 and Rab38, has been implicated in trafficking of the Salmonella-containing vacuole (SCV) in infected epithelial cells.^{15, 16} Rab32 and Rab38 participate in the generation and traffic of melanosomes,¹⁷ while Rab29 regulates retrograde endolysosome-to-Golgi trafficking of the mannose-6-phosphate receptor (MPR) in epithelial and neuronal cells.^{18, 19} Based on the implication of Rab29 in typhoid toxin trafficking to the plasma membrane from the SCV,¹⁵ where Rab4 and Rab11 have also been observed,²⁰ here we assessed the role of Rab29 in the regulation of TCR recycling. The results identify Rab29 as a novel regulator of vesicular trafficking in T cells, acting as a complex with IFT20 and the Rabs Rab8 and Rab11 to control TCR recycling and IS assembly. We also show that Rab29 participates in a similar pathway to control primary cilium growth and the ciliary localization of the receptor Smoothened, undescoring the homologies between cilium and IS.     

Evidence that the small GTPase Rab8 is involved in melanosome traffic and dendrite extension in B16 melanoma cells

One of the major activities of melanocytes in skin is to produce melanin and transport it via dendrites to neighboring keratinocytes. Here, we present evidence that Rab8, a member of the small GTPase superfamily, is present in purified melanosomal fractions, and is upregulated by pigmentogenic agents like melanocyte-stimulating hormone/isobutylmethyl xanthine (MSH/IBMX) and ultraviolet radiation B (UVB). Confocal immunofluorescence microscopic studies revealed that Rab8 is colocalized with Mel5, a melanosomal protein, at the trans-Golgi area and in the cytoplasmic vesicles of B16 cells. During MSH/IBMX treatment, while a number of dendrites with numerous processes are formed, colocalization is extended towards the tips of protrusions. Since process formation is supported by cytoskeletal assembly as well as membrane transport, we tested the colocalization of Rab8 with actin filaments in B16 cells. Rab8, indeed, colocalized with phalloidin, mostly at the periphery, but when irradiated with UVB, cells were rounded instead of dendritic, and colocalization was found predominantly at the cytoplasmic area. Further, suppression of Rab8 expression by its antisense oligonucleotide revealed the reduction in staining intensity of Rab8 but not of Mel5, dendrite formation and melanosome transport towards the tips of the dendrites in B16 melanoma cells. Taken together, it is suggestive that Rab8, in B16 melanoma cells, might have a role in melanosome traffic and dendrite extension, both in constitutive and regulated fashion.This investigation was supported in part by Grants-In-Aid for Scientific Research from the Ministry of Education, Science, and Culture, Japan (grant 15591176)     

Syndecan-1 ectodomain shedding is regulated by the small GTPase Rab5

The ectodomain shedding of syndecan-1, a major cell surface heparan sulfate proteoglycan, modulates molecular and cellular processes central to the pathogenesis of inflammatory diseases. Syndecan-1 shedding is a highly regulated process in which outside-in signaling accelerates the proteolytic cleavage of syndecan-1 ectodomains at the cell surface. Several extracellular agonists that induce syndecan-1 shedding and metalloproteinases that cleave syndecan-1 ectodomains have been identified, but the intracellular mechanisms that regulate syndecan-1 shedding are largely unknown. Here we examined the role of the syndecan-1 cytoplasmic domain in the regulation of agonist-induced syndecan-1 shedding. Our results showed that the syndecan-1 cytoplasmic domain is essential because mutation of invariant cytoplasmic Tyr residues abrogates ectodomain shedding, but not because it is Tyr phosphorylated upon shedding stimulation. Instead, our data showed that the syndecan-1 cytoplasmic domain binds to Rab5, a small GTPase that regulates intracellular trafficking and signaling events, and this interaction controls the onset of syndecan-1 shedding. Syndecan-1 cytoplasmic domain bound specifically to Rab5 and preferentially to inactive GDP-Rab5 over active GTP-Rab5, and shedding stimulation induced the dissociation of Rab5 from the syndecan-1 cytoplasmic domain. Moreover, the expression of dominant-negative Rab5, unable to exchange GDP for GTP, interfered with the agonist-induced dissociation of Rab5 from the syndecan-1 cytoplasmic domain and significantly inhibited syndecan-1 shedding induced by several distinct agonists. Based on these data, we propose that Rab5 is a critical regulator of syndecan-1 shedding that serves as an on-off molecular switch through its alternation between the GDP-bound and GTP-bound forms.     

Tyr-phosphorylation signals translocate RIN3, the small GTPase Rab5-GEF, to early endocytic vesicles

The small GTPase Rab5 plays a key role in early endocytic pathway, and its activation requires guanine-nucleotide exchange factors (GEFs). Rab5-GEFs share a conserved VPS9 domain for the GEF action, and RIN3 containing additional domains, such as Src-homology 2, RIN-family homology (RH), and Ras-association (RA), was identified as a new Rab5-GEF. However, precise functions of the additional domains and the activation mechanism of RIN3 remain unknown. Here, we found tyrosine-phosphorylation signals are involved in the Rab5-GEF activation. Treatment of HeLa cells with pervanadate translocates RIN3 from cytoplasm to the Rab5-positive vesicles. This RIN3 translocation was applied to various mutants lacking each domain of RIN3. Our present results suggest that a Ras GTPase(s) activated by tyrosine-phosphorylation signals interacts with the inhibitory RA domain, resulting in an active conformation of RIN3 as a Rab5-GEF and that RIN-unique RH domain constitutes a Rab5-binding region for the progress of GEF action.     

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.     

Rab24 is an atypical member of the Rab GTPase family. Deficient GTPase activity, GDP dissociation inhibitor interaction, and prenylation of Rab24 expressed in cultured cells

The function of Rab24 is currently unknown, but other members of the Rab GTPase family are known to participate in various protein trafficking pathways. Rab proteins are thought to cycle on and off vesicle membranes in conjunction with changes in their guanine nucleotide state. The present studies indicate that Rab24 possesses several unusual characteristics that distinguish it from other Rab proteins. 1) Based on [(32)P]orthophosphate labeling of protein-bound nucleotide, Rab24 exists predominantly in the GTP state when expressed in cultured cells. The low GTPase activity is related to the presence of serine instead of glutamine at the position cognate to Ras Gln-61. 2) Posttranslational geranylgeranylation of Rab24, determined by metabolic labeling or detergent partitioning assays, is inefficient when compared with other Rabs ending with the common CXC and CC carboxyl-terminal motifs. This is partly due to the presence of two histidines distal to the target cysteines, but also involves other unidentified features. 3) Most of the Rab24 in the cytoplasmic compartment of cultured cells is not associated with Rab GDP dissociation inhibitors. These findings indicate that, if Rab24 functions in vesicular transport processes, it may operate through a novel mechanism that does not depend on GTP hydrolysis or GDP dissociation inhibitor-mediated recycling.     

Rab8, a small GTPase involved in vesicular traffic between the TGN and the basolateral plasma membrane

Small GTP-binding proteins of the rab family have been implicated as regulators of membrane traffic along the biosynthetic and endocytic pathways in eukaryotic cells. We have investigated the localization and function of rab8, closely related to the yeast YPT1/SEC4 gene products. Confocal immunofluorescence microscopy and immunoelectron microscopy on filter-grown MDCK cells demonstrated that, rab8 was localized to the Golgi region, vesicular structures, and to the basolateral plasma membrane. Two-dimensional gel electrophoresis showed that rab8p was highly enriched in immuno-isolated basolateral vesicles carrying vesicular stomatitis virus-glycoprotein (VSV-G) but was absent from vesicles transporting the hemagglutinin protein (HA) of influenza virus to the apical cell surface. Using a cytosol dependent in vitro transport assay in permeabilized MDCK cells we studied the functional role of rab8 in biosynthetic membrane traffic. Transport of VSV-G from the TGN to the basolateral plasma membrane was found to be significantly inhibited by a peptide derived from the hypervariable COOH-terminal region of rab8, while transport of the influenza HA from the TGN to the apical surface and ER to Golgi transport were unaffected. We conclude that rab8 plays a role in membrane traffic from the TGN to the basolateral plasma membrane in MDCK cells.     

The crystal structure of the small GTPase Rab11b reveals critical differences relative to the Rab11a isoform

Rab GTPases constitute the largest family of small monomeric GTPases, including over 60 members in humans. These GTPases share conserved residues related to nucleotide binding and hydrolysis, and main sequence divergences lie in the carboxyl termini. They cycle between inactive (GDP-bound) and active (GTP-bound) forms and the active site regions, termed Switch I and II, undergo the larger conformational changes between the two states. The Rab11 subfamily members, comprising Rab11a, Rab11b, and Rab25, act in recycling of proteins from the endosomes to the plasma membrane, in transport of molecules from the trans-Golgi network to the plasma membrane and in phagocytosis. In this work, we describe Rab11b-GDP and Rab11b-GppNHp crystal structures solved to 1.55 and 1.95 angstroms resolution, respectively. Although Rab11b shares 90% amino acid identity to Rab11a, its crystal structure shows critical differences relative to previously reported Rab11a structures. Inactive Rab11a formed dimers with unusually ordered Switch regions and missing the magnesium ion at the nucleotide binding site. In this work, inactive Rab11b crystallized as a monomer showing a flexible Switch I and a magnesium ion which is coordinated by four water molecules, the phosphate beta of GDP (beta-P) and the invariant S25. S20 from the P-loop and S42 from the Switch I are associated to GTP hydrolysis rate. In the active structures, S20 interacts with the gamma-P oxygen in Rab11b-GppNHp but does not in Rab11a-GppNHp and the Q70 side chain is found in different positions. In the Rab11a-GTPgammaS structure, S40 is closer to S25 and S42 does not interact with the gamma-P oxygen. These differences indicate that the Rab11 isoforms may possess different GTP hydrolysis rates. In addition, the Switch II of inactive Rab11b presents a 3(10)-helix (residues 69-73) that disappears upon activation. This 3(10)-helix is not found in the Rab11a-GDP structure, which possesses a longer alpha2 helix, spanning from residue 73 to 82 alpha-helix 5.     

A role for the small GTPase Rab6 in assembly of human cytomegalovirus

In human-cytomegalovirus (HCMV)-infected cells, the localization of the viral protein pp150 to the virus assembly compartment (AC) is dependent on its direct interaction with the cellular protein Bicaudal D1 through a dynein- and microtubule-dependent mechanism. We found that the small GTPase Rab6 also interacts indirectly with pp150 through its interaction with Bicaudal D1. Inhibition of Rab6 activity in HCMV-infected cells interrupted the intracellular trafficking of pp150, significantly reducing infectious virus production without affecting the formation of the AC, arguing for an important function for this cellular GTPase in the intracellular localization of pp150 during virus assembly.     

Amino acid residues in the Ras-like GTPase Rab3A that specify sensitivity to factors that regulate the GTP/GDP cycling of Rab3A.

Two cellular factors have been described, Rab3A-GAP (GTPase-activating protein) and Rab3A-GRF (guanine nucleotide releasing factor) which, respectively, accelerate the intrinsic GTPase activity of, or the rate of dissociation of GDP from, the Ras-related GTP-binding protein, p25 Rab3A. Mutational analysis of p25 Rab3A was undertaken to define amino acid residues important for interaction with these factors. Mutations in residues 51-59, which correspond to the effector domain of p21 Ras, completely abolished sensitivity of p25 Rab3A to Rab3A-GRF and decreased the affinity of p25 Rab3A for Rab3A-GRF. Surprisingly, only one mutant in this region was Rab3A-GAP-insensitive, while the others retained partial, complete, or significantly increased GAP responsiveness. Mutations in the first G-domain had only modest effects on intrinsic GTPase activity and little effect on either Rab3A-GRF or Rab3A-GAP interactions. Truncation of 34 residues from the carboxyl terminus had no effect Rab3A-GAP sensitivity but facilitated Rab3A-GRF stimulation. Mutation T36N, analogous to the dominant inhibitory mutation T17N in Ras, which has been hypothesized to sequester an upstream activator of Ras, conferred a 10-fold higher affinity upon p25 Rab3A for Rab3A-GRF.     

Identification of a Rab GTPase-activating protein cascade that controls recycling of the Rab5 GTPase Vps21 from the vacuole

Transport within the endocytic pathway depends on a consecutive function of the endosomal Rab5 and the late endosomal/lysosomal Rab7 GTPases to promote membrane recycling and fusion in the context of endosomal maturation. We previously identified the hexameric BLOC-1 complex as an effector of the yeast Rab5 Vps21, which also recruits the GTPase-activating protein (GAP) Msb3. This raises the question of when Vps21 is inactivated on endosomes. We provide evidence for a Rab cascade in which activation of the Rab7 homologue Ypt7 triggers inactivation of Vps21. We find that the guanine nucleotide exchange factor (GEF) of Ypt7 (the Mon1-Ccz1 complex) and BLOC-1 both localize to the same endosomes. Overexpression of Mon1-Ccz1, which generates additional Ypt7-GTP, or overexpression of activated Ypt7 promotes relocalization of Vps21 from endosomes to the endoplasmic reticulum (ER), which is indicative of Vps21 inactivation. This ER relocalization is prevented by loss of either BLOC-1 or Msb3, but it also occurs in mutants lacking endosome–vacuole fusion machinery such as the HOPS tethering complex, an effector of Ypt7. Importantly, BLOC-1 interacts with the HOPS on vacuoles, suggesting a direct Ypt7-dependent cross-talk. These data indicate that efficient Vps21 recycling requires both Ypt7 and endosome–vacuole fusion, thus suggesting extended control of a GAP cascade beyond Rab interactions.     

A core complex of BBS proteins cooperates with the GTPase Rab8 to promote ciliary membrane biogenesis

Primary cilium dysfunction underlies the pathogenesis of Bardet-Biedl syndrome (BBS), a genetic disorder whose symptoms include obesity, retinal degeneration, and nephropathy. However, despite the identification of 12 BBS genes, the molecular basis of BBS remains elusive. Here we identify a complex composed of seven highly conserved BBS proteins. This complex, the BBSome, localizes to nonmembranous centriolar satellites in the cytoplasm but also to the membrane of the cilium. Interestingly, the BBSome is required for ciliogenesis but is dispensable for centriolar satellite function. This ciliogenic function is mediated in part by the Rab8 GDP/GTP exchange factor, which localizes to the basal body and contacts the BBSome. Strikingly, Rab8(GTP) enters the primary cilium and promotes extension of the ciliary membrane. Conversely, preventing Rab8(GTP) production blocks ciliation in cells and yields characteristic BBS phenotypes in zebrafish. Our data reveal that BBS may be caused by defects in vesicular transport to the cilium.     

IFT25 links the signal-dependent movement of Hedgehog components to intraflagellar transport

The intraflagellar transport (IFT) system is required for building primary cilia, sensory organelles that cells use to respond to their environment. IFT particles are composed of about 20 proteins, and these proteins are highly conserved across ciliated species. IFT25, however, is absent from some ciliated organisms, suggesting that it may have a unique role distinct from ciliogenesis. Here, we generate an Ift25 null mouse and show that IFT25 is not required for ciliary assembly but is required for proper Hedgehog signaling, which in mammals occurs within cilia. Mutant mice die at birth with multiple phenotypes, indicative of Hedgehog signaling dysfunction. Cilia lacking IFT25 have defects in the signal-dependent transport of multiple Hedgehog components including Patched-1, Smoothened, and Gli2, and fail to activate the pathway upon stimulation. Thus, IFT function is not restricted to building cilia where signaling occurs, but also plays a separable role in signal transduction events.     

Diacylglycerol-activated Hmunc13 serves as an effector of the GTPase Rab34

Hmunc13 is a cytosolic diacylglycerol (DAG)-binding protein, which is upregulated in renal cortical tubule and mesangial cells by hyperglycemia. In response to DAG activation, hmunc13 translocates to the Golgi. To investigate how this may relate to its function, we used a bacterial two-hybrid screen to look for hmunc13-interacting proteins. Full-length Rab34 was specifically isolated from a human kidney cDNA library. Co-expression of the two proteins confirmed Rab34 as a Golgi-associated protein, which was immunoprecipitated from cell lysates by hmunc13. Glutathione S-transferase fusion proteins of WT, Q111L (GTP bound), and T66N (GDP bound) mutants were created, and their GTP-binding activity verified by radioactive overlay assay. Binding of hmunc13 was observed with Q111L, barely detectable with T66N and enhanced with Rab34WT loaded with GTPgammaS compared with GDP loaded. Deletion of munc homolgy domain (MHD)-2, eliminated the hmunc13/Rab34 interaction. The Q111L mutant localized to the Golgi apparatus, but T66N was cytosolic. Localization of both mutants and Rab34WT was unchanged by DAG activation. The data suggest that DAG activation of hmunc13 causes it to be translocated to the Golgi, where it binds to GTP-bound Rab34 via MHD-2. Because Rab34 is known to regulate intracellular lysosome positioning, we propose that hmunc13 serves as an effector of Rab34, mediating lysosome-Golgi trafficking.     

Two distinct effectors of the small GTPase Rab5 cooperate in endocytic membrane fusion.

Using the yeast two-hybrid system, we have identified a novel 62 kDa coiled-coil protein that specifically interacts with the GTP-bound form of Rab5, a small GTPase that regulates membrane traffic in the early endocytic pathway. This protein shares 42% sequence identity with Rabaptin-5, a previously identified effector of Rab5, and we therefore named it Rabaptin-5beta. Like Rabaptin-5, Rabaptin-5beta displays heptad repeats characteristic of coiled-coil proteins and is recruited on the endosomal membrane by Rab5 in a GTP-dependent manner. However, Rabaptin-5beta has features that distinguish it from Rabaptin-5. The relative expression levels of the two proteins varies in different cell types. Rabaptin-5beta does not heterodimerize with Rabaptin-5, and forms a distinct complex with Rabex-5, the GDP/GTP exchange factor for Rab5. Immunodepletion of the Rabaptin-5beta complex from cytosol only partially inhibits early endosome fusion in vitro, whereas the additional depletion of the Rabaptin-5 complex has a stronger inhibitory effect. Fusion activity can mostly be recovered by addition of the Rabaptin-5 complex alone, but maximal fusion efficiency requires the presence of both Rabaptin-5 and Rabaptin-5beta complexes. Our results suggest that Rab5 binds to at least two distinct effectors which cooperate for optimal endocytic membrane docking and fusion.