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.     

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.     

Molecular dissection of Rab11 binding from coiled-coil formation in the Rab11-FIP2 C-terminal domain

The Rab11-family interacting protein (Rab11-FIP) group of effector proteins contain a highly conserved region in their C-termini that bind the GTPase, Rab11. Rab11 belongs to the largest family of small GTPases and is believed to regulate vesicle docking with target membranes and vesicle fusion. The amino acid sequence of the Rab11-FIP proteins predicts coiled-coil formation in the conserved C-terminal domain. In this study on Rab11-FIP2, we found experimental evidence for the coiled-coil and then defined the minimal structured core using limited proteolysis. We also showed that the Rab11-FIP2 coiled-coil domain forms a parallel homodimer in solution using cross-linking and mutagenesis and sedimentation equilibrium experiments. Various constructs representing the C-terminal domain of Rab11-FIP2 were characterized by circular dichroism, and their affinity with Rab11 was measured using isothermal titration calorimetry. The longest construct was both well-structured and bound Rab11. A construct truncated at the N-terminus was poorly structured but retained the same affinity for binding to Rab11. Conformational changes were also demonstrated upon complex formation between Rab11 and Rab11-FIP2. A construct truncated at the C-terminus, which was the minimal coiled-coil domain defined by limited proteolysis, did not retain the ability to interact with Rab11, although it was as well-structured as the longer peptide. These data show that coiled-coil formation and Rab11 binding are separable functions of the C-terminal domain of Rab11-FIP2. The dissection of Rab11 binding from the formation of defined structure in a coiled-coil provides a potential mechanism for regulating Rab11-dependent endosomal trafficking.     

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.     

Phosphorylation provides a negative mode of regulation for the yeast Rab GTPase Sec4p

The Rab family of Ras-related GTPases are part of a complex signaling circuitry in eukaryotic cells, yet we understand little about the mechanisms that underlie Rab protein participation in such signal transduction networks, or how these networks are integrated at the physiological level. Reversible protein phosphorylation is widely used by cells as a signaling mechanism. Several phospho-Rabs have been identified, however the functional consequences of the modification appear to be diverse and need to be evaluated on an individual basis. In this study we demonstrate a role for phosphorylation as a negative regulatory event for the action of the yeast Rab GTPase Sec4p in regulating polarized growth. Our data suggest that the phosphorylation of the Rab Sec4p prevents interactions with its effector, the exocyst component Sec15p, and that the inhibition may be relieved by a PP2A phosphatase complex containing the regulatory subunit Cdc55p.     

Isoprenylation of Rab proteins possessing a C-terminal CaaX motif

Rab proteins are small GTPases highly related to the yeast Ypti and Sec4 proteins involved in secretion. The Rab proteins were found associated with membranes of different compartments along the secretory and endocytic pathways. They share distinct C-terminal cysteine motifs required for membrane association. Unlike the other Rab proteins, Rab8, Rab11 and Rab13 terminate with a C-terminal CaaX motif similar to those of Ras/Rho proteins. This report demonstrates that Rab8 and Rab13 proteins are isoprenylated in vivo and geranylgeranylated in vitro. Rab11 associates in vitro geranylgeranylpyrophosphate and farnesylpyrophosphate. Our study shows that the CaaX motif is required for isoprenylation.     

Exocyst requirement for endocytic traffic directed toward the apical and basolateral poles of polarized MDCK cells

The octameric exocyst complex is associated with the junctional complex and recycling endosomes and is proposed to selectively tether cargo vesicles directed toward the basolateral surface of polarized Madin-Darby canine kidney (MDCK) cells. We observed that the exocyst subunits Sec6, Sec8, and Exo70 were localized to early endosomes, transferrin-positive common recycling endosomes, and Rab11a-positive apical recycling endosomes of polarized MDCK cells. Consistent with its localization to multiple populations of endosomes, addition of function-blocking Sec8 antibodies to streptolysin-O-permeabilized cells revealed exocyst requirements for several endocytic pathways including basolateral recycling, apical recycling, and basolateral-to-apical transcytosis. The latter was selectively dependent on interactions between the small GTPase Rab11a and Sec15A and was inhibited by expression of the C-terminus of Sec15A or down-regulation of Sec15A expression using shRNA. These results indicate that the exocyst complex may be a multipurpose regulator of endocytic traffic directed toward both poles of polarized epithelial cells and that transcytotic traffic is likely to require Rab11a-dependent recruitment and modulation of exocyst function, likely through interactions with Sec15A.     

Structure-Function Analyses of the Interactions between Rab11 and Rab14 Small GTPases with Their Shared Effector Rab Coupling Protein (RCP)

Rab GTPases recruit effector proteins, via their GTP-dependent switch regions, to distinct subcellular compartments. Rab11 and Rab25 are closely related small GTPases that bind to common effectors termed the Rab11 family of interacting proteins (FIPs). The FIPs are organized into two subclasses (class I and class II) based on sequence and domain organization, and both subclasses contain a highly conserved Rab-binding domain at their C termini. Yeast two-hybrid and biochemical studies have revealed that the more distantly related Rab14 also interacts with class I FIPs. Here, we perform detailed structural, thermodynamic, and cellular analyses of the interactions between Rab14 and one of the class I FIPs, the Rab-coupling protein (RCP), to clarify the molecular aspects of the interaction. We find that Rab14 indeed binds to RCP, albeit with reduced affinity relative to conventional Rab11-FIP and Rab25-FIP complexes. However, in vivo, Rab11 recruits RCP onto biological membranes. Furthermore, biophysical analyses reveal a noncanonical 1:2 stoichiometry between Rab14-RCP in dilute solutions, in contrast to Rab11/25 complexes. The structure of Rab14-RCP reveals that Rab14 interacts with the canonical Rab-binding domain and also provides insight into the unusual properties of the complex. Finally, we show that both the Rab coupling protein and Rab14 function in neuritogenesis.     

Rab coupling protein (RCP), a novel Rab4 and Rab11 effector protein.

Rab4 and Rab11 are small GTPases belonging to the Ras superfamily. They both function as regulators along the receptor recycling pathway. We have identified a novel 80-kDa protein that interacts specifically with the GTP-bound conformation of Rab4, and subsequent work has shown that it also interacts strongly with Rab11. We name this protein Rab coupling protein (RCP). RCP is predominantly membrane-bound and is expressed in all cell lines and tissues tested. It colocalizes with early endosomal markers including Rab4 and Rab11 as well as with the transferrin receptor. Overexpression of the carboxyl-terminal region of RCP, which contains the Rab4- and Rab11-interacting domain, results in a dramatic tubulation of the transferrin compartment. Furthermore, expression of this mutant causes a significant reduction in endosomal recycling without affecting ligand uptake or degradation in quantitative assays. RCP is a homologue of Rip11 and therefore belongs to the recently described Rab11-FIP family.     

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.     

A Rab8 guanine nucleotide exchange factor-effector interaction network regulates primary ciliogenesis

Primary cilia are microtubule-based solitary membrane projections on the cell surface that play important roles in signaling and development. Recent studies have demonstrated that polarized vesicular trafficking involving the small GTPase Rab8 and its guanine nucleotide exchange factor Rabin8 is essential for primary ciliogenesis. In this study, we show that a highly conserved region of Rabin8 is pivotal for its activation as a guanine nucleotide exchange factor for Rab8. In addition, in its activated conformation, Rabin8 interacts with Sec15, a subunit of the exocyst and downstream effector of Rab8. Expression of constitutively activated Rab8 promotes the association of Sec15 with Rabin8. Using immunofluorescence microscopy, we found that Sec15 co-localized with Rab8 along the primary cilium. Inhibition of Sec15 function in cells led to defects in primary ciliogenesis. The Rabin8-Rab8-Sec15 interaction may couple the activation of Rab8 to the recruitment of the Rab8 effector and is involved in the regulation of vesicular trafficking for primary cilium formation.     

The novel Rab11-FIP/Rip/RCP family of proteins displays extensive homo- and hetero-interacting abilities

The Rab11-FIP/Rip/RCP proteins are a recently described novel protein family, whose members interact with Rab GTPases that function in endosomal recycling. To date, five such proteins have been described in humans, all of which interact with Rab11, and one (RCP) also interacts with Rab4. Here, we characterise several of these proteins with respect to their ability to interact with Rab4, as well as their ability to self-interact, and to interact with each other. We now demonstrate that two of the family members-pp75/Rip11 and Rab11-FIP3 do not bind Rab4 and show that several members of the family can self-interact and interact with each other. These interactions primarily involve their C-terminal end which includes the Rab binding domain (RBD) that is contained within a predicted coiled-coil, or ERM motif. We identify a new (sixth) member of the protein family, which we propose to name Rab11-FIP4, and report the family evolutionary complexity and chromosomal distribution. Furthermore, we propose that the ability of these proteins to bind each other will be important in effecting membrane trafficking events by forming protein 'platforms,' regulated by Rab11 and/or Rab4 activity.     

Class I Rab11‐family interacting proteins are binding targets for the Rab14 GTPase

Background information. Rab11 and Rab14 are two related Rab GTPases that are believed to function in endosomal recycling and Golgi/endosome transport processes. We, and others, have identified a group of proteins that interact with Rab11 and function as Rab11 effectors, known as the Rab11‐FIPs (family interacting proteins). This protein family has been sub‐classified into two groups—class I FIPs [FIP2, RCP (Rab coupling protein) and Rip11 (Rab11‐interacting protein)] and class II FIPs (FIP3 and FIP4). Results. In the present study we identify the class I FIPs as dual Rab‐binding proteins by demonstrating that they also interact with Rab14 in a GTP‐dependent manner. We show that these interactions are specific for the class I FIPs and that they occur via their C‐terminal regions, which encompass the previously described RBD (Rab11‐binding domain). Furthermore, we show that Rab14 significantly co‐localizes with the TfnR (transferrin receptor) and that Rab14 Q70L co‐localizes with Rab11a and with the class I FIPs on the ERC (endosomal recycling compartment) during interphase. Additionally, we show that during cytokinesis Rab14 localizes to the cleavage furrow/midbody. Conclusions. The data presented in the present study, which identifies the class I FIPs as the first putative effector proteins for the Rab14 GTPase, indicates greater complexity in the Rab‐binding specificity of the class I FIP proteins.     

Babesia gibsoni: molecular cloning and characterization of Rab6 and Rab11 homologues

Members of the Rab subfamily of GTPases have been implicated as important components in vesicle trafficking in the eukaryotes, individual Rab proteins have a remarkable degree of specific subcellular localization. As a first step towards developing a set of compartment specific probes for studying protein trafficking in Babesia-infected erythrocyte, here we describe the cloning and characterization of Rab6 and Rab11 gene homologues in Babesia gibsoni (BgRab6 and BgRab11). The deduced amino acid sequence of both BgRab6 and BgRab11 contained the highly conserved GTP-binding consensus sequence and C-terminal cysteines. Northern blotting analysis of total RNA hybridized a 1.3 kb band on both BgRab6 and BgRab11 probed blots consistent with the expected size. Using a GTP-binding assay we demonstrated that Escherichia coli expressed recombinant BgRab6 and BgRab11 were able to bind GTP. BgRab6 and BgRab11 represent the first two molecular markers of B. gibsoni.     

Rab11A controls the biogenesis of Birbeck granules by regulating Langerin recycling and stability

The extent to which Rab GTPases, Rab-interacting proteins, and cargo molecules cooperate in the dynamic organization of membrane architecture remains to be clarified. Langerin, a recycling protein accumulating in the Rab11-positive compartments of Langerhans cells, induces the formation of Birbeck granules (BGs), which are membrane subdomains of the endosomal recycling network. We investigated the role of Rab11A and two members of the Rab11 family of interacting proteins, Rip11 and RCP, in Langerin traffic and the biogenesis of BGs. The overexpression of a dominant-negative Rab11A mutant or Rab11A depletion strongly influenced Langerin traffic and stability and the formation of BGs, whereas modulation of other Rab proteins involved in dynamic regulation of the endocytic-recycling pathway had no effect. Impairment of Rab11A function led to a missorting of Langerin to lysosomal compartments, but inhibition of Langerin degradation by chloroquine did not restore the formation of BGs. Loss of RCP, but not of Rip11, also had a modest, but reproducible effect on Langerin stability and BG biogenesis, pointing to a role for Rab11A-RCP complexes in these events. Our results show that Rab11A and Langerin are required for BG biogenesis, and they illustrate the role played by a Rab GTPase in the formation of a specialized subcompartment within the endocytic-recycling system.     

The yeast lgl family member Sro7p is an effector of the secretory Rab GTPase Sec4p

Rab guanosine triphosphatases regulate intracellular membrane traffic by binding specific effector proteins. The yeast Rab Sec4p plays multiple roles in the polarized transport of post-Golgi vesicles to, and their subsequent fusion with, the plasma membrane, suggesting the involvement of several effectors. Yet, only one Sec4p effector has been documented to date: the exocyst protein Sec15p. The exocyst is an octameric protein complex required for tethering secretory vesicles, which is a prerequisite for membrane fusion. In this study, we describe the identification of a second Sec4p effector, Sro7p, which is a member of the lethal giant larvae tumor suppressor family. Sec4-GTP binds to Sro7p in cell extracts as well as to purified Sro7p, and the two proteins can be coimmunoprecipitated. Furthermore, we demonstrate the formation of a ternary complex of Sec4-GTP, Sro7p, and the t-SNARE Sec9p. Genetic data support our conclusion that Sro7p functions downstream of Sec4p and further imply that Sro7p and the exocyst share partially overlapping functions, possibly in SNARE regulation.     

Rab11-FIP4 interacts with Rab11 in a GTP-dependent manner and its overexpression condenses the Rab11 positive compartment in HeLa cells

We have recently identified Rab11-FIP4 as the sixth member of the Rab11-FIP family of Rab11 interacting proteins. Here, we demonstrate that Rab11-FIP4 interacts with Rab11 in a GTP-dependent manner and that its C-terminal region allows the protein to self-interact and interact with pp75/Rip11, Rab11-FIP2, and Rab11-FIP3. However, Rab11-FIP4 does not appear to interact directly with Rab coupling protein (RCP). We investigated the subcellular localisation of Rab11-FIP4 in HeLa cells and show that it colocalises extensively with transferrin and with Rab11. Furthermore, when overexpressed, it causes a condensation of the Rab11 compartment in the perinuclear region. We demonstrate that the carboxy-terminal region of Rab11-FIP4 (Rab11-FIP4(C-ter)) is necessary and sufficient for its endosomal membrane association. Expression of Rab11-FIP4(C-ter) causes a dispersal of the Rab11 compartment towards the cell periphery and does not inhibit transferrin recycling in HeLa cells. It is likely that Rab11-FIP4 serves as a Rab11 effector in a Rab11 mediated function other than transferrin recycling.     

Structural basis for Rab11-mediated recruitment of FIP3 to recycling endosomes

The Rab11 GTPase regulates recycling of internalized plasma membrane receptors and is essential for completion of cytokinesis. A family of Rab11 interacting proteins (FIPs) that conserve a C-terminal Rab-binding domain (RBD) selectively recognize the active form of Rab11. Normal completion of cytokinesis requires a complex between Rab11 and FIP3. Here, we report the crystal structure and mutational analysis of a heterotetrameric complex between constitutively active Rab11 and a FIP3 construct that includes the RBD. Two Rab11 molecules bind to dyad symmetric sites at the C terminus of FIP3, which forms a non-canonical coiled-coiled dimer with a flared C terminus and hook region. The RBD overlaps with the coiled coil and extends through the C-terminal hook. Although FIP3 engages the switch and interswitch regions of Rab11, the mode of interaction differs significantly from that of other Rab-effector complexes. In particular, the switch II region undergoes a large structural rearrangement from an ordered but non-complementary active conformation to a remodeled conformation that facilitates the interaction with FIP3. Finally, we provide evidence that FIP3 can form homo-oligomers in cells, and that a critical determinant of Rab11 binding in vitro is necessary for FIP3 recruitment to recycling endosomes during cytokinesis.     

Molecular characterization of Rab11 interactions with members of the family of Rab11-interacting proteins

The Rab11 subfamily of GTPases plays an important role in vesicle trafficking from endosomes to the plasma membrane. At least six Rab11 effectors (family of Rab11-interacting proteins (FIPs)) have been shown to interact with Rab11 and are hypothesized to regulate various membrane trafficking pathways such as transferrin recycling, cytokinesis, and epidermal growth factor trafficking. In this study, we characterized interactions of FIPs with the Rab11 GTPase using isothermal titration calorimetric studies and mutational analysis. Our data suggest that FIPs cannot differentiate between GTP-bound Rab11a and Rab11b in vitro (50-100 nm affinity) and in vivo. We also show that, although FIPs interact with the GDP-bound form of Rab11 in vitro, the binding affinity (>1000 nm) is not sufficient for FIP and GDP-bound Rab11 interactions to occur in vivo. Mutational analysis revealed that both the conserved hydrophobic patch and Tyr628 are important for the GTP-dependent binding of Rab11 to FIPs. The entropy and enthalpy analyses suggest that binding to Rab11a/b may induce conformational changes in FIPs.