Distinct Rab-binding domains mediate the interaction of Rabaptin-5 with GTP-bound Rab4 and Rab5.

Rabaptin-5 functions as an effector for the small GTPase Rab5, a regulator of endocytosis and early endosome fusion. We have searched for structural determinants that confer functional specificity on Rabaptin-5. Here we report that native cytosolic Rabaptin-5 is present in a homodimeric state and dimerization depends upon the presence of its coiled-coil predicted sequences. A 73 residue C-terminal region of Rabaptin-5 is necessary and sufficient both for the interaction with Rab5 and for Rab5-dependent recruitment of the protein on early endosomes. Surprisingly, we uncovered the presence of an additional Rab-binding domain at the N-terminus of Rabaptin-5. This domain mediates the direct interaction with the GTP-bound form of Rab4, a small GTPase that has been implicated in recycling from early endosomes to the cell surface. Based on these results, we propose that Rabaptin-5 functions as a molecular linker between two sequentially acting GTPases to coordinate endocytic and recycling traffic.     

Dimerization properties of Rabaptin-5 and its isoforms

Rabaptin-5 plays an important role in intracellular membrane traffic acting as an effector molecule of small GTPases Rab5 and Rab4. It was previously demonstrated that Rabaptin-5 exists as a part of a large protein complex in vivo and is able to form dimers in vitro. Data of X-ray structural analysis suggest that dimerization of Rabaptin-5 is an important feature required for its interaction with Rab5 GTPase. Recently several isoforms of Rabaptin-5 characterized by various deletions in the polypeptide chains have been identified. These isoforms might exhibit functional properties that differ from those of Rabaptin-5. In this study, we have investigated dimerization properties of delta and gamma isoforms of Rabaptin-5. In addition, we have provided the first direct evidence for Rabaptin-5 dimerization in cells.     

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.     

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.     

Apoptotic Cleavage of Rabaptin-5-like Proteins and a Model for Rabaptin-5 Inactivation in Apoptosis

Intracellular membrane transport from the plasma membrane is one of the processes affected in apoptotic cells. Apoptotic inhibition of endosomal transport occurs due to cleavage of Rabaptin-5, an effector of small GTPase Rab5, which results in inhibition of early endosome fusion. Recently several novel Rabaptin-5-like proteins were identified. We investigated whether Rabaptin-5-like proteins, Rabaptin-5? and Rabaptin-5?, are also cleaved in apoptosis and found that both proteins are cleaved in apoptotic cell extracts by caspase-3-related proteases. This suggests that functional inactivation of these proteins is necessary for apoptotic cell death. We also mapped a novel, N-terminal, putative Rab5 binding site in Rabaptin-5-like proteins, which becomes physically separated from the previously known C-terminal Rab5 binding site after apoptotic cleavage of these proteins. Presence of the second Rab5 binding site provides a new insight into Rabaptin-5 function in early endosome fusion and a mechanistic model for functional inactivation of Rabaptin-5 in apoptosis.     

Physical and functional interaction of KV10.1 with Rabaptin-5 impacts ion channel trafficking

K_V10.1 is a potassium channel expressed in brain and implicated in tumor progression. We have searched for proteins interacting with K_V10.1 and identified Rabaptin-5, an effector of the Rab5 GTPase. Both proteins co-localize on large early endosomes induced by Rab5 hyperactivity. Silencing of Rabaptin-5 induces down-regulation of recycling of K_V10.1 channel in transfected cells and reduction of K_V10.1 current density in cells natively expressing K_V10.1, indicating a role of Rabaptin-5 in channel trafficking. K_V10.1 co-localizes, but does not physically interact, with Rab7 and Rab11. Our data highlights the complex control of the amount of K_V10.1 channels on the cell surface.Structured summary of protein interactionsRabaptin-5 physically interacts with Kv10.1 by anti bait coimmunoprecipitation (View interaction)Rabaptin-5 physically interacts with Rabaptin-5 by two hybrid (View interaction)Kv10.1 physically interacts with Kv10.1 by two hybrid (View interaction)Kv10.1 physically interacts with Rabaptin-5 by anti bait coimmunoprecipitation (View Interaction: 1, 2)RAB11 and Kv10.1 colocalize by fluorescence microscopy (View interaction)Kv10.1 and Rabaptin-5 colocalize by fluorescence microscopy (View interaction)Kv10.1 physically interacts with Rabaptin-5 by two hybrid (View Interaction: 1, 2)Kv10.1 and RAB7 colocalize by fluorescence microscopy (View interaction)     

Rabaptin-5-independent membrane targeting and Rab5 activation by Rabex-5 in the cell

Rabex-5 is a guanine nucleotide exchange factor (GEF) for Rab5. Here, we report the identification of a novel functional domain of Rabex-5 that is essential for its membrane targeting and Rab5 GEF activity in vivo. The data show that full-length Rabex-5 efficiently activates Rab5 in the cell. However, the GEF domain itself (residues 135-399) is inactive in this respect, despite its activity in vitro. Generation and characterization of a series of Rabex-5 constructs reveal that the GEF domain is unable to target to early endosomes and that a sequence N-terminal to the GEF domain can restore its early endosomal targeting and its ability to activate Rab5 in the cell. This region (residues 81-135) is termed membrane-binding motif, which together with the downstream helical bundle domain (residues 135-230) forms an early endosomal targeting (EET) domain necessary and sufficient for association with early endosomes. Furthermore, several active Rabex-5 constructs do not contain the Rabaptin-5-binding domain in the C-terminal region. Thus, Rabex-5 can target to early endosomes via the EET domain and activate Rab5 in a Rabaptin-5-independent manner in vivo. We discuss a model to reconcile these in vivo data with previous in vitro results on Rabex-5 function and its interaction with Rabaptin-5.     

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.     

The ubiquitous dermokine delta activates Rab5 function in the early endocytic pathway

The expression of the recently identified dermokine (Dmkn) gene leads to four families of proteins with as yet unknown functions. The secreted α, β and γ isoforms share an epidermis-restricted expression pattern, whereas the δ isoform is intracellular and ubiquitous. To get an insight into Dmknδ function, we performed yeast two-hybrid screening and identified the small GTPases Rab5 as partners for Dmknδ. The Rab5 proteins are known to regulate membrane docking and fusion in the early endocytic pathway. GST pull-down assays confirmed the direct interaction between Rab5 and Dmknδ. Transient expression of Dmknδ in HeLa cells led to the formation of punctate structures colocalized with endogenous Rab5 and clathrin, indicating Dmknδ involvement in the early steps of endocytosis. Dmknδ indeed colocalized with transferrin at early stages of endocytosis, but did not modulate its endocytosis or recycling kinetics. We also showed that Dmknδ was able to bind both inactive (GDP-bound) and active (GTP-bound) forms of Rab5 in vitro but preferentially targeted GDP-bound form in HeLa cells. Interestingly, Dmknδ expression rescued the Rab5S34N-mediated inhibition of endosome fusion. Moreover, Dmknδ caused the enlargement of vesicles positive for Rab5 by promoting GTP loading onto the small GTPase. Together our data reveal that Dmknδ activates Rab5 function and thus is involved in the early endosomal trafficking.     

Gamma-adaptin interacts directly with Rabaptin-5 through its ear domain

In yeast two-hybrid screening using gamma1-adaptin, a subunit of the AP-1 adaptor complex of clathrin-coated vesicles derived from the trans-Golgi network (TGN), as bait, we found that it could interact with Rabaptin-5, an effector of Rab5 and Rab4 that regulates membrane docking with endosomes. Further two-hybrid analysis revealed that the interaction occurs between the ear domain of gamma1-adaptin and the COOH-terminal coiled-coil region of Rabaptin-5. Pull down assay with a fusion protein between glutathione S-transferase and the ear domain of gamma1-adaptin and coimmunoprecipitation analysis revealed that the interaction occurs in vitro and in vivo. Immunocytochemical analysis showed that gamma1-adaptin and Rabaptin-5 colocalize to a significant extent on perinuclear structures, probably on recycling endosomes, and are redistributed into the cytoplasm upon treatment with brefeldin A. These results suggest that the gamma1-adaptin-Rabaptin-5 interaction may play a role in membrane trafficking between the TGN and endosomes.     

The p85alpha subunit of phosphatidylinositol 3'-kinase binds to and stimulates the GTPase activity of Rab proteins

Rab5 and Rab4 are small monomeric GTPases localized on early endosomes and function in vesicle fusion events. These Rab proteins regulate the endocytosis and recycling or degradation of activated receptor tyrosine kinases such as the platelet-derived growth factor receptor (PDGFR). The p85alpha subunit of phosphatidylinositol 3'-kinase contains a BH domain with sequence homology to GTPase activating proteins (GAPs), but has not previously been shown to possess GAP activity. In this report, we demonstrate that p85alpha has GAP activity toward Rab5, Rab4, Cdc42, Rac1 and to a lesser extent Rab6, with little GAP activity toward Rab11. Purified recombinant Rab5 and p85alpha can bind directly to each other and not surprisingly, the p85alpha-encoded GAP activity is present in the BH domain. Because p85alpha stays bound to the PDGFR during receptor endocytosis, p85alpha will also be localized to the same early endosomal compartment as Rab5 and Rab4. Taken together, the physical co-localization and the ability of p85alpha to preferentially stimulate the down-regulation of Rab5 and Rab4 GTPases suggests that p85alpha regulates how long Rab5 and Rab4 remain in their GTP-bound active state. Cells expressing BH domain mutants of p85 show a reduced rate of PDGFR degradation as compared with wild type p85 expressing cells. These cells also show sustained activation of the mitogen-activated protein kinase and Akt pathways. Thus, the p85alpha protein may play a role in the down-regulation of activated receptors through its temporal control of the GTPase cycles of Rab5 and Rab4.     

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.     

Delayed Onset of Positive Feedback Activation of Rab5 by Rabex-5 and Rabaptin-5 in Endocytosis

Background

Rabex-5 is a guanine nucleotide exchange factor (GEF) that specifically activates Rab5, i.e., converting Rab5-GDP to Rab5-GTP, through two distinct pathways to promote endosome fusion and endocytosis. The direct pathway involves a pool of membrane-associated Rabex-5 that targets to the membrane via an early endosomal targeting (EET) domain. The indirect pathway, on the other hand, involves a cytosolic pool of Rabex-5/Rabaptin-5 complex. The complex is recruited to the membrane via Rabaptin-5 binding to Rab5-GTP, suggesting a positive feedback mechanism. The relationship of these two pathways for Rab5 activation in the cell is unclear.

Methodology/Principal Findings

We dissect the relative contribution of each pathway to Rab5 activation via mathematical modeling and kinetic analysis in the cell. These studies show that the indirect pathway constitutes a positive feedback loop for converting Rab5-GDP to Rab5-GTP on the endosomal membrane and allows sensitive regulation of endosome fusion activity by the levels of Rab5 and Rabex-5 in the cell. The onset of this positive feedback effect, however, contains a threshold, which requires above endogenous levels of Rab5 or Rabex-5 in the cell. We term this novel phenomenon “delayed response”. The presence of the direct pathway reduces the delay by increasing the basal level of Rab5-GTP, thus facilitates the function of the Rabex-5/Rabaptin-5-mediated positive feedback loop.

Conclusion

Our data support the mathematical model. With the model''s guidance, the data reveal the affinity of Rabex-5/Rabaptin-5/Rab5-GTP interaction in the cell, which is quantitatively related to the Rabex-5 concentration for the onset of the indirect positive feedback pathway. The presence of the direct pathway and increased Rab5 concentration can reduce the Rabex-5 concentration required for the onset of the positive feedback loop. Thus the direct and indirect pathways cooperate in the regulation of early endosome fusion.     

Direct interaction of EEA1 with Rab5b.

The early endosomal autoantigen EEA1 is essential for early endosomal membrane fusion. It binds to endosomes via a C-terminal domain (EEA1-CT). To identify proteins interacting with EEA1-CT, we screened a human brain library in the yeast two-hybrid system. Fourteen clones reacted strongly with EEA1-CT. Sequencing of these clones revealed that they all contained the ORF of the small GTPase, Rab5b. Further two-hybrid analysis suggested that Rab5b also interacts with the N-terminus of EEA1 (EEA1-NT). The interaction of both EEA1-CT and EEA1-NT with Rab5b was confirmed biochemically, and was found to be GTP dependent. Confocal immunofluorescence microscopy indicated that EEA1 colocalizes with Rab5b on early endosomes. Although EEA1-CT and EEA1-NT interacted strongly with wild-type Rab5b in the two-hybrid system, we detected no interaction with wild-type Rab5a, even though GTPase-deficient mutants of both Rab5a and Rab5b interacted equally well with EEA1. This difference could not be explained by differences in intrinsic GTPase activities, as these were found to be very similar. Instead, we speculate that yeast may contain a GTPase-activating protein (GAP) activity that stimulates Rab5a but not Rab5b. In contrast, pig brain cytosol was found to contain a GAP activity that stimulates the GTPase activity of Rab5b in preference to that of Rab5a. These data provide evidence that EEA1 interacts with both Rab5a and Rab5b, and that the GTPase activities of the two proteins are differentially regulated in vivo.     

Divalent interaction of the GGAs with the Rabaptin-5-Rabex-5 complex

Cargo transfer from trans-Golgi network (TGN)-derived transport carriers to endosomes involves a still undefined set of tethering/fusion events. Here we analyze a molecular interaction that may play a role in this process. We demonstrate that the GGAs, a family of Arf-dependent clathrin adaptors involved in selection of TGN cargo, interact with the Rabaptin-5-Rabex-5 complex, a Rab4/Rab5 effector regulating endosome fusion. These interactions are bipartite: GGA-GAE domains recognize an FGPLV sequence (residues 439-443) in a predicted random coil of Rabaptin-5 (a sequence also recognized by the gamma1- and gamma2-adaptin ears), while GGA-GAT domains bind to the C-terminal coiled-coils of Rabaptin-5. The GGA-Rabaptin-5 interaction decreases binding of clathrin to the GGA-hinge domain, and expression of green fluorescent protein (GFP)-Rabaptin-5 shifts the localization of endogenous GGA1 and associated cargo to enlarged early endosomes. These observations thus identify a binding sequence for GAE/gamma-adaptin ear domains and reveal a functional link between proteins regulating TGN cargo export and endosomal tethering/fusion events.     

The intracellular trafficking of the G protein-coupled receptor TPbeta depends on a direct interaction with Rab11

Intracellular trafficking pathways of cell surface receptors following their internalization are the subject of intense research efforts. However, the mechanisms by which they recycle back to the cell surface are still poorly defined. We have recently demonstrated that the small Rab11 GTPase protein is a determinant factor in controlling the recycling to the cell surface of the beta-isoform of the thromboxane A2 receptor (TPbeta) following its internalization. Here, we demonstrate with co-immunoprecipitation studies in HEK293 cells that there is a Rab11-TPbeta association occurring in the absence of agonist, which is not modulated by stimulation of TPbeta. We show with purified TPbeta intracellular domains fused to GST and HIS-Rab11 proteins that Rab11 interacts directly with the first intracellular loop and the C-tail of TPbeta. Amino acids 335-344 of the TPbeta C-tail were determined to be essential for the interaction of Rab11 with this receptor domain. This identified sequence appears to be important in directing the intracellular trafficking of the receptor from the Rab5-positive intracellular compartment to the perinuclear recycling endosome. Interestingly, our data indicate that TPbeta interacts with the GDP-bound form, and not the GTP-bound form, of Rab11 which is necessary for recycling of the receptor back to the cell surface. To our knowledge, this is the first demonstration of a direct interaction between Rab11 and a transmembrane receptor.     

D-AKAP2 Interacts with Rab4 and Rab11 through Its RGS Domains and Regulates Transferrin Receptor Recycling

Dual-specific A-kinase-anchoring protein 2 (D-AKAP2/AKAP10), which interacts at its carboxyl terminus with protein kinase A and PDZ domain proteins, contains two tandem regulator of G-protein signaling (RGS) domains for which the binding partners have remained unknown. We show here that these RGS domains interact with Rab11 and GTP-bound Rab4, the first demonstration of RGS domains binding small GTPases. Rab4 and Rab11 help regulate membrane trafficking through the endocytic recycling pathways by recruiting effector proteins to specific membrane domains. Although D-AKAP2 is primarily cytosolic in HeLa cells, a fraction of the protein localizes to endosomes and can be recruited there to a greater extent by overexpression of Rab4 or Rab11. D-AKAP2 also regulates the morphology of the Rab11-containing compartment, with co-expression causing accumulation of both proteins on enlarged endosomes. Knockdown of D-AKAP2 by RNA interference caused a redistribution of both Rab11 and the constitutively recycling transferrin receptor to the periphery of cells. Knockdown also caused an increase in the rate of transferrin recycling, suggesting that D-AKAP2 promotes accumulation of recycling proteins in the Rab4/Rab11-positive endocytic recycling compartment.     

A novel binding protein composed of homophilic tetramer exhibits unique properties for the small GTPase Rab5.

The small GTPase Rab family, which cycles between GTP-bound active and GDP-bound inactive states, plays an important role in membrane trafficking. Among them, Rab5 is involved in early endocytic pathway, and several Rab5-binding proteins have been identified as regulators or effectors to coordinate the docking and fusion processes of endocytic vesicles. We describe a novel binding protein exhibiting unique biochemical properties for Rab5. The Rab5-binding protein enhances GDP-GTP exchange reaction on Rab5 but preferentially interacts with its GTP-bound form. Gel filtration and immunoprecipitation analyses indicate that the Rab5-binding protein functions as a tetramer composed of anti-parallel linkage of two parallel dimers. These results suggest that the newly identified protein may function as an upstream activator and/or downstream effector for Rab5 in endocytic pathway. Possible roles of the quaternary structure have been discussed in terms of the Rab5-mediated signaling.     

Myosin5a tail associates directly with Rab3A-containing compartments in neurons

Myosin-Va (Myo5a) is a motor protein associated with synaptic vesicles (SVs) but the mechanism by which it interacts has not yet been identified. A potential class of binding partners are Rab GTPases and Rab3A is known to associate with SVs and is involved in SV trafficking. We performed experiments to determine whether Rab3A interacts with Myo5a and whether it is required for transport of neuronal vesicles. In vitro motility assays performed with axoplasm from the squid giant axon showed a requirement for a Rab GTPase in Myo5a-dependent vesicle transport. Furthermore, mouse recombinant Myo5a tail revealed that it associated with Rab3A in rat brain synaptosomal preparations in vitro and the association was confirmed by immunofluorescence imaging of primary neurons isolated from the frontal cortex of mouse brains. Synaptosomal Rab3A was retained on recombinant GST-tagged Myo5a tail affinity columns in a GTP-dependent manner. Finally, the direct interaction of Myo5a and Rab3A was determined by sedimentation velocity analytical ultracentrifugation using recombinant mouse Myo5a tail and human Rab3A. When both proteins were incubated in the presence of 1 mm GTPγS, Myo5a tail and Rab3A formed a complex and a direct interaction was observed. Further analysis revealed that GTP-bound Rab3A interacts with both the monomeric and dimeric species of the Myo5a tail. However, the interaction between Myo5a tail and nucleotide-free Rab3A did not occur. Thus, our results show that Myo5a and Rab3A are direct binding partners and interact on SVs and that the Myo5a/Rab3A complex is involved in transport of neuronal vesicles.