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.     

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.     

Rab22 controls NGF signaling and neurite outgrowth in PC12 cells

Rab22 is a small GTPase that is localized on early endosomes and regulates early endosomal sorting. This study reports that Rab22 promotes nerve growth factor (NGF) signaling-dependent neurite outgrowth and gene expression in PC12 cells by sorting NGF and the activated/phosphorylated receptor (pTrkA) into signaling endosomes to sustain signal transduction in the cell. NGF binding induces the endocytosis of pTrkA into Rab22-containing endosomes. Knockdown of Rab22 via small hairpin RNA (shRNA) blocks NGF-induced pTrkA endocytosis into the endosomes and gene expression (VGF) and neurite outgrowth. Overexpression of human Rab22 can rescue the inhibitory effects of the Rab22 shRNA, suggesting a specific Rab22 function in NGF signal transduction, rather than off-target effects. Furthermore, the Rab22 effector, Rabex-5, is necessary for NGF-induced neurite outgrowth and gene expression, as evidenced by the inhibitory effect of shRNA-mediated knockdown of Rabex-5. Disruption of the Rab22-Rabex-5 interaction via overexpression of the Rab22-binding domain of Rabex-5 in the cell also blocks NGF-induced neurite outgrowth, suggesting a critical role of Rab22-Rabex-5 interaction in the biogenesis of NGF-signaling endosomes to sustain the signal for neurite outgrowth. These data provide the first evidence for an early endosomal Rab GTPase as a positive regulator of NGF signal transduction and cell differentiation.     

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.     

RabGEFs are a major determinant for specific Rab membrane targeting

Eukaryotic cells critically depend on the correct regulation of intracellular vesicular trafficking to transport biological material. The Rab subfamily of small guanosine triphosphatases controls these processes by acting as a molecular on/off switch. To fulfill their function, active Rab proteins need to localize to intracellular membranes via posttranslationally attached geranylgeranyl lipids. Each member of the manifold Rab family localizes specifically to a distinct membrane, but it is unclear how this specific membrane recruitment is achieved. Here, we demonstrate that Rab-activating guanosine diphosphate/guanosine triphosphate exchange factors (GEFs) display the minimal targeting machinery for recruiting Rabs from the cytosol to the correct membrane using the Rab-GEF pairs Rab5A–Rabex-5, Rab1A-DrrA, and Rab8-Rabin8 as model systems. Specific mistargeting of Rabex-5/DrrA/Rabin8 to mitochondria led to catalytic recruitment of Rab5A/Rab1A/Rab8A in a time-dependent manner that required the catalytic activity of the GEF. Therefore, RabGEFs are major determinants for specific Rab membrane targeting.     

Nerve growth factor-mediated neurite outgrowth via regulation of Rab5

Nerve growth factor (NGF) induces neurite outgrowth and differentiation in a process that involves NGF binding to its receptor TrkA and endocytosis of the NGF-TrkA complex into signaling endosomes. Here, we find that biogenesis of signaling endosomes requires inactivation of Rab5 to block early endosome fusion. Expression of dominant-negative Rab5 mutants enhanced NGF-mediated neurite outgrowth, whereas a constitutively active Rab5 mutant or Rabex-5 inhibited this process. Consistently, inactivation of Rab5 sustained TrkA activation on the endosomes. Furthermore, NGF treatment rapidly decreased cellular level of active Rab5-GTP, as shown by pull-down assays. This Rab5 down-regulation was mediated by RabGAP5, which was shown to associate with TrkA by coimmunoprecipitation assays. Importantly, RNA interference of RabGAP5 as well as a RabGAP5 truncation mutant containing the TrkA-binding domain blocked NGF-mediated neurite outgrowth, indicating a requirement for RabGAP5 in this process. Thus, NGF signaling down-regulates Rab5 activity via RabGAP5 to facilitate neurite outgrowth and differentiation.     

Lpg0393 of Legionella pneumophila Is a Guanine-Nucleotide Exchange Factor for Rab5, Rab21 and Rab22

Legionella pneumophila, a human intracellular pathogen, encodes about 290 effector proteins that are translocated into host cells through a secretion machinery. Some of these proteins have been shown to manipulate or subvert cellular processes during infection, but functional roles of a majority of them remain unknown. Lpg0393 is a newly identified Legionella effector classified as a hypothetical protein. Through X-ray crystallographic analysis, we show that Lpg0393 contains a Vps9-like domain, which is structurally most similar to the catalytic core of human Rabex-5 that activates the endosomal Rab proteins Rab5, Rab21 and Rab22. Consistently, Lpg0393 exhibited a guanine-nucleotide exchange factor activity toward the endosomal Rabs. This work identifies the first example of a bacterial guanine-nucleotide exchange factor that is active towards the Rab5 sub-cluster members, implying that the activation of these Rab proteins might be advantageous for the intracellular survival of Legionella.     

Rab5 regulates motility of early endosomes on microtubules

The small GTPase Rab5 regulates membrane docking and fusion in the early endocytic pathway. Here we reveal a new role for Rab5 in the regulation of endosome interactions with the microtubule network. Using Rab5 fused to green fluorescent protein we show that Rab5-positive endosomes move on microtubules in vivo. In vitro, Rab5 stimulates both association of early endosomes with microtubules and early-endosome motility towards the minus ends of microtubules. Moreover, similarly to endosome membrane docking and fusion, Rab5-dependent endosome movement depends on the phosphatidylinositol-3-OH kinase hVPS34. Thus, Rab5 functionally links regulation of membrane transport, motility and intracellular distribution of early endosomes.     

Regulation of retromer recruitment to endosomes by sequential action of Rab5 and Rab7

The retromer complex mediates retrograde transport of transmembrane cargo from endosomes to the trans-Golgi network (TGN). Mammalian retromer is composed of a sorting nexin (SNX) dimer that binds to phosphatidylinositol 3-phosphate–enriched endosomal membranes and a vacuolar protein sorting (Vps) 26/29/35 trimer that participates in cargo recognition. The mammalian SNX dimer is necessary but not sufficient for recruitment of the Vps26/29/35 trimer to membranes. In this study, we demonstrate that the guanosine triphosphatase Rab7 contributes to this recruitment. The Vps26/29/35 trimer specifically binds to Rab7–guanosine triphosphate (GTP) and localizes to Rab7-containing endosomal domains. Interference with Rab7 function causes dissociation of the Vps26/29/35 trimer but not the SNX dimer from membranes. This blocks retrieval of mannose 6-phosphate receptors to the TGN and impairs cathepsin D sorting. Rab5-GTP does not bind to the Vps26/29/35 trimer, but perturbation of Rab5 function causes dissociation of both the SNX and Vps26/29/35 components from membranes through inhibition of a pathway involving phosphatidylinositol 3-kinase. These findings demonstrate that Rab5 and Rab7 act in concert to regulate retromer recruitment to endosomes.     

Regulation of intracellular trafficking of the EGF receptor by Rab5 in the absence of phosphatidylinositol 3-kinase activity

Rab5 and phosphatidylinositol 3-kinase (PI3K) have been proposed to co-regulate receptor endocytosis by controlling early endosome fusion. However, in this report we demonstrate that inhibition of epidermal growth factor (EGF)-stimulated PI3K activity by expression of the kinase-deficient PI3K p110 subunit (p110Δkin) does not block the lysosomal targeting and degradation of the EGF receptor (EGFR). Moreover, inhibition of total PI3K activity by wortmannin or LY294002 significantly enlarges EGFR-containing endosomes and dissociates the early-endosomal autoantigen EEA1 from membrane fractions. However, this does not block the lysosomal targeting and degradation of EGFR. In contrast, transfection of cells with mutant Rab5 S34N or microinjection of anti-Rabaptin5 antibodies inhibits EGFR endocytosis. Our results, therefore, demonstrate that PI3K is not universally required for the regulation of receptor intracellular trafficking. The present work suggests that the intracellular trafficking of EGFR is controlled by a novel endosome fusion pathway that is regulated by Rab5 in the absence of PI3K, rather than by the previously defined endosome fusion pathway that is co-regulated by Rab5 and PI3K.     

Huntingtin-HAP40 complex is a novel Rab5 effector that regulates early endosome motility and is up-regulated in Huntington's disease

The molecular mechanisms underlying the targeting of Huntingtin (Htt) to endosomes and its multifaceted role in endocytosis are poorly understood. In this study, we have identified Htt-associated protein 40 (HAP40) as a novel effector of the small guanosine triphosphatase Rab5, a key regulator of endocytosis. HAP40 mediates the recruitment of Htt by Rab5 onto early endosomes. HAP40 overexpression caused a drastic reduction of early endosomal motility through their displacement from microtubules and preferential association with actin filaments. Remarkably, endogenous HAP40 was up-regulated in fibroblasts and brain tissue from human patients affected by Huntington's disease (HD) as well as in STHdhQ(111) striatal cells established from a HD mouse model. These cells consistently displayed altered endosome motility and endocytic activity, which was restored by the ablation of HAP40. In revealing an unexpected link between Rab5, HAP40, and Htt, we uncovered a new mechanism regulating cytoskeleton-dependent endosome dynamics and its dysfunction under pathological conditions.     

Ubiquitin binding and conjugation regulate the recruitment of Rabex-5 to early endosomes

Rab GTPases and ubiquitination are critical regulators of transmembrane cargo sorting in endocytic and lysosomal targeting pathways. The endosomal protein Rabex-5 intersects these two layers of regulation by being both a guanine nucleotide exchange factor (GEF) for Rab5 and a substrate for ubiquitin (Ub) binding and conjugation. The ability of trafficking machinery components to bind ubiquitinated proteins is known to have a function in cargo sorting. Here, we demonstrate that Ub binding is essential for the recruitment of Rabex-5 from the cytosol to endosomes, independently of its GEF activity and of Rab5. We also show that monoubiquitinated Rabex-5 is enriched in the cytosol. These observations are consistent with a model whereby a cycle of Ub binding and monoubiquitination regulates the association of Rabex-5 with endosomes.     

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.     

Basolateral Endocytic Recycling Requires RAB-10 and AMPH-1 Mediated Recruitment of RAB-5 GAP TBC-2 to Endosomes

The small GTPase RAB-5/Rab5 is a master regulator of the early endosome, required for a myriad of coordinated activities, including the degradation and recycling of internalized cargo. Here we focused on the recycling function of the early endosome and the regulation of RAB-5 by GAP protein TBC-2 in the basolateral C. elegans intestine. We demonstrate that downstream basolateral recycling regulators, GTPase RAB-10/Rab10 and BAR domain protein AMPH-1/Amphiphysin, bind to TBC-2 and help to recruit it to endosomes. In the absence of RAB-10 or AMPH-1 binding to TBC-2, RAB-5 membrane association is abnormally high and recycling cargo is trapped in early endosomes. Furthermore, the loss of TBC-2 or AMPH-1 leads to abnormally high spatial overlap of RAB-5 and RAB-10. Taken together our results indicate that RAB-10 and AMPH-1 mediated down-regulation of RAB-5 is an important step in recycling, required for cargo exit from early endosomes and regulation of early endosome–recycling endosome interactions.     

Developmentally regulated GTP-binding protein 2 coordinates Rab5 activity and transferrin recycling

The small GTPase Rab5 regulates the early endocytic pathway of transferrin (Tfn), and Rab5 deactivation is required for Tfn recycling. Rab5 deactivation is achieved by RabGAP5, a GTPase-activating protein, on the endosomes. Here we report that recruitment of RabGAP5 is insufficient to deactivate Rab5 and that developmentally regulated GTP-binding protein 2 (DRG2) is required for Rab5 deactivation and Tfn recycling. DRG2 was associated with phosphatidylinositol 3-phosphate–containing endosomes. It colocalized and interacted with EEA1 and Rab5 on endosomes in a phosphatidylinositol 3-kinase–dependent manner. DRG2 depletion did not affect Tfn uptake and recruitment of RabGAP5 and Rac1 to Rab5 endosomes. However, it resulted in impairment of interaction between Rab5 and RabGAP5, Rab5 deactivation on endosomes, and Tfn recycling. Ectopic expression of shRNA-resistant DRG2 rescued Tfn recycling in DRG2-depleted cells. Our results demonstrate that DRG2 is an endosomal protein and a key regulator of Rab5 deactivation and Tfn recycling.     

Selective membrane recruitment of EEA1 suggests a role in directional transport of clathrin-coated vesicles to early endosomes

The molecular mechanisms ensuring directionality of endocytic membrane trafficking between transport vesicles and target organelles still remain poorly characterized. We have been investigating the function of the small GTPase Rab5 in early endocytic transport. In vitro studies have demonstrated a role of Rab5 in two membrane fusion events: the heterotypic fusion between plasma membrane-derived clathrin-coated vesicles (CCVs) and early endosomes and in the homotypic fusion between early endosomes. Several Rab5 effectors are required in homotypic endosome fusion, including EEA1, which mediates endosome membrane docking, as well as Rabaptin-5 x Rabex-5 complex and phosphatidylinositol 3-kinase hVPS34. In this study we have examined the localization and function of Rab5 and its effectors in heterotypic fusion in vitro. We report that the presence of active Rab5 is necessary on both CCVs and early endosomes for a heterotypic fusion event to occur. This process requires EEA1 in addition to the Rabaptin-5 complex. However, whereas Rab5 and Rabaptin-5 are symmetrically distributed between CCVs and early endosomes, EEA1 is recruited selectively onto the membrane of early endosomes. Our results suggest that EEA1 is a tethering molecule that provides directionality to vesicular transport from the plasma membrane to the early endosomes.     

The Rab5 guanine nucleotide exchange factor Rabex-5 binds ubiquitin (Ub) and functions as a Ub ligase through an atypical Ub-interacting motif and a zinc finger domain

Rabex-5, the mammalian orthologue of yeast Vps9p, is a guanine nucleotide exchange factor for Rab5. Rabex-5 forms a tight complex with Rabaptin-5, a multivalent adaptor protein that also binds to Rab4, Rab5, and to domains present in gamma-adaptins and the Golgi-localized, gamma-ear-containing, ARF-binding proteins (GGAs). Rabaptin-5 augments the Rabex-5 exchange activity, thus generating GTP-bound, membrane-associated Rab5 that, in turn, binds Rabaptin-5 and stabilizes the Rabex-5.Rabaptin-5 complex on endosomes. Although the Rabex-5.Rabaptin-5 complex is critical to the regulation of endosomal fusion, the structural determinants of this interaction are unknown. Likewise, the possible binding and covalent attachment of ubiquitin to Rabex-5, two modifications that are critical to the function of yeast Vps9p in endosomal transport, have not been studied. In this study, we identify the 401-462 and 551-661 coiled-coils as the regions in Rabex-5 and Rabaptin-5, respectively, that interact with one another. We also demonstrate that Rabex-5 undergoes ubiquitination and binds ubiquitin, though not via its proposed C-terminal CUE-like domain. Instead, the N-terminal region of Rabex-5 (residues 1-76), comprising an A20-like Cys2/Cys2 zinc finger and an adjacent alpha-helix, is important for ubiquitin binding and ubiquitination. Importantly, we demonstrate that the Rabex-5 zinc finger displays ubiquitin ligase (E3) activity. These observations extend our understanding of the regulation of Rabex-5 by Rabaptin-5. Moreover, the demonstration that Rabex-5 is a ubiquitin ligase that binds ubiquitin and undergoes ubiquitination indicates that its role in endosome fusion may be subject to additional regulation by ubiquitin-dependent modifications.     

Differential requirements of Rab5 and Rab7 for endocytosis of influenza and other enveloped viruses

Enveloped viruses often enter cells via endocytosis; however, specific endocytic trafficking pathway(s) for many viruses have not been determined. Here we demonstrate, through the use of dominant-negative Rab5 and Rab7, that influenza virus (Influenza A/WSN/33 (H1N1) and A/X-31 (H3N2)) requires both early and late endosomes for entry and subsequent infection in HeLa cells. Time-course experiments, monitoring viral ribonucleoprotein colocalization with endosomal markers, indicated that influenza exhibits a conventional endocytic uptake pattern – reaching early endosomes after approximately 10 min, and late endosomes after 40 min. Detection with conformation-specific hemagglutinin antibodies indicated that hemagglutinin did not reach a fusion-competent form until the virus had trafficked beyond early endosomes. We also examined two other enveloped viruses that are also pH-dependent for entry – Semliki Forest virus and vesicular stomatitis virus. In contrast to influenza virus, infection with both Semliki Forest virus and vesicular stomatitis virus was inhibited only by the expression of dominant negative Rab5 and not by dominant negative Rab7, indicating an independence of late endosome function for infection by these viruses. As a whole, these data provide a definitive characterization of influenza virus endocytic trafficking and show differential requirements for endocytic trafficking between pH-dependent enveloped viruses .     

Rabex-5 Protein Regulates Dendritic Localization of Small GTPase Rab17 and Neurite Morphogenesis in Hippocampal Neurons

Small GTPase Rab17 has recently been shown to regulate dendritic morphogenesis of mouse hippocampal neurons; however, the exact molecular mechanism of Rab17-mediated dendritogenesis remained to be determined, because no guanine nucleotide exchange factor (GEF) for Rab17 had been identified. In this study we screened for the Rab17-GEF by performing yeast two-hybrid assays with a GDP-locked Rab17 mutant as bait and found that Rabex-5 and ALS2, both of which were originally described as Rab5-GEFs, interact with Rab17. We also found that expression of Rabex-5, but not of ALS2, promotes translocation of Rab17 from the cell body to the dendrites of developing mouse hippocampal neurons. The shRNA-mediated knockdown of Rabex-5 or its known downstream target Rab5 in hippocampal neurons inhibited morphogenesis of both axons and dendrites, whereas knockdown of Rab17 affected dendrite morphogenesis alone. Based on these findings, we propose that Rabex-5 regulates neurite morphogenesis of hippocampal neurons by activating at least two downstream targets, Rab5, which is localized in both axons and dendrites, and Rab17, which is localized in dendrites alone.     

Sequential roles for phosphatidylinositol 3-phosphate and Rab5 in tethering and fusion of early endosomes via their interaction with EEA1

Early endosome antigen 1 (EEA1) is a 170-kDa polypeptide required for endosome fusion in mammalian cells. The COOH terminus of EEA1 contains a FYVE domain that interacts specifically with phosphatidylinositol 3-phosphate (PtdIns-3-P) and a Rab5 GTPase binding region adjacent to the FYVE domain. The dual interaction of EEA1 with both PtdIns-3-P and Rab5 has been hypothesized to provide the specificity required to target EEA1 to early endosomes. To test this hypothesis, we generated truncated (amino acids 1277--1411) and full-length EEA1 constructs containing point mutations in the COOH terminus that impair Rab5 but not PtdIns-3-P binding. These constructs localized to endosomes in intact cells as efficiently as their wild-type counterparts. Furthermore, overexpression of the truncated constructs, both wild-type and mutated, impaired the function of endogenous EEA1 resulting in the accumulation of small, untethered endosomes. These results suggest that association with Rab5 is not necessary for the initial binding and tethering functions of EEA1. A role for Rab5 binding was revealed, however, upon comparison of endosomes in cells expressing full-length wild-type or mutated EEA1. The mutant full-length EEA1 caused the accumulation of endosome clusters and suppressed the enlargement of endosomes caused by a persistently active form of Rab5 (Rab5Q79L). In contrast, expression of wild-type EEA1 with Rab5Q79L enhanced this enlargement. Thus, endosome tethering depends on the interaction of EEA1 with PtdIns-3-P, and its interaction with Rab5 appears to regulate subsequent fusion.