Vps9p CUE domain ubiquitin binding is required for efficient endocytic protein traffic

Rab5 GTPases are key regulators of protein trafficking through the early stages of the endocytic pathway. The yeast Rab5 ortholog Vps21p is activated by its guanine nucleotide exchange factor Vps9p. Here we show that Vps9p binds ubiquitin and that the CUE domain is necessary and sufficient for this interaction. Vps9p ubiquitin binding is required for efficient endocytosis of Ste3p but not for the delivery of the biosynthetic cargo carboxypeptidase Y to the vacuole. In addition, Vps9p is itself monoubiquitylated. Ubiquitylation is dependent on a functional CUE domain and Rsp5p, an E3 ligase that participates in cell surface receptor endocytosis. These findings define a new ubiquitin binding domain and implicate ubiquitin as a modulator of Vps9p function in the endocytic pathway.     

Regulation of early endosomes across eukaryotes: Evolution and functional homology of Vps9 proteins

Endocytosis is a crucial process in eukaryotic cells. The GTPases Rab 5, 21 and 22 that mediate endocytosis are ancient eukaryotic features and all available evidence suggests retained conserved function. In animals and fungi, these GTPases are regulated in part by proteins possessing Vps9 domains. However, the diversity, evolution and functions of Vps9 proteins beyond animals or fungi are poorly explored. Here we report a comprehensive analysis of the Vps9 family of GTPase regulators, combining molecular evolutionary data with functional characterization in the non‐opisthokont model organism Trypanosoma brucei. At least 3 subfamilies, Alsin, Varp and Rabex5 + GAPVD1, are found across eukaryotes, suggesting that all are ancient features of regulation of endocytic Rab protein function. There are examples of lineage‐specific Vps9 subfamily member expansions and novel domain combinations, suggesting diversity in precise regulatory mechanisms between individual lineages. Characterization of the Rabex5 + GAPVD1 and Alsin orthologues in T. brucei demonstrates that both proteins are involved in endocytosis, and that simultaneous knockdown prevents membrane recruitment of Rab5 and Rab21, indicating conservation of function. These data demonstrate that, for the Vps9‐domain family at least, modulation of Rab function is mediated by evolutionarily conserved protein‐protein interactions.      

Ubiquitin binding by the CUE domain promotes endosomal localization of the Rab5 GEF Vps9

Vps9 and Muk1 are guanine nucleotide exchange factors (GEFs) in Saccharomyces cerevisiae that regulate membrane trafficking in the endolysosomal pathway by activating Rab5 GTPases. We show that Vps9 is the primary Rab5 GEF required for biogenesis of late endosomal multivesicular bodies (MVBs). However, only Vps9 (but not Muk1) is required for the formation of aberrant class E compartments that arise upon dysfunction of endosomal sorting complexes required for transport (ESCRTs). ESCRT dysfunction causes ubiquitinated transmembrane proteins to accumulate at endosomes, and we demonstrate that endosomal recruitment of Vps9 is promoted by its ubiquitin-binding CUE domain. Muk1 lacks ubiquitin-binding motifs, but its fusion to the Vps9 CUE domain allows Muk1 to rescue endosome morphology, cargo trafficking, and cellular stress-tolerance phenotypes that result from loss of Vps9 function. These results indicate that ubiquitin binding by the CUE domain promotes Vps9 function in endolysosomal membrane trafficking via promotion of localization.     

Vps9p is a guanine nucleotide exchange factor involved in vesicle-mediated vacuolar protein transport.

Vacuolar protein sorting (vps) mutants of Saccharomyces cerevisiae missort and secrete vacuolar hydrolases. The gene affected in one of these mutants, VPS21, encodes a member of the Sec4/Ypt/Rab family of small GTPases. Rab proteins play an essential role in vesicle-mediated protein transport. Using both yeast two-hybrid assays and chemical cross-linking, we have identified another VPS gene product, Vps9p, that preferentially interacts with a mutant form of Vps21p-S21N that binds GDP but not GTP. In vitro purified Vps9p was found to stimulate GDP release from Vps21p in a dose-dependent manner. Vps9p also stimulated GTP association as a result of facilitated GDP release. However, Vps9p did not stimulate guanine nucleotide exchange of GTP-bound Vps21p or GTP hydrolysis. We tested the ability of Vps9p to stimulate the intrinsic guanine nucleotide exchange activity of Rab5, which is a mammalian sequence homologue of Vps21p, and Ypt7p, which is another yeast Rab protein involved in vacuolar protein transport. Rab5, but not Ypt7p was responsive to Vps9p, which indicates that Vps9p recognizes sequence variation among Rab proteins. We conclude that Vps9p is a novel guanine nucleotide exchange factor that is specific for Vps21p/Rab5. Since there are no obvious Vps9p sequence homologues in yeast, Vps9p may also possess unique regulatory functions required for vacuolar protein transport.     

Rab5-activating protein 6, a novel endosomal protein with a role in endocytosis

Rab GTPases are regulators of membrane trafficking that cycle between active (GTP-bound) and inactive (GDP-bound) states. In this study, we report the identification of a new human Rab5 guanine nucleotide exchange factor (GEF), which we have named RAP6 (Rab5-activating protein 6). RAP6 contains a Rab5 GEF and a Ras GAP domain. We show that the Vps9 domain is sufficient for the interaction of RAP6 with GDP-bound Rab5 and that RAP6 stimulates Rab5 guanine nucleotide exchange. We also find that the Ras GAP domain of RAP6 shows GAP activity for Ras. Immunofluorescence experiments reveal that RAP6 is associated with plasma membrane and small intracellular vesicles that also contain Rab5. Additionally, the overexpression of RAP6 affects both fluid phase and receptor-mediated endocytosis. This study is the first to show that RAP6 is a novel regulator of endocytosis that exhibits GEF activity specific for Rab5 and GAP activity specific for Ras.     

VPS9a, the common activator for two distinct types of Rab5 GTPases, is essential for the development of Arabidopsis thaliana

Rab5, a subfamily of Rab GTPases, regulates a variety of endosomal functions as a molecular switch. Arabidopsis thaliana has two different types of Rab5-member GTPases: conventional type, ARA7 and RHA1, and a plant-specific type, ARA6. We found that only one guanine nucleotide exchange factor (GEF), named VPS9a, can activate all Rab5 members to GTP-bound forms in vitro in spite of their diverged structures. In the vps9a-1 mutant, whose GEF activity is completely lost, embryogenesis was arrested at the torpedo stage. Green fluorescent protein (GFP)-ARA7 and ARA6-GFP were diffused in cytosol like GDP-fixed mutants of Rab5 in vps9a-1, indicating that both types of GTPase are regulated by VPS9a. In the leaky vps9a-2 mutant, elongation of the primary root was severely affected. Overexpression of the GTP-fixed form of ARA7 suppressed the vps9a-2 mutation, but overexpression of ARA6 had no apparent effects. These results indicate that the two types of plant Rab5 members are functionally differentiated, even though they are regulated by the same activator, VPS9a.     

Rin-like, a novel regulator of endocytosis, acts as guanine nucleotide exchange factor for Rab5a and Rab22

RIN proteins serve as guanine nucleotide exchange factors for Rab5a. They are characterized by the presence of a RIN homology domain and a C-terminal Vps9 domain. Currently three family members have been described and analyzed. Here we report the identification of a novel RIN family member, Rin-like (Rinl), that represents a new interaction partner of the receptor tyrosine kinase MuSK, which is an essential key regulator of neuromuscular synapse development. Rinl is localized to neuromuscular synapses but shows the highest expression in thymus and spleen. Rinl preferentially binds to nucleotide-free Rab5a and catalyzes the exchange of GDP for GTP. Moreover, Rinl also binds GDP-bound Rab22 and increases the GDP/GTP exchange implicating Rinl in endocytotic processes regulated by Rab5a and Rab22. Interestingly, Rinl shows a higher catalytic rate for Rab22 compared to Rab5a. Rinl is closely associated with the cytoskeleton and thus contributes to the spatial control of Rab5a and Rab22 signaling at actin-positive compartments. Most importantly, overexpression of Rinl affects fluid-phase as well as EGFR endocytosis.     

Evidence for sequential action of Rab5 and Rab7 GTPases in prevacuolar organelle partitioning

GTPases of the Rab5 and Rab7 families were shown to control vacuolar sorting but their specific subcellular localization is controversial in plants. Here, we show that both the canonical as well as the plant-specific Rab5 reside at the newly discovered 'late prevacuolar compartment' (LPVC) while Rab7 partitions to the vacuolar membrane when expressed at low levels. Higher expression levels of wild-type Rab5 GTPases but not Rab7 lead to dose-dependent inhibition of biosynthetic vacuolar transport. In the case of Ara6, this included aberrant co-localization with markers for earlier post-Golgi compartments including the trans-Golgi network. However, nucleotide-free mutants of all three GTPases (Rha1, Ara6 and Rab7) cause stronger dose-dependent inhibition of vacuolar sorting. In addition, nucleotide-free Rha1 led to a later maturation defect and co-localization of markers for the prevacuolar compartment (PVC) and the LPVC. The corresponding Rab7 mutant strongly inhibited vacuolar delivery without merging of PVC and LPVC markers. Evidence for functional differentiation of the Rab5 family members is underlined by the fact that mutant Rha1 expression can be suppressed by increasing wild-type Rha1 levels while mutant Ara6 specifically titrates the nucleotide exchange factor Vps9. A model describing the sequential action of Rab5 and Rab7 GTPases is presented in the light of the current observations.     

Ras-activated endocytosis is mediated by the Rab5 guanine nucleotide exchange activity of RIN1.

RIN1 was originally identified by its ability to inhibit activated Ras and likely participates in multiple signaling pathways because it binds c-ABL and 14-3-3 proteins, in addition to Ras. RIN1 also contains a region homologous to the catalytic domain of Vps9p-like Rab guanine nucleotide exchange factors (GEFs). Here, we show that this region is necessary and sufficient for RIN1 interaction with the GDP-bound Rabs, Vps21p, and Rab5A. RIN1 is also shown to stimulate Rab5 guanine nucleotide exchange, Rab5A-dependent endosome fusion, and EGF receptor-mediated endocytosis. The stimulatory effect of RIN1 on all three of these processes is potentiated by activated Ras. We conclude that Ras-activated endocytosis is facilitated, in part, by the ability of Ras to directly regulate the Rab5 nucleotide exchange activity of RIN1.     

A Vps21 endocytic module regulates autophagy

In autophagy, the double-membrane autophagosome delivers cellular components for their degradation in the lysosome. The conserved Ypt/Rab GTPases regulate all cellular trafficking pathways, including autophagy. These GTPases function in modules that include guanine-nucleotide exchange factor (GEF) activators and downstream effectors. Rab7 and its yeast homologue, Ypt7, in the context of such a module, regulate the fusion of both late endosomes and autophagosomes with the lysosome. In yeast, the Rab5-related Vps21 is known for its role in early- to late-endosome transport. Here we show an additional role for Vps21 in autophagy. First, vps21∆ mutant cells are defective in selective and nonselective autophagy. Second, fluorescence and electron microscopy analyses show that vps21∆ mutant cells accumulate clusters of autophagosomal structures outside the vacuole. Third, cells with mutations in other members of the endocytic Vps21 module, including the GEF Vps9 and factors that function downstream of Vps21, Vac1, CORVET, Pep12, and Vps45, are also defective in autophagy and accumulate clusters of autophagosomes. Finally, Vps21 localizes to PAS. We propose that the endocytic Vps21 module also regulates autophagy. These findings support the idea that the two pathways leading to the lysosome—endocytosis and autophagy—converge through the Vps21 and Ypt7 GTPase modules.     

Vrl1 relies on its VPS9‐domain to play a role in autophagy in Saccharomyces cerevisiae

Autophagy is an intracellular degradation process involving many Atg proteins, which are recruited hierarchically to regulate this process. Rab/Ypt GTPases and their activators, guanine nucleotide exchange factors (GEFs), which are critical for regulating vesicle trafficking, are also involved in autophagy. Previously, we reported that yeast Vps21 and its GEF Vps9 are required for autophagy. Later, a third yeast VPS9‐domain‐containing protein, V AR P‐l ike 1 (Vrl1), which was identified as a mutant in major laboratory strains, had partially overlapping functions with Vps9 in trafficking. In this study, we showed that Vrl1 performed roles in autophagy, and its VPS9‐domain was crucial for its role in autophagy. We found that localization of Vrl1 differed from the other two VPS9‐domain‐containing proteins, Vps9 and Muk1, and only Vrl1 changed from multipoint to diffusion after starvation. Like Vps9, Vrl1 suppressed autophagic defects caused by the VPS9 deletion. We further showed that these VPS9‐domain‐containing proteins, Vps9, Muk1, and Vrl1, all co‐localized with Atg8 on autophagosomes in cells blocked in any late step of starvation‐induced autophagy, with Vrl1 most often co‐localizing with Atg8. A small portion (<25%) of these VPS9‐domain‐containing proteins were degraded through autophagy. However, a large portion (>60%) of Vrl1 decreased independently of autophagy. We propose that Vrl1 may regulate autophagy in a similar way as Vps9, and the level of Vrl1 partly decreases through both autophagy‐dependent and ‐independent routes.     

RINL, guanine nucleotide exchange factor Rab5-subfamily, is involved in the EphA8-degradation pathway with odin

The Rab family of small guanosine triphosphatases (GTPases) plays a vital role in membrane trafficking. Its active GTP-bound state is driven by guanine nucleotide-exchange factors (GEFs). Ras and Rab interactor (or Ras interaction/interference)-like (RINL), which contains a conserved VPS9 domain critical for GEF action, was recently identified as a new Rab5 subfamily GEF in vitro. However, its detailed function and interacting molecules have not yet been fully elucidated. Here we found that RINL has GEF activity for the Rab5 subfamily proteins by measuring their GTP-bound forms in cultured cells. We also found that RINL interacts with odin, a member of the ankyrin-repeat and sterile-alpha motif (SAM) domain-containing (Anks) protein family. In addition, the Eph tyrosine kinase receptor EphA8 formed a ternary complex with both RINL and odin. Interestingly, RINL expression in cultured cells reduced EphA8 levels in a manner dependent on both its GEF activity and interaction with odin. In addition, knockdown of RINL increased EphA8 level in HeLa cells. Our findings suggest that RINL, as a GEF for Rab5 subfamily, is implicated in the EphA8-degradation pathway via its interaction with odin.     

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.     

Role of Varp,a Rab21 exchange factor and TI‐VAMP/VAMP7 partner,in neurite growth

The vesicular soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE) tetanus neurotoxin‐insensitive vesicle‐associated membrane protein (TI‐VAMP/VAMP7) was previously shown to mediate an exocytic pathway involved in neurite growth, but its regulation is still largely unknown. Here we show that TI‐VAMP interacts with the Vps9 domain and ankyrin‐repeat‐containing protein (Varp), a guanine nucleotide exchange factor (GEF) of the small GTPase Rab21, through a specific domain herein called the interacting domain (ID). Varp, TI‐VAMP and Rab21 co‐localize in the perinuclear region of differentiating hippocampal neurons and transiently in transport vesicles in the shaft of neurites. Silencing the expression of Varp by RNA interference or expressing ID or a form of Varp deprived of its Vps9 domain impairs neurite growth. Furthermore, the mutant form of Rab21, defective in GTP hydrolysis, enhances neurite growth. We conclude that Varp is a positive regulator of neurite growth through both its GEF activity and its interaction with TI‐VAMP.     

Vps9 Family Protein Muk1 Is the Second Rab5 Guanosine Nucleotide Exchange Factor in Budding Yeast

VPS9 domains can act as guanosine nucleotide exchange factors (GEFs) against small G proteins of the Rab5 family. Saccharomyces cerevisiae vps9Δ mutants have trafficking defects considerably less severe than multiple deletions of the three cognate Rab5 paralogs (Vps21, Ypt52, and Ypt53). Here, we show that Muk1, which also contains a VPS9 domain, acts as a second GEF against Vps21, Ypt52, and Ypt53. Muk1 is partially redundant with Vps9 in vivo, with vps9Δ muk1Δ double mutant cells displaying hypersensitivity to temperature and ionic stress, as well as profound impairments in endocytic and Golgi endosome trafficking, including defects in sorting through the multivesicular body. Cells lacking both Vps9 and Muk1 closely phenocopy double and triple knock-out strains lacking Rab5 paralogs. Microscopy and overexpression experiments demonstrate that Vps9 and Muk1 have distinct localization determinants. These experiments establish Muk1 as the second Rab5 GEF in budding yeast.     

The Msb3/Gyp3 GAP controls the activity of the Rab GTPases Vps21 and Ypt7 at endosomes and vacuoles

Fusion of organelles in the endomembrane system depends on Rab GTPases that interact with tethering factors before lipid bilayer mixing. In yeast, the Rab5 GTPase Vps21 controls fusion and membrane dynamics between early and late endosomes. Here we identify Msb3/Gyp3 as a specific Vps21 GTPase-activating protein (GAP). Loss of Msb3 results in an accumulation of Vps21 and one of its effectors Vps8, a subunit of the CORVET complex, at the vacuole membrane in vivo. In agreement, Msb3 forms a specific transition complex with Vps21, has the highest activity of all recombinant GAPs for Vps21 in vitro, and is found at vacuoles despite its predominant localization to bud tips and bud necks at the plasma membrane. Surprisingly, Msb3 also inhibits vacuole fusion, which can be rescued by the Ypt7 GDP-GTP exchange factor (GEF), the Mon1-Ccz1 complex. Consistently, msb3 vacuoles fuse more efficiently than wild-type vacuoles in vitro, suggesting that GAP can also act on Ypt7. Our data indicate that GAPs such as Msb3 can act on multiple substrates in vivo at both ends of a trafficking pathway. This ensures specificity of the subsequent GEF-mediated activation of the Rab that initiates the next transport event.     

Characterization of RIN3 as a guanine nucleotide exchange factor for the Rab5 subfamily GTPase Rab31

The small GTPase Rab5, which cycles between GDP-bound inactive and GTP-bound active forms, plays essential roles in membrane budding and trafficking in the early endocytic pathway. Rab5 is activated by various vacuolar protein sorting 9 (VPS9) domain-containing guanine nucleotide exchange factors. Rab21, Rab22, and Rab31 (members of the Rab5 subfamily) are also involved in the trafficking of early endosomes. Mechanisms controlling the activation Rab5 subfamily members remain unclear. RIN (Ras and Rab interactor) represents a family of multifunctional proteins that have a VPS9 domain in addition to Src homology 2 (SH2) and Ras association domains. We investigated whether RIN family members act as guanine nucleotide exchange factors (GEFs) for the Rab5 subfamily on biochemical and cell morphological levels. RIN3 stimulated the formation of GTP-bound Rab31 in cell-free and in cell GEF activity assays. RIN3 also formed enlarged vesicles and tubular structures, where it colocalized with Rab31 in HeLa cells. In contrast, RIN3 did not exhibit any apparent effects on Rab21. We also found that serine to alanine substitutions in the sequences between SH2 and RIN family homology domain of RIN3 specifically abolished its GEF action on Rab31 but not Rab5. We examined whether RIN3 affects localization of the cation-dependent mannose 6-phosphate receptor (CD-MPR), which is transported between trans-Golgi network and endocytic compartments. We found that RIN3 partially translocates CD-MPR from the trans-Golgi network to peripheral vesicles and that this is dependent on its Rab31-GEF activity. These results indicate that RIN3 specifically acts as a GEF for Rab31.     

Engagement of the Small GTPase Rab31 Protein and Its Effector,Early Endosome Antigen 1, Is Important for Trafficking of the Ligand-bound Epidermal Growth Factor Receptor from the Early to the Late Endosome

Rab31 is a member of the Rab5 subfamily of Rab GTPases. Although localized largely to the trans-Golgi network, it shares common guanine nucleotide exchange factors and effectors with other Rab5 subfamily members that have been implicated in endocytic membrane traffic. We investigated whether Rab31 also has a role in the trafficking of the ligand-bound EGF receptor (EGFR) internalized through receptor-mediated endocytosis. We found that loss of Rab31 inhibits, but overexpression enhances, EGFR trafficking to the late endosomes and that the effect of Rab31 silencing could be specifically rescued by overexpression of a silencing-resistant form of Rab31. Rab31 was found to interact with the EGFR by coimmunoprecipitation and affinity pulldown analyses, and the primarily trans-Golgi network-localized Rab31 has increased colocalization with the EGFR in A431 cells 30 min after pulsing with EGF. A glycerol gradient sedimentation assay suggested that Rab31 is sequestered into a high molecular weight complex after stimulation with EGF, as was early endosome antigen 1 (EEA1), a factor responsible for endosomal tethering and fusion events. We found that loss of EEA1 reduced the interaction between Rab31 and the EGFR and abrogated the effect of Rab31 overexpression on the trafficking of the EGFR. Likewise, loss of GAPex5, a Rab31 guanine nucleotide exchange factor that has a role in ubiquitination and degradation of the EGFR, reduced the interaction of Rab31 with the EGFR and its effect on EGFR trafficking. Taken together, our results suggest that Rab31 is an important regulator of endocytic trafficking of the EGFR and functions in an EGFR trafficking complex that includes EEA1 and GAPex5.     

Vps9 domain-containing proteins: activators of Rab5 GTPases from yeast to neurons

Endocytosis of cell surface receptors plays an important role in regulating cell signaling cascades. In some cases, internalization of an activated receptor attenuates the signaling process, while in other cases the clustering of activated receptors on early endosomal structures has been proposed to be essential for fully activating signaling cascades. Regulating the movement of receptors and other signaling proteins through the endocytic pathway, therefore, has a direct impact on cellular homeostasis. The small GTPase Rab5 is a crucial regulatory component of the endocytic pathway. Activation of Rab5 is mediated by GDP-GTP exchange factors (GEFs) that generate the Rab5-GTP complex. A large number of proteins have been identified that contain a specific, highly conserved domain (Vps9) that catalyzes nucleotide exchange on Rab5, linking the regulation of cell signaling cascades with intracellular receptor trafficking through the endocytic pathway.     

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.