Involvement of the Ras-Ras-activated Rab5 guanine nucleotide exchange factor RIN2-Rab5 pathway in the hepatocyte growth factor-induced endocytosis of E-cadherin

E-cadherin is a key cell-cell adhesion molecule at adherens junctions (AJs) and undergoes endocytosis when AJs are disrupted by the action of an extracellular signal, such as hepatocyte growth factor (HGF)/scatter factor. Rab5 small G protein has been implicated in the HGF-induced endocytosis of E-cadherin, but the molecular mechanism for the regulation of Rab5 activity remains unknown. We first studied this mechanism by using the cell-free assay system for the endocytosis of E-cadherin of the AJ-enriched fraction from rat livers. HGF induced activation of Ras small G protein, which then bound to RIN2, a Rab5 GDP/GTP exchange factor with the Vps9p-like guanine nucleotide exchange factor and Ras association domains, and activated it. Activated RIN2 then activated Rab5, eventually inducing the endocytosis of E-cadherin. We then studied whether RIN2 was involved in the HGF-induced endocytosis of E-cadherin in intact Madin-Darby canine kidney cells. RIN2 localized at the cell-cell adhesion sites, and its guanine nucleotide exchange factor activity was required for the HGF-induced endocytosis of E-cadherin in Madin-Darby canine kidney cells. These results indicate that RIN2 connects Ras to Rab5 in the HGF-induced endocytosis of E-cadherin.     

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.     

Alsin is a Rab5 and Rac1 guanine nucleotide exchange factor

ALS2 is the gene mutated in a recessive juvenile form of amyotrophic lateral sclerosis (ALS2). ALS2 encodes a large protein termed alsin, which contains a number of predicted cell signaling and protein trafficking sequence motifs. To gain insight into the overall function of alsin and to begin to evaluate its role in motor neuron maintenance, we examined the subcellular localization of alsin and the biochemical activities associated with its individual subdomains. We found that the Vps9p domain of alsin has Rab5 guanine nucleotide exchange activity. In addition, alsin interacted specifically with and acted as a guanine nucleotide exchange factor for Rac1. Immunofluorescence and fractionation experiments in both fibroblasts and neurons revealed that alsin is a cytosolic protein, with a significant portion associated with small, punctate membrane structures. Many of these membrane structures also contained Rab5 or Rac1. Upon overexpression of full-length alsin, the overexpressed material was largely cytosolic, indicating that the association with membrane structures could be saturated. We also found that alsin was present in membrane ruffles and lamellipodia. These data suggest that alsin is involved in membrane transport events, potentially linking endocytic processes and actin cytoskeleton remodeling.     

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.     

EphA8-ephrinA5 signaling and clathrin-mediated endocytosis is regulated by Tiam-1, a Rac-specific guanine nucleotide exchange factor

Recent studies indicate that endocytosis of Eph-ephrin complexes may be one of the mechanisms by which a high affinity cell-cell adhesion is converted to a repulsive interaction. In this study, we show that EphA8 undergoes clathrin-mediated endocytosis upon treatment with ephrin-A5, and that EphA8 is associated tightly with Tiam-1, a Rac-specific guanine nucleotide exchange factor. Analysis of EphA8 deletion mutants revealed that a juxtamembrane region in EphA8 is critically involved in endocytosis of EphA8-ephrinA5 complexes. An EphA8 mutant lacking this juxtamembrane portion was defective for endocytosis with ephrinA5, and also displayed a weak association with Tiam-1. Expression of an endocytosis-defective version of EphA8 resulted in a low level of Rac activity in response to ephrin-A5 stimulation. More importantly, down-regulation of Tiam-1 resulted in inefficient endocytosis of EphA8-ephrinA5 complexes. These results suggest that Tiam-1 plays a role in clathrin-dependent endocytosis of EphA8-ephrinA5 complexes.     

Rab3GEP is the non-redundant guanine nucleotide exchange factor for Rab27a in melanocytes

Rab GTPases regulate discrete steps in vesicular transport pathways. Rabs require activation by specific guanine nucleotide exchange factors (GEFs) that stimulate the exchange of GDP for GTP. Rab27a controls motility and regulated exocytosis of secretory granules and related organelles. In melanocytes, Rab27a regulates peripheral transport of mature melanosomes by recruiting melanophilin and myosin Va. Here, we studied the activation of Rab27a in melanocytes. We identify Rab3GEP, previously isolated as a GEF for Rab3a, as the non-redundant Rab27a GEF. Similar to Rab27a-deficient ashen melanocytes, Rab3GEP-depleted cells show both clustering of melanosomes in the perinuclear area and loss of the Rab27a effector Mlph. Consistent with a role as an activator, levels of Rab27a-GTP are decreased in cells lacking Rab3GEP. Recombinant Rab3GEP exhibits guanine nucleotide exchange activity against Rab27a and Rab27b in vitro, in addition to its previously documented activity against Rab3. Our results indicate promiscuity in Rab GEF action and suggest that members of related but functionally distinct Rab subfamilies can be controlled by common activators.     

Intersectin 1L guanine nucleotide exchange activity is regulated by adjacent src homology 3 domains that are also involved in endocytosis

Intersectin 1L is a scaffolding protein involved in endocytosis that also has guanine nucleotide exchange activity for Cdc42. In the context of the full-length protein, the catalytic exchange activity of the DH domain is repressed. Here we use biochemical methods to dissect the mechanism for this inhibition. We demonstrate that the intersectin 1L SH3 domains, which bind endocytic proteins, directly inhibit the activity of the DH domain in assays for both binding and exchange of Cdc42. This inhibitory mechanism seems to act through steric hindrance of Cdc42 binding by an intramolecular interaction between the intersectin 1L SH3 domain region and the adjacent DH domain. Surprisingly, the mode of SH3 domain binding is other than through the proline peptide binding pocket. The dual role of the SH3 domains in endocytosis and repression of exchange activity suggests that the intersectin 1L exchange activity is regulated by endocytosis. We show that the endocytic protein, dynamin, competes for binding to the SH3 domains with the neural Wiskott-Aldrich Syndrome protein, an actin filament nucleation protein that is a substrate for activated Cdc42. Swapping of SH3 domain binding partners might act as a switch controlling the actin nucleation activity of intersectin 1L.     

Structural basis of guanine nucleotide exchange mediated by the T-cell essential Vav1

The guanine nucleotide exchange factor (GEF) Vav1 plays an important role in T-cell activation and tumorigenesis. In the GEF superfamily, Vav1 has the ability to interact with multiple families of Rho GTPases. The structure of the Vav1 DH-PH-CRD/Rac1 complex to 2.6 Å resolution reveals a unique intramolecular network of contacts between the Vav1 cysteine-rich domain (CRD) and the C-terminal helix of the Vav1 Dbl homology (DH) domain. These unique interactions stabilize the Vav1 DH domain for its intimate association with the Switch II region of Rac1 that is critical for the displacement of the guanine nucleotide. Small angle x-ray scattering (SAXS) studies support this domain arrangement for the complex in solution. Further, mutational analyses confirms that the atypical CRD is critical for maintaining both optimal guanine nucleotide exchange activity and broader specificity of Vav family GEFs. Taken together, the data outline the detailed nature of Vav1's ability to contact a range of Rho GTPases using a novel protein-protein interaction network.     

A Rab1 mutant affecting guanine nucleotide exchange promotes disassembly of the Golgi apparatus

The Golgi apparatus is a dynamic organelle whose structure is sensitive to vesicular traffic and to cell cycle control. We have examined the potential role for rab1a, a GTPase previously associated with ER to Golgi and intra-Golgi transport, in the formation and maintenance of Golgi structure. Bacterially expressed, recombinant rab1a protein was microinjected into rat embryonic fibroblasts, followed by analysis of Golgi morphology by fluorescence and electron microscopy. Three recombinant proteins were tested: wild-type rab, mutant rab1a(S25N), a constitutively GDP-bound form (Nuoffer, C., H. W. Davidson, J. Matteson, J. Meinkoth, and W. E. Balch, 1994. J. Cell Biol. 125: 225- 237), and mutant rab1a(N124I) defective in guanine nucleotide binding. Microinjection of wild-type rab1a protein or a variety of negative controls (injection buffer alone or activated ras protein) did not affect the appearance of the Golgi, as visualized by immunofluorescence of alpha-mannosidase II (Man II), used as a Golgi marker. In contrast, microinjection of the mutant forms promoted the disassembly of the Golgi stacks into dispersed vesicular structures visualized by immunofluorescence. When S25N-injected cells were analyzed by EM after immunoperoxidase labeling, Man II was found in isolated ministacks and large vesicular elements that were often surrounded by numerous smaller unlabeled vesicles resembling carrier vesicles. Golgi disassembly caused by rab1a mutants differs from BFA-induced disruption, since beta- COP remains membrane associated, and Man II does not redistribute to the ER. BFA can still cause these residual Golgi elements to fuse and disperse, albeit at a slower rate. Moreover, BFA recovery is incomplete in the presence of rab1 mutants or GTP gamma S. We conclude that GTP exchange and hydrolysis by GTPases, specifically rab1a, are required to form and maintain normal Golgi stacks. The similarity of Golgi disassembly seen with rab1a mutants to that occurring during mitosis, may point to a molecular basis involving rab1a for fragmentation of the Golgi apparatus during cell division.     

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.     

Rab5-family guanine nucleotide exchange factors bind retromer and promote its recruitment to endosomes

The retromer complex facilitates the sorting of integral membrane proteins from the endosome to the late Golgi. In mammalian cells, the efficient recruitment of retromer to endosomes requires the lipid phosphatidylinositol 3-phosphate (PI3P) as well as Rab5 and Rab7 GTPases. However, in yeast, the role of Rabs in recruiting retromer to endosomes is less clear. We identified novel physical interactions between retromer and the Saccharomyces cerevisiae VPS9-domain Rab5-family guanine nucleotide exchange factors (GEFs) Muk1 and Vps9. Furthermore, we identified a new yeast VPS9 domain-containing protein, VARP-like 1 (Vrl1), which is related to the human VARP protein. All three VPS9 domain–containing proteins show localization to endosomes, and the presence of any one of them is necessary for the endosomal recruitment of retromer. We find that expression of an active VPS9-domain protein is required for correct localization of the phosphatidylinositol 3-kinase Vps34 and the production of endosomal PI3P. These results suggest that VPS9 GEFs promote retromer recruitment by establishing PI3P-enriched domains at the endosomal membrane. The interaction of retromer with distinct VPS9 GEFs could thus link GEF-dependent regulatory inputs to the temporal or spatial coordination of retromer assembly or function.     

Mobilization of late-endosomal cholesterol is inhibited by Rab guanine nucleotide dissociation inhibitor

Cholesterol entering cells in low-density lipoproteins (LDL) via receptor-mediated endocytosis is transported to organelles of the late endocytic pathway for degradation of the lipoprotein particles. The fate of the free cholesterol released remains poorly understood, however. Recent observations suggest that late-endosomal cholesterol sequestration is regulated by the dynamics of lysobisphosphatidic acid (LBPA)-rich membranes [1]. Genetic studies have pinpointed a protein, Niemann-Pick C-1 (NPC-1), that is required for the mobilization of late-endosomal/lysosomal cholesterol by an unknown mechanism [2]. Here, we report the removal of accumulated cholesterol by overexpression of the NPC-1 protein in NPC-1-deficient fibroblasts from patients with Niemann-Pick disease, and in normal fibroblasts upon release of a progesterone-induced block of cholesterol transport. We show that late-endosomal/lysosomal cholesterol mobilization is specifically inhibited by microinjection of Rab GDP-dissociation inhibitor (Rab-GDI). Moreover, clearance of the cholesterol deposits by NPC-1 in patients' fibroblasts is accompanied by the redistribution of LBPA and of a lysosomal hydrolase that utilizes the mannose-6-phosphate receptor. Our results reveal, for the first time, the involvement of a specific molecular component of the membrane-trafficking machinery in cholesterol transport and the coupling of late-endosomal cholesterol egress to the trafficking of other lipid and protein cargo.     

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.     

Role of the guanine nucleotide exchange factor Ost in negative regulation of receptor endocytosis by the small GTPase Rac1

The Rho family of GTPases has been implicated in the regulation of intracellular vesicle trafficking. Here, we investigated the mechanism underlying the negative regulation of clathrin-mediated endocytosis of cell surface receptors mediated by the Rho family protein Rac1. Contrary to previous reports, only the activated mutant of Rac1, but not other Rho family members including RhoA and Cdc42, suppressed internalization of the transferrin receptor. On the other hand, down-regulation of Rac1 expression by RNA interference resulted in enhanced receptor internalization, suggesting that endogenous Rac1 in fact functions as a negative regulator. We identified a guanine nucleotide exchange factor splice variant designated Ost-III, which contains a unique C-terminal region including an Src homology 3 domain, as a regulator of Rac1 involved in the inhibition of receptor endocytosis. In contrast, other splice variants Ost-I and Ost-II exerted virtually no effect on receptor endocytosis. We also examined subcellular localization of synaptojanin 2, a putative Rac1 effector implicated in negative regulation of receptor endocytosis. Each Ost splice variant induced distinct subcellular localization of synaptojanin 2, depending on Rac1 activation. Furthermore, we isolated gamma-aminobutyric acid type A receptor-associated protein (GABARAP) as a protein that binds to the C-terminal region of Ost-III. When ectopically expressed, GABARAP was co-localized with Ost-III and potently suppressed the Ost-III-dependent Rac1 activation and the inhibition of receptor endocytosis. Lipid modification of GABARAP was necessary for the suppression of Ost-III. These results are discussed in terms of subcellular region-specific regulation of the Rac1-dependent signaling pathway that negatively regulates clathrin-mediated endocytosis.     

Gapex-5, a Rab31 guanine nucleotide exchange factor that regulates Glut4 trafficking in adipocytes

Insulin stimulates glucose uptake by promoting translocation of the Glut4 glucose transporter from intracellular storage compartments to the plasma membrane. In the absence of insulin, Glut4 is retained intracellularly; the mechanism underlying this process remains uncertain. Using the TC10-interacting protein CIP4 as bait in a yeast two-hybrid screen, we cloned a RasGAP and VPS9 domain-containing protein, Gapex-5/RME-6. The VPS9 domain is a guanine nucleotide exchange factor for Rab31, a Rab5 subfamily GTPase implicated in trans-Golgi network (TGN)-to-endosome trafficking. Overexpression of Rab31 blocks insulin-stimulated Glut4 translocation, whereas knockdown of Rab31 potentiates insulin-stimulated Glut4 translocation and glucose uptake. Gapex-5 is predominantly cytosolic in untreated cells; its overexpression promotes intracellular retention of Glut4 in adipocytes. Insulin recruits the CIP4/Gapex-5 complex to the plasma membrane, thus reducing Rab31 activity and permitting Glut4 vesicles to translocate to the cell surface, where Glut4 docks and fuses to transport glucose into the cell.     

Molecular basis for Rac1 recognition by guanine nucleotide exchange factors.

Rho GTPases are activated by a family of guanine nucleotide exchange factors (GEFs) known as Dbl family proteins. The structural basis for how GEFs recognize and activate Rho GTPases is presently ill defined. Here, we utilized the crystal structure of the DH/PH domains of the Rac-specific GEF Tiam1 in complex with Rac1 to determine the structural elements of Rac1 that regulate the specificity of this interaction. We show that residues in the Rac1 beta2-beta3 region are critical for Tiam1 recognition. Additionally, we determined that a single Rac1-to-Cdc42 mutation (W56F) was sufficient to abolish Rac1 sensitivity to Tiam1 and allow recognition by the Cdc42-specific DH/PH domains of Intersectin while not impairing Rac1 downstream activities. Our findings identified unique GEF specificity determinants in Rac1 and provide important insights into the mechanism of DH/PH selection of GTPase targets.     

Collagen phagocytosis is regulated by the guanine nucleotide exchange factor Vav2

Collagen phagocytosis is a crucial alpha2beta1-integrin-dependent process that mediates extracellular matrix remodeling by fibroblasts. We showed previously that after initial contact with collagen, activated Rac1 accelerates collagen phagocytosis but the Rac guanine nucleotide exchange factors (GEFs) that regulate Rac are not defined. We examined here the GEFs that regulate collagen phagocytosis in mouse fibroblasts. Collagen binding enhanced Rac1 activity (5-20 min) but not Cdc42 or RhoA activity. Analysis of collagen bead-associated proteins showed enrichment with Vav2, which correlated temporally with increased Rac1 activity. Knockdown of Vav2 prevented Rac activation, recruitment of Rac1 to collagen bead binding sites, and collagen bead binding, but knockdown of Sos-1 or beta-Pix had no effect on Rac activation or collagen binding. Vav2 was associated with the nucleotide-free Rac1 mutant (G15ARac1) after collagen binding. Collagen bead binding promoted phosphorylation of Vav2, which temporally correlated with Rac1 activation and which required Src kinase activity. Blockage of Src activity prevented collagen bead-induced Rac activation and collagen bead binding. Collectively these data indicate that Vav2 regulates the Rac1 activity associated with the binding step of collagen phagocytosis.     

Allosteric behavior in transducin activation mediated by rhodopsin. Initial rate analysis of guanine nucleotide exchange

Photolyzed rhodopsin acts in a catalytic manner to mediate the exchange of GTP for GDP bound to transducin. We have analyzed the steady-state kinetics of this activation process in order to determine the molecular mechanism of interactions between rhodopsin, transducin, and guanine nucleotides. Initial velocities (Vo) of the exchange reaction catalyzed by rhodopsin were measured for various transducin concentrations at several fixed levels of the GTP analog, [35S]guanosine 5'-(3-O-thio)triphosphate (GTP gamma S). The initial rate data analysis rigorously demonstrates that rhodopsin mediates the activation of transducin by a double-displacement catalytic mechanism. The Michaelis-Menten curves determined as a function of [transducin] reveal remarkable allosteric behavior; analysis of this data yields a Hill coefficient of 2. Lineweaver-Burk plots of Vo-1 versus [transducin]-1 display curvilinearity indicative of positive cooperativity and a series of parallel lines are generated by plotting Vo-1 as a function of [transducin]-2. The plots of Vo-1 versus [GTP gamma S]-1 show no evidence of allosterism and are a parallel series. Furthermore, the allosteric behavior observed in the activation of transducin is also witnessed in the rhodopsin-catalyzed guanine nucleotide exchange of the G protein's purified alpha subunit in the absence of the beta X gamma subunit complex. The latter observation implies that the molecular basis for allosterism in the activation process resides in the interactions between the photoreceptor and transducin's alpha subunit.     

Homo-oligomerization of ALS2 through its unique carboxyl-terminal regions is essential for the ALS2-associated Rab5 guanine nucleotide exchange activity and its regulatory function on endosome trafficking

Mutations in the ALS2 gene have been known to account for a juvenile recessive form of amyotrophic lateral sclerosis (ALS2), a rare juvenile recessive form of primary lateral sclerosis, and a form of hereditary spastic paraplegia (HSP), indicating that the ALS2 protein is essential for the maintenance of motor neurons. Recently, we have demonstrated that the ALS2 protein specifically binds to the small GTPase Rab5 and acts as a GEF (guanine nucleotide exchange factor) for Rab5. We have also shown that its Rab5GEF-requisite domain resides within the C-terminal 640-amino acid region spanning membrane occupation and recognition nexus motifs and the vacuolar protein sorting 9 domain. Transiently expressed ALS2 localized onto early endosomal compartments and stimulated endosome fusions in neuronal and non-neuronal cells in an Rab5GEF activity-dependent manner. These results indicate that the C-terminal region of ALS2 plays a crucial role in endosomal dynamics by its Rab5GEF activity. Here we delineate a molecular feature of the ALS2-associated function through the C-terminal region-mediated homo-oligomerization. A yeast two-hybrid screen for interacting proteins with the ALS2 C-terminal portion identified ALS2 itself. ALS2 forms a homophilic oligomer through its distinct C-terminal regions. This homo-oligomerization is crucial for the Rab5GEF activity in vitro and the ALS2-mediated endosome enlargement in the cells. Taken together, these results indicate that oligomerization of the ALS2 protein is one of the fundamental features for its physiological function involving endosome dynamics in vivo.