Hypomyelination and developmental delay associated with VPS11 mutation in Ashkenazi-Jewish patients.

BACKGROUND: The genetic heterogeneity of developmental delay and cognitive impairment is vast. The endocytic network is essential for neural development and synaptic plasticity by regulating the sorting of numerous transmembrane proteins. Disruption of the pathway can lead to neuronal pathology. Endosomal biogenesis relies on two Rab proteins, Rab5 and Rab7, which bind to two hexameric tethering complexes, the endosomal class C core vacuole/endosome tethering complex (CORVET) and the late endosomal/lysosomal homotypic fusion and protein sorting complex (HOPS). Both complexes consist of four core proteins and differ by their specific Rab-binding proteins. OBJECTIVES: To identify the molecular basis of a neurological disease, which consists of global developmental stagnation at 3-8 months, increasing appendicular spasticity, truncal hypotonia and acquired microcephaly, with variable seizure disorder, accompanied by thin corpus callosum, paucity of white matter and delayed myelination in eight patients from four unrelated Ashkenazi-Jewish (AJ) families. METHODS: Exome analysis, homozygosity mapping and Mup1-GFP transport assay in mutant yeast. RESULTS: Homozygosity for a missense mutation, p.Cys846Gly, in one of the endosomal biogenesis core proteins, VPS11, was identified in all the patients. This was shown to be a founder mutation with a carrier frequency of 0.6% in the AJ population. The homologous yeast mutant had moderate impairment of fusion of the late endosome to the vacuole in Mup1-GFP transport assay. CONCLUSIONS: We speculate that in neuronal cells, impairment of fusion of the late endosome to the vacuole would attenuate the degradation of plasma membrane receptors, thereby underlying the progressive neuronal phenotype in our patients. The VPS11 p.Cys846Gly mutation should be added to the AJ carrier screening panel.

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.

Tracking of the dynamic localization of the Rab-specific HOPS subunits reveal their distinct interaction with Ypt7 and vacuoles.

Endosomal and vacuole fusion depends on the two homologous tethering complexes CORVET and HOPS. HOPS binds the activated Rab GTPase Ypt7 via two distinct subunits, Vps39 and Vps41. To understand the participation and possible polarity of Vps41 and Vps39 during tethering, we used an in vivo approach. For this, we established the ligand-induced relocalization to the plasma membrane, using the Mon1-Ccz1 GEF complex that activates Ypt7 on endosomes. We then employed slight overexpression to compare the mobility of the HOPS-specific Vps41 and Vps39 subunits during this process. Our data indicate an asymmetry in the Rab-specific interaction of the two HOPS subunits: Vps39 is more tightly bound to the vacuole, and relocalizes the entire vacuole to the plasma membrane, whereas Vps41 behaved like the more mobile subunit. This is due to their specific Rab binding, as the mobility of both subunits was similar in ypt7∆ cells. In contrast, both HOPS subunits were far less mobile if tagged endogenously, suggesting that the entire HOPS complex is tightly bound to the vacuole in vivo. Similar results were obtained for the endosomal association of CORVET, when we followed its Rab-specific subunit Vps8. Our data provide in vivo evidence for distinct Rab specificity within HOPS, which may explain its function during tethering, and indicate that these tethering complexes are less mobile within the cell than previously anticipated.

The Vps39-like TRAP1 is an effector of Rab5 and likely the missing Vps3 subunit of human CORVET.

Membrane fusion in the endocytic pathway is mediated by a protein machinery consistent of Rab GTPases, tethering factors and SNAREs. In yeast, the endosomal CORVET and lysosomal HOPS tethering complexes share 4 of their 6 subunits. The 2 additional subunits in each complex - Vps3 and Vps8 for CORVET, and the homologous Vps39 and Vps41 for HOPS - bind directly to Rab5 and Rab7, respectively. In humans, all subunits for HOPS have been described. However, human CORVET remains poorly characterized and a homolog of Vps3 is still missing. Here we characterize 2 previously identified Vps39 isoforms, hVps39-1/hVam6/TLP and hVps39-2/TRAP1, in yeast and HEK293 cells. None of them can compensate the loss of the endogenous yeast Vps39, though the specific interaction of hVps39-1 with the virus-specific LT protein was reproduced. Both human Vps39 proteins show a cytosolic localization in yeast and mammalian cells. However, hVps39-2/TRAP1 strongly co-localizes with co-expressed Rab5 and interacts directly with Rab5-GTP in vitro. We conclude that hVps39-2/TRAP1 is an endosomal protein and an effector of Rab5, suggesting a role of the protein as a subunit of the putative human CORVET complex.

The BLOC-1 complex promotes endosomal maturation by recruiting the Rab5 GTPase-activating protein Msb3.

Membrane microcompartments of the early endosomes serve as a sorting and signaling platform, where receptors are either recycled back to the plasma membrane or forwarded to the lysosome for destruction. In metazoan cells, three complexes, termed BLOC-1 to -3, mediate protein sorting from the early endosome to lysosomes and lysosome-related organelles. We now demonstrate that BLOC-1 is an endosomal Rab-GAP (GTPase-activating protein) adapter complex in yeast. The yeast BLOC-1 consisted of six subunits, which localized interdependently to the endosomes in a Rab5/Vps21-dependent manner. In the absence of BLOC-1 subunits, the balance between recycling and degradation of selected cargoes was impaired. Additionally, our data show that BLOC-1 is both a Vps21 effector and an adapter for its GAP Msb3. BLOC-1 and Msb3 interacted in vivo, and both mutants resulted in a redistribution of active Vps21 to the vacuole surface. We thus conclude that BLOC-1 controls the lifetime of active Rab5/Vps21 and thus endosomal maturation along the endocytic pathway.

The CORVET complex promotes tethering and fusion of Rab5/Vps21-positive membranes.

Membrane fusion along the endocytic pathway occurs in a sequence of tethering, docking, and fusion. At endosomes and vacuoles, the CORVET (class C core vacuole/endosome tethering) and HOPS (homotypic fusion and vacuole protein sorting) tethering complexes require their organelle-specific Rabs for localization and function. Until now, despite the absence of experimental evidence, it has been assumed that CORVET is a membrane-tethering factor. To test this theory and understand the mechanistic analogies with the HOPS complex, we set up an in vitro system, and establish CORVET as a bona-fide tether for Vps21-positive endosome/vacuole membranes. Purified CORVET binds to SNAREs and Rab5/Vps21-GTP. We then demonstrate that purified CORVET can specifically tether Vps21-positive membranes. Tethering via CORVET is dose-dependent, stimulated by the GEF Vps9, and inhibited by Msb3, the Vps21-GAP. Moreover, CORVET supports fusion of isolated membranes containing Vps21. In agreement with its role as a tether, overexpressed CORVET drives Vps21, but not the HOPS-specific Ypt7 into contact sites between vacuoles, which likely represent vacuole-associated endosomes. We therefore conclude that CORVET is a tethering complex that promotes fusion of Rab5-positive membranes and thus facilitates receptor down-regulation and recycling at the late endosome.

Functional separation of endosomal fusion factors and the class C core vacuole/endosome tethering (CORVET) complex in endosome biogenesis.

Transport along the endolysosomal system requires multiple fusion events at early and late endosomes. Deletion of several endosomal fusion factors, including the Vac1 tether and the Class C core vacuole/endosome tethering (CORVET) complex-specific subunits Vps3 and Vps8, results in a class D vps phenotype. As these mutants have an apparently similar defect in endosomal transport, we asked whether CORVET and Vac1 could still act in distinct tethering reactions. Our data reveal that CORVET mutants can be rescued by Vac1 overexpression in the endocytic pathway but not in CPY or Cps1 sorting to the vacuole. Moreover, when we compared the ultrastructure, CORVET mutants were most similar to deletions of the Rab Vps21 and its guanine nucleotide exchange factor Vps9 and different from vac1 deletion, indicating separate functions. Likewise, CORVET still localized to endosomes even in the absence of Vac1, whereas Vac1 localization became diffuse in CORVET mutants. Importantly, CORVET localization requires the Rab5 homologs Vps21 and Ypt52, whereas Vac1 localization is strictly Vps21-dependent. In this context, we also uncover that Muk1 can compensate for loss of Vps9 in CORVET localization, indicating that two Rab5 guanine nucleotide exchange factors operate in the endocytic pathway. Overall, our study reveals a unique role of CORVET in the sorting of biosynthetic cargo to the vacuole/lysosome.

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.

Rab GTPases and tethering in the yeast endocytic pathway.

Within eukaryotic cells, Rab GTPases control the maturation of early to late endosomes and their subsequent fusion with the vacuole. Within this ExtraView, we will focus on our recent findings regarding the activation of the Rab7 homolog Ypt7 in yeast and its interplay with the two multisubunit tethering complexes CORVET and HOPS.

Defined subunit arrangement and rab interactions are required for functionality of the HOPS tethering complex.

Within the endomembrane system of eukaryotic cells, multisubunit tethering complexes together with their corresponding Rab-GTPases coordinate vesicle tethering and fusion. Here, we present evidence that two homologous hexameric tethering complexes, the endosomal CORVET (Class C core vacuole/endosome transport) and the vacuolar HOPS (homotypic vacuole fusion and protein sorting) complex, have similar subunit topologies. Both complexes contain two Rab-binding proteins at one end, and the Sec1/Munc18-like Vps33 at the opposite side, suggesting a model on membrane bridging via Rab-GTP and SNARE binding. In agreement, HOPS activity can be reconstituted using purified subcomplexes containing the Rab and Vps33 module, but requires all six subunits for activity. At the center of HOPS and CORVET, the class C proteins Vps11 and Vps18 connect the two parts, and Vps11 binds both HOPS Vps39 and CORVET Vps3 via the same binding site. As HOPS Vps39 is also found at endosomes, our data thus suggest that these tethering complexes follow defined but distinct assembly pathways, and may undergo transition by simple subunit interchange.