Mutations in VIPAR cause an arthrogryposis, renal dysfunction and cholestasis syndrome phenotype with defects in epithelial polarization (original) (raw)
Related papers
The EMBO Journal, 1993
Communicated by D.Meyer The Vps15 protein kinase and the Vps34 phosphatidylinositol 3-kinase (PI 3-kinase) are required for the sorting of soluble hydrolases to the yeast vacuole. Overproduction of Vps34p suppresses the growth and vacuolar protein sorting defects associated with vpslS kinase domain mutants, suggesting that Vpsl5p and Vps34p functionally interact. Subceliular fractionation and sucrose density gradients indicate that Vpsl5p is responsible for the association of Vps34p with an intracellular membrane fraction. Chemical cross-linking and native immunoprecipitation experiments demonstrate that Vpsl5p and Vps34p interact as components of a hetero-oligomeric protein complex. In addition, we show that an intact Vps15 protein kinase domain is required for activation of the Vps34 PI 3-kinase, suggesting that the Vps34 lipid kinase is regulated by a Vpsl5p-mediated protein phosphorylation event. We propose that Vpsl5p and Vps34p function together as components of a membrane-associated signal transduction complex that regulates intracellular protein trafficking decisions through protein and lipid phosphorylation events.
Structural basis of Vps33A recruitment to the human HOPS complex by Vps16
Proceedings of the National Academy of Sciences, 2013
The multisubunit homotypic fusion and vacuole protein sorting (HOPS) membrane-tethering complex is required for late endosome-lysosome and autophagosome-lysosome fusion in mammals. We have determined the crystal structure of the human HOPS subunit Vps33A, confirming its identity as a Sec1/Munc18 family member. We show that HOPS subunit Vps16 recruits Vps33A to the human HOPS complex and that residues 642–736 are necessary and sufficient for this interaction, and we present the crystal structure of Vps33A in complex with Vps16(642–736). Mutations at the binding interface disrupt the Vps33A–Vps16 interaction both in vitro and in cells, preventing recruitment of Vps33A to the HOPS complex. The Vps33A–Vps16 complex provides a structural framework for studying the association between Sec1/Munc18 proteins and tethering complexes.
Self-Assembly of VPS41 Promotes Sorting Required for Biogenesis of the Regulated Secretory Pathway
The regulated release of polypeptides has a central role in physiology, behavior, and development, but the mechanisms responsible for production of the large dense core vesicles (LDCVs) capable of regulated release have remained poorly understood. Recent work has implicated cytosolic adaptor protein AP-3 in the recruitment of LDCV membrane proteins that confer regulated release. However, AP-3 in mammals has been considered to function in the endolysosomal pathway and in the biosynthetic pathway only in yeast. We now find that the mammalian homolog of yeast VPS41, a member of the homotypic fusion and vacuole protein sorting (HOPS) complex that delivers biosynthetic cargo to the endocytic pathway in yeast, promotes LDCV formation through a common mechanism with AP-3, indicating a conserved role for these proteins in the biosynthetic pathway. VPS41 also self-assembles into a lattice, suggesting that it acts as a coat protein for AP-3 in formation of the regulated secretory pathway.
2000
Sorting nexin (SNX) 1 and SNX2 are mammalian orthologs of Vps5p, a yeast protein that is a subunit of a large multimeric complex, termed the retromer complex, involved in retrograde transport of proteins from endosomes to the trans-Golgi network. We report the cloning and characterization of human orthologs of three additional components of the complex: Vps26p, Vps29p, and Vps35p. The close structural similarity between the yeast and human proteins suggests a similarity in function. We used both yeast two-hybrid assays and expression in mammalian cells to define the binding interactions among these proteins. The data suggest a model in which hVps35 serves as the core of a multimeric complex by binding directly to hVps26, hVps29, and SNX1. Deletional analyses of hVps35 demonstrate that amino acid residues 1-53 and 307-796 of hVps35 bind to the coiled coil-containing domain of SNX1. In contrast, hVps26 binds to amino acid residues 1-172 of hVps35, whereas hVps29 binds to amino acid residues 307-796 of hVps35. Furthermore, hVps35, hVps29, and hVps26 have been found in membrane-associated and cytosolic compartments. Gel filtration chromatography of COS7 cell cytosol showed that both recombinant and endogenous hVps35, hVps29, and hVps26 coelute as a large complex (ϳ220 -440 kDa). In the absence of hVps35, neither hVps26 nor hVps29 is found in the large complex. These data provide the first insights into the binding interactions among subunits of a putative mammalian retromer complex.
VPS18 recruits VPS41 to the human HOPS complex via a RING–RING interaction
Biochemical Journal, 2017
Eukaryotic cells use conserved multisubunit membrane tethering complexes, including CORVET (class C core vacuole/endosome tethering) and HOPS (homotypic fusion and vacuole protein sorting), to control the fusion of endomembranes. These complexes have been extensively studied in yeast, but to date there have been far fewer studies of metazoan CORVET and HOPS. Both of these complexes comprise six subunits: a common four-subunit core and two unique subunits. Once assembled, these complexes function to recognise specific endosomal membrane markers and facilitate SNARE-mediated membrane fusion. CORVET promotes the homotypic fusion of early endosomes, while HOPS promotes the fusion of lysosomes to late endosomes and autophagosomes. Many of the subunits of both CORVET and HOPS contain putative C-terminal zinc-finger domains. Here, the contribution of these domains to the assembly of the human CORVET and HOPS complexes has been examined. Using biochemical techniques, we demonstrate that the...
Termination of Isoform-Selective Vps21/Rab5 Signaling at Endolysosomal Organelles by Msb3/Gyp3
Traffic through endosomes and lysosomes is controlled by small G-proteins of the Rab5 and Rab7 families. Like humans, Saccharomyces cerevisiae has three Rab5s (Vps21, Ypt52 and Ypt53) and one Rab7 (Ypt7). Here, we elucidate the functional roles and regulation of the yeast Rab5s. Using GFP-tagged cargoes, a novel quantitative multivesicular body (MVB) sorting assay, and electron microscopy, we show that MVB biogenesis and thus MVB cargo sorting is severely impaired in vps21Δ ypt52Δ double mutants. Ypt53, the third Rab5 paralog, is hardly expressed during normal growth but its transcription is strongly induced by cellular stress through the calcineurin-Crz1 pathway. The requirement for Rab5 activity in stress tolerance facilitated identification of Msb3/Gyp3 as the principal Rab5 GAP (GTPase accelerating protein). In vitro GAP assays verified that Vps21 is a preferred Gyp3 target. Moreover, we demonstrate that Gyp3 spatially restricts active Vps21 to intermediate endosomal compartments by preventing Vps21 accumulation on lysosomal vacuoles. Gyp3, therefore, operates as a compartmental insulator that helps to define the spatial domain of Vps21 signaling in the endolysosomal pathway. Membrane traffic through the secretory and endolysoso-mal systems is highly dynamic. Within these systems small G proteins of the Arf and Rab families regulate compartmental identity and control rates of membrane influx and egress from each compartment (1–4). Rabs are anchored to membranes through C-terminal prenyl anchors. In their inactive GDP-bound state, Rabs can be extracted from membranes and relocated by the cytoplas-mic chaperone GDI (GDP dissociation inhibitor). Activated GTP-bound Rabs and Arfs bind effector proteins and complexes that execute diverse functions in membrane traffic including vesicle budding, transport, and fusion. Rab signaling is stringently regulated. Like other small G proteins within the Ras superfamily, Rabs bind GDP tenaciously and are activated through catalyzed exchange of GTP for GDP mediated by guanosine nucleotide exchange factors (GEFs). Rabs generally have slow intrinsic GTPase activity and are inactivated by GTPase accelerating proteins (GAPs). All known Rab GAPs contain TBC domains. In ternary Rab-GTP-GAP complexes, the TBC domain supplies catalytic Arg and Gln residues which stabilize the hydrolytic transition intermediate (5,6). Hence, the integrated activities of GEFs, GAPs and chaperones define the spatial and temporal domains of Rab signaling and activity. Anterograde transport from early to late endosomes, and then to terminal lysosomes, is principally controlled by members of the Rab5 and Rab7 families, with Rab5 paralogs operating at earlier compartments and Rab7 at later compartments (7). Recent studies also suggest that Rab5 and Rab7 small G proteins coordinate retrograde traffic to the Golgi through interactions with the retromer complex (8,9). Saccharomyces cerevisiae, like humans, has three Rab5 paralogs that reside on endosomes (Vps21, Ypt52 and Ypt53), and one Rab7 (Ypt7) found mainly on the vacuolar lysosome (10–13). No clear role for Ypt53 has been established, and the relative roles of Vps21, Ypt52 and Ypt53 in multivesicular body (MVB) biogenesis and cargo sorting have not been fully characterized. During maturation of the late endosome Rab5 is rather abruptly replaced by Rab7 in a process termed endosomal Rab conversion (14). The acquisition of Rab7 is thought to confer competence for fusion with terminal endolysosomal compartments. An important waypoint within the endolysosomal system is the late endosomal MVB. At the MVB, ESCRTs (endosomal sorting complexes required for transport) package specific cargo into intraluminal vesicles (ILVs) that sequester cargo away from the cytoplasm (15,16). When mature MVBs fuse with lysosomes, ILVs and their cargos are dumped into the lysosome lumen and destroyed, while lipids and transmembrane proteins not packaged into ILVs are delivered to the lysosomal limiting membrane. The relative timing and regulatory dependencies of ESCRT activity, Rab7 activation and Rab5 inactivation are not fully understood. Rab7 recruitment and signaling require the activities of the Vps-C effector complexes HOPS and CORVET (14,17) and the GEF complex Ccz1-Mon1 (18,19). In contrast, it has been unclear when and where GAPs negatively regulate Rab5 in the endolysosomal Rab cascade. Multiple yeast GAPs have been reported to target www.traffic.dk 1411