RESUMO
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.
Assuntos
Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas rab de Ligação ao GTP/química , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/química , Proteínas rab5 de Ligação ao GTP/metabolismo , Endocitose , Endossomos/metabolismo , Lisossomos/metabolismo , Fusão de Membrana , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas SNARE/química , Proteínas SNARE/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Vacúolos/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/genéticaRESUMO
Protein and lipid transport along the endolysosomal system of eukaryotic cells depends on multiple fusion and fission events. Over the past few years, the molecular constituents of both fission and fusion machineries have been identified. Here, we focus on the mechanism of membrane fusion at endosomes, vacuoles and lysosomes, and in particular on the role of the two homologous tethering complexes called CORVET and HOPS. Both complexes are heterohexamers; they share four subunits, interact with Rab GTPases and soluble NSF attachment protein receptors (SNAREs) and can tether membranes. Owing to the presence of specific subunits, CORVET is a Rab5 effector complex, whereas HOPS can bind efficiently to late endosomes and lysosomes through Rab7. Based on the recently described overall structure of the HOPS complex and a number of in vivo and in vitro analyses, important insights into their function have been obtained. Here, we discuss the general function of both complexes in yeast and in metazoan cells in the context of endosomal biogenesis and maturation.
Assuntos
Endossomos/fisiologia , Lisossomos/fisiologia , Fusão de Membrana , Complexos Multiproteicos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Animais , Transporte Biológico , Membrana Celular/fisiologia , Humanos , Ligação Proteica , Proteínas SNARE/metabolismo , Leveduras/metabolismo , proteínas de unión al GTP Rab7RESUMO
Membrane fusion within the eukaryotic endomembrane system depends on the initial recognition of Rab GTPase on transport vesicles by multisubunit tethering complexes and subsequent coupling to SNARE-mediated fusion. The conserved vacuolar/lysosomal homotypic fusion and vacuole protein sorting (HOPS) tethering complex combines both activities. Here we present the overall structure of the fusion-active HOPS complex. Our data reveal a flexible ≈30-nm elongated seahorse-like structure, which can adopt contracted and elongated shapes. Surprisingly, both ends of the HOPS complex contain a Rab-binding subunit: Vps41 and Vps39. The large head contains in addition to Vps41 the SNARE-interacting Vps33, whereas Vps39 is found in the bulky tip of its tail. Vps11 and Vps18 connect head and tail. Our data suggest that HOPS bridges Ypt7-positive membranes and chaperones SNAREs at fusion sites.
Assuntos
Fusão de Membrana , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Vacúolos/metabolismo , Sítios de Ligação , Proteínas de Fluorescência Verde/metabolismo , Complexos Multiproteicos/isolamento & purificação , Complexos Multiproteicos/ultraestrutura , Ligação Proteica , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Eletricidade Estática , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Organelles of the endomembrane system need to counterbalance fission and fusion events to maintain their surface-to-volume ratio. At the late mammalian endosome, the Rab GTPase Rab7 is a major regulator of fusion, whereas the homologous yeast protein Ypt7 seems to be restricted to the vacuole surface. Here, we present evidence that Ypt7 is recruited to and acts on late endosomes, where it affects multiple trafficking reactions. We show that overexpression of Ypt7 results in expansion and massive invagination of the vacuolar membrane, which requires cycling of Ypt7 between GDP- and GTP-bound states. Invaginations are blocked by ESCRT, CORVET and retromer mutants, but not by autophagy or AP-3 mutants. We also show that Ypt7-GTP specifically binds to the retromer cargo-recognition subcomplex, which--like its cargo Vps10--is found on the vacuole upon Ypt7 overproduction. Our data suggest that Ypt7 functions at the late endosome to coordinate retromer-mediated recycling with the fusion of late endosomes with vacuoles.