title: Sequential analysis of trans-SNARE formation in intracellular membrane fusion.
abstract: SNARE complexes are required for membrane fusion in the endomembrane system. They contain coiled-coil bundles of four helices, three (Q(a), Q(b), and Q(c)) from target (t)-SNAREs and one (R) from the vesicular (v)-SNARE. NSF/Sec18 disrupts these cis-SNARE complexes, allowing reassembly of their subunits into trans-SNARE complexes and subsequent fusion. Studying these reactions in native yeast vacuoles, we found that NSF/Sec18 activates the vacuolar cis-SNARE complex by selectively displacing the vacuolar Q(a) SNARE, leaving behind a Q(bc)R subcomplex. This subcomplex serves as an acceptor for a Q(a) SNARE from the opposite membrane, leading to Q(a)-Q(bc)R trans-complexes. Activity tests of vacuoles with diagnostic distributions of inactivating mutations over the two fusion partners confirm that this distribution accounts for a major share of the fusion activity. The persistence of the Q(bc)R cis-complex and the formation of the Q(a)-Q(bc)R trans-complex are both sensitive to the Rab-GTPase inhibitor, GDI, and to mutations in the vacuolar tether complex, HOPS (HOmotypic fusion and vacuolar Protein Sorting complex). This suggests that the vacuolar Rab-GTPase, Ypt7, and HOPS restrict cis-SNARE disassembly and thereby bias trans-SNARE assembly into a preferred topology.
authors.affiliation: Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas, United States of America.
authors.forename: Kannan
authors.initials: K
authors.lastname: Alpadi
authors.forename: Aditya
authors.initials: A
authors.lastname: Kulkarni
authors.forename: Veronique
authors.initials: V
authors.lastname: Comte
authors.forename: Monique
authors.initials: M
authors.lastname: Reinhardt
authors.forename: Andrea
authors.initials: A
authors.lastname: Schmidt
authors.forename: Sarita
authors.initials: S
authors.lastname: Namjoshi
authors.forename: Andreas
authors.initials: A
authors.lastname: Mayer
authors.forename: Christopher
authors.initials: C
authors.lastname: Peters
mesh_terms: D000483:Alleles; D007425:Intracellular Membranes / Q000378:metabolism*; D007700:Kinetics; D008561:Membrane Fusion; D008954:Models, Biological; D046912:Multiprotein Complexes / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010084:Oxidation-Reduction; D011485:Protein Binding; D055550:Protein Stability; D050600:SNARE Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D014617:Vacuoles / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000378:metabolism
chemical_list: D046912:Multiprotein Complexes; D050600:SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; D020691:rab GTP-Binding Proteins