Sequential analysis of trans-SNARE formation in intracellular membrane fusion

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 bcr subcomplex. this subcomplex serves as an acceptor for a q a snare from the opposite membrane,leading to q a-q bcr 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 bcr cis-complex and the formation of the q a-q bcr 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. © 2012 alpadi et al.