SNARE complexes mediate membrane fusion in the endomembrane system. They consist of coiled-coil bundles of four helices designated as Qa, Qb, Qc and R. A critical intermediate in the fusion pathway is the trans-SNARE complex generated by the assembly of SNAREs residing in opposing membranes. Mechanistic details of trans-SNARE complex formation and topology in a physiological system remain largely unresolved. Our studies on native yeast vacuoles revealed that SNAREs alone are insufficient to form trans-SNARE complexes and that additional factors, potentially tethering complexes and Rab GTPases, are required for the process. Here we report a novel finding that a HOPS tethering complex dimer catalyzes Rab GTPase-dependent formation of a topologically preferred QbQcR-Qa trans-SNARE complex.

SM proteins stabilize cis-SNARE complexes leading to a specific preferred topology for trans-SNARE formation.

SNARE complexes are required for membrane fusion in the endomembrane system. They contain coiled-coil bundles of four helices, three (Qa, Qb, and Qc) 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 Qa SNARE, leaving behind a QbcR subcomplex. This subcomplex serves as an acceptor for a Qa SNARE from the opposite membrane, leading to Qa-QbcR 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 QbcR cis-complex and the formation of the Qa-QbcR 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.

Author Summary

Cellular components often travel between organelles in vesicular entities. This intracellular traffic usually involves production of a vesicle containing cargo from one organelle membrane, movement of the vesicle to its destination, and then fusion of the vesicle with the target organelle. Thus, membrane fusion is a fundamental process required for these intracellular trafficking events. SNARE proteins and SM proteins mediate this fusion process. SNAREs form complexes that are either located on the same membrane or vesicle (called cis-SNARE complexes) or bridge two membrane compartments or vesicles (trans-SNARE complexes). The cis-SNARE complexes must be activated before trans-SNARE complexes can form and allow the membranes to fuse. We investigated the mechanism of cis-SNARE activation and trans-SNARE formation by studying the fusion of highly purified yeast vacuoles. We found that cis-SNARE activation involves the selective removal of a single SNARE protein from a pre-existing cis-SNARE complex, which is replaced by a similar SNARE originating from the other fusion partner. The activated cis-SNARE complexes depended on SM proteins for their stability. Thus, we have shown that the preferred topology of trans-SNARE formation is determined by cis-SNARE–SM protein interactions.