Mechanisms of Rab- and Rab effector-dependent membrane tethering, docking, and fusion

Yeast vacuole fusion requires 4 SNAREs, the Rab GTPase Ypt7p, vacuolar lipids, the SNARE complex disassembly machinery Sec17p and Sec18p, and the heterohexameric HOPS complex. Vacuole fusion occurs in stages: cis-SNARE complexes (bound to one membrane) are disassembled by Sec17p and Sec18p during priming, initial vacuole-vacuole contacts are made during tethering, trans-SNARE complexes are formed during docking, and finally, lipids and contents mix during fusion. Prior to this thesis, it was known that the vacuolar SNAREs alone, like SNAREs from other organelles, can drive lipid mixing between pairs of proteoliposomes if the three vacuolar Q-SNAREs are on one proteoliposome and the R-SNARE is on the other. This thesis describes mechanistic insight attained from studies of the tethering, docking, and lipid mixing of chemically defined proteoliposomes bearing not only SNAREs but also various combinations of Ypt7p, vacuolar lipids, Sec17p, Sec18p, and HOPS.;The initial HOPS-dependent proteoliposome fusion reactions required vacuolar lipids but did not require the Rab Ypt7p. This thesis reports that phosphorylation of HOPS by the vacuolar kinase Yck3p blocks HOPS binding to vacuolar lipids, making HOPS membrane association and the ensuing proteoliposome fusion depend on the presence of Ypt7p. In accord with this finding in the reconstituted fusion reaction, the inactivation of Ypt7p by the GTPase-activating protein Gyp1-46p only blocks the fusion of purified vacuoles when Yck3p is present and active. Thus, while Ypt7p may contribute to other fusion functions, its central role is to bind HOPS to the membrane.;Proteoliposome clustering can be used to study the tethering and docking stages of membrane fusion. This thesis presents model subreactions of clustering and fusion that reveal that HOPS is the direct agent of tethering. The Rab and vacuole lipids contribute to tethering by supporting the membrane association of HOPS. HOPS indirectly facilitates trans-SNARE complex formation by tethering membranes, as the synthetic liposome tethering factor polyethylene glycol can also stimulate trans-SNARE complex formation and fusion. SNAREs further stabilize the associations of HOPS-tethered membranes. HOPS then protects newly formed trans-SNARE complexes from disassembly by Sec17p/Sec18p.