Membrane fusion is central to cell growth, protein secretion, organelle biogenesis, and neurotransmission. We have established yeast vacuole homotypic fusion for studying membrane fusion mechanisms- with excellent genetics, organelle isolation, organelle cytology, and a rapid quantitative in vitro assay. Docked vacuoles are drawn together, each with a disc-like regions of closely apposed membrane. The proteins and lipids needed for docking and fusion are highly enriched at the periphery of these apposed discs, the """"""""vertex ring domain"""""""". Fusion occurs around these vertex rings. Vacuole fusion requires SNAREs, SM and NSF/SNAP chaperones of SNAREs, Rab/Ypt proteins, Rho GTPases, actin and its regulatory proteins, regulatory lipids (sterol, diacylglycerol, and 3- and 4- phosphoinositides), and calcium, each implicated in other fusion reactions as well. To investigate their interrelationships, We have purified the soluble :and peripheral membrane proteins which support vacuole fusion, assembled antibody, lipid ligand, and drug inhibitors, and characterized the stages of the reaction: priming (Sec18/17/ATP-driven SNARE disassembly, phosphoinositide synthesis), docking (consisting of tethering, vertex ring enrichment of selective proteins and lipids, and trans-pairing of SNAREs), Ca2+ efflux, and fusion (compartment mixing). We now propose to: 1. Discover the rest of the proteins and lipids of the vertex ring and map their assembly relationships. 2. Develop assays of the bound-nucleotide and functional relationships of the 4 GTPases of vacuole fusion. 3. Examine the receptors, regulation, and functions of remodeling of vacuole-bound actin. 4. Uncover the channel of Ca2+ efflux from the vacuole and its downstream effectors. 5. Establish biochemical assays of the function of the heterohexameric HOPS complex, which integrates Ypt/Rab activation, phosphoinositide binding, and SM-mediated SNARE complex assembly. 6. Exploit additional functional assays, including lipid mixing and patch-clamping docked vacuoles, and seek reconstitution of vacuole fusion subreactions.
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