Project Summary/Abstract Membrane fusion is essential for cell growth, hormone secretion, neurotransmission, and cell invasion by pathogens. Membrane fusion mechanisms are conserved from yeast to humans. We have developed yeast vacuole fusion as a model system, identifying genes for membrane fusion, establishing an in vitro fusion assay with purified vacuoles, and purifying each relevant protein and lipid for reconstitution into proteoliposomes which faithfully reconstitute each aspect of fusion. These studies have revealed novel elements, most recently: 1. A dazzling array of proteins and lipids which cooperate for orderly lipid bilayer strain and rearrangement, giving fusion without lysis. 2. The assembly of complexes among membrane-bound proteins termed "SNAREs" isn't spontaneous, as heretofore believed. Their assembly is actually catalyzed by a large hexameric complex termed HOPS, which recognizes each of the individual SNAREs and assembles them in active intermediates, poised for rapid fusion. 3. Chaperones to the SNAREs, termed NSF/Sec18 and aSNAP/Sec17, which had been believed to only function to disassemble SNARE complexes after fusion, also promote fusion. 4. Lipids have a vital and active role in fusion. Each of these mechanistic insights will be pursued; our goals for the next 5 years are to understand the intermediates of HOPS-catalyzed SNARE assembly, their structures, the roles of chaperones Sec17/Sec18, and how these proteins trigger the lipid rearrangements of fusion. The importance of understanding this pathway is underscored by the central role of HOPS in the invasion of human cells by pathogenic viruses and bacteria.