Eukaryotic cells are compartmentalized by membrane-bound organelles, which communicate with each other through the transport of cargo vesicles that culminates in membrane fusion. We use vacuolar lysosomes isolated from the yeast Saccharomyces cerevisiae to examine membrane fusion. Vacuolar fusion requires a group of regulatory lipids that includes phosphoinositides, ergosterol, phosphatidic acid and diacylglycerol. Fusion occurs at membrane microdomains where regulatory lipids interdependently assemble with the proteins that catalyze fusion including SNAREs, the Rab GTPase Ypt7 the tethering complex HOPS, and actin. Because the lipid bilayer undergoes constant remodeling through lipid modification, we hypothesize that the metabolic interconversion of lipid species is integrally coupled to membrane fusion. In this proposal we examine the relationship between the lipid composition of vacuolar membranes and the function of SNAREs as well as the Rab7/Ypt7 nucleotide exchange factor Mon1. We plan to elucidate the role of lipid composition and the regulation of vacuole fusion by pursuing the following aims: 1 - Examine the function of SNARE trans-membrane domains during fusion and how these domains are controlled by the local lipid environment; 2 - Determine the role of lipid composition on kinetics of affinities of SNARE complex formation; 3 - Examine the relationship between the lipid phosphatidylinositol 3-phosphate and the recruitment and activation of the Rab7/Ypt7 nucleotide exchange factor Mon1. PUBLIC HEALTH RELAVANCE: Membrane fusion is essential for cell viability, neurotransmission, the secretion of antibodies and hormones, blood clotting, and the destruction of microbes. Dysregulation of lipid metabolism/modification is tied t many diseases including Charcot- Marie-Tooth disease, obesity, type-2 diabetes, and heart disease. In each case, membrane trafficking and fusion is deleteriously affected, yet the mechanisms that link lipid modification and the regulation of the fusion machinery remain unknown. The proposed study will define how the composition of the vacuolar membrane regulates SNARE and Mon1 function and will give us clues about the relationship between lipid modification and human health and disease. PUBLIC HEALTH RELEVANCE: Dysfunctional membrane fusion can have severe consequences to human health, and defects are found in many genetic and infectious diseases that are too numerous to list here. However, suffice it to say that proper membrane fusion is essential for cell viability and cellular functions that include the release of neurotransmitters, hormones and antibodies. Therefore, understanding the fundamental mechanisms of membrane fusion is critical if we are to understand the diseases that target this machinery.