The membrane anchored SNARE proteins mediate fusion of vesicle to target membranes during protein maturation and secretion. This proposal describes experiments to kinetically measure membrane fusion intermediates during fusion of flipped SNARE cells, which express SNAREs on their outer surfaces. Electrophysiological capacitance measurements will be developed to monitor flipped SNARE cell-cell fusion, as well as with reconstituted liposomes:cell fusion to kinetically map measure fusion pore formation, and propagation. Additionally, fusion events mediated by external facing SNAREs will allow separation from cellular components, and the effects of the fusion regulating proteins Munc13, Munc18, synaptotagmin, and complexin will be quantified. Finally, the cooperativity of SNARE complex formation will be measured, by correlating fusion efficiency to SNARE surface expression, leading to a Hill coefficient of fusion. An understanding of vesicle fusion, and development of quantitive assays is essential as mutations in SNARE proteins are associated with developmental and congenital diseases. ? ?
