The maintenance of distinct organelles within the eukaryotic cytosol is essential for survival. The exchange of material between these organelles requires the merger of two phospholipids membranes. All known forms of intracellular membrane fusion, including synaptic vesicle fusion, involve a highly conserved family of proteins termed SNAREs (Soluble N-ethyl maleimide sensitive factor Attachment Protein Receptors). Auxiliary SNARE binding proteins are known to regulate membrane fusion events, such as the Ca2+ binding protein synaptotagmin. Crystal structures of some of the key players involved in this process have been solved, such as the synaptic SNARE complex and the cytoplasmic domain of synaptotagmin. In vitro bulk liposome-liposome fusion experiments have established that SNARES and synaptotagmin constitute a minimal, albeit inefficient, fusion machinery, but they have done little to reveal the underlying molecular mechanism, both in terms of sequential and spatial interactions between proteins and lipids during fusion. We propose to study the molecular mechanism of Ca2+-triggered synaptic vesicle fusion by single molecule fluorescence methods. Our previous work has provided the framework for the proposed studies. Specifically, we propose to study correlations between protein-protein, protein-lipid interactions and fusion, to study the effect of post-translational modifications of SNARE proteins and of the lipid/cholesterol composition on fusion, to study the interactions of SNAREs and synaptotagmin at the interface between docked membranes, and to study the molecular mechanism of SNARE complex disassembly by the ATPase N- ethylmalemeide-sensitive factor (NSF). These in vitro studies will be complemented by in vivo studies using PC12 cells. We anticipate that our in vitro system can be extended to include other factors in order to obtain a reconstituted system that may eventually approach the properties of the synaptic vesicle fusion machinery in the neuron. Such a system could serve as an efficient model system for novel drug discovery. ? ? ? ?
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