Protein-Protein Interactions of the Neuronal Synapse: An in vitro Investigation of the Interactions Between Synaptotagmin I and the SNARE Proteins

Epp, Natalie Erin, Department of Chemistry, University of Virginia
Cafiso, David, Department of Chemistry, University of Virginia
Bryant, Robert, Department of Chemistry, University of Virginia
Landers, James, Department of Chemistry, University of Virginia

Synaptic vesicle fusion is the fundamental mechanism underlying neurotransmission in the brain. It is a highly regulated process, which involves the coordination of hundreds of proteins, and is mediated by calcium. At the heart of this process are Synaptotagmin I and the SNARE proteins. Synaptotagmin I, a synaptic vesicle protein, binds calcium ions through calcium binding loops on its two C2 domains. This has led to its wide spread acceptance as the calcium sensor for neuronal exocytosis. The SNARE complex, composed of Syntaxin 1A, SNAP 25, and Synaptobrevin 2 is a coiled coil which is thought to drive the fusion of synaptic vesicles to the presynaptic membrane. Despite the critical importance of both the SNARE complex and Synaptotagmin to synaptic vesicle fusion, little is know about how they interact to coordinate their functions. This study uses EPR spectroscopy and Site Directed Spin Labeling to investigate the nature of the interactions between Synaptotagmin I and the SNARE proteins under various conditions. The EPR data show that the C2B polybasic region, sites 325 and 327, of Synaptotagmin bind to SNARE complexes reconstituted in POPC:POPS (3:1) vesicles in the absence of calcium. In the presence of calcium the interaction was disrupted. These findings suggest that Synaptotagmin I can not simultaneously bind membranes and the SNARE complex. Sites 325 and 327 were also found to interact with a soluble fragment of Syntaxin 1A in a calcium dependent manner. These findings challenge many of the present models of this system. Although the scope of this study is narrow, these´╗┐x findings may have important implications for the understanding of synaptic vesicle exocytosis.

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MS (Master of Science)
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