Regulated exocytosis involves the fusion of secretory vesicles with the plasma membrane to release hormones and neurotransmitters from endocrine and neural cells in response to a rise in Ca2+ concentrations. The machinery that senses this increase in Ca2+ to trigger fusion remains unidentified. The proteins SNAP25, syntaxin, and VAMP (termed SNAREs) frorm a stable SNARE complex and have been found to be critical for regulated exocytosis, but their exact roles remain unclear. Exocytosis of docked vesicles proceeds through ATP-dependent priming and Ca2+-dependent fusion steps. Studies with botulinum neurotoxins have revealed that the C-terminus of SNPA25 is required at a late Ca2+-dependent stage of exocytosis immediately prior to membrane fusion. We propose that the C-terminus of SNPA25 plays an essential role in the Ca2+-sensing machinery of regulated exocytosis by mediated Ca2+-dependent interactions between synaptotagmin and SNARE complexes. We can reconstitute the post-docking stages of exocytosis in a permeabilized cell assay. Using transfection methods, we can replace endogenous SNAP25 in PC12 cells with mutant SNAP25. By generating mutations in the C-terminus of SNAP25, we will study the role of this portion of SNAP25 in secretion and complex assembly.
Our aims are to 1) determine the in vivo role of the C-terminus of SNAP25 in the Ca2+-regulation of exocytosis using our permeabilized cell assay, and 2) determine the C-terminal residues of SNAP25 critical for function using in vitro assays to assess Ca2+- dependent synaptotagmin binding to SNARE complexes. This work will provide insight into the CA2+-sensing machinery of regulated exocytosis and elucidate the role of specific SNAP25 residues in its interactions with synaptotagmin and the SNARE complex.
