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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Mental Health (NIMH)
Type
Individual Predoctoral NRSA for M.D./Ph.D. Fellowships (ADAMHA) (F30)
Project #
7F30MH012694-02
Application #
6391778
Study Section
Special Emphasis Panel (ZRG1-MDCN-7 (02))
Program Officer
Goldschmidts, Walter L
Project Start
2001-05-01
Project End
Budget Start
2001-06-01
Budget End
2002-05-31
Support Year
2
Fiscal Year
2001
Total Cost
$37,308
Indirect Cost
Name
University of Chicago
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
225410919
City
Chicago
State
IL
Country
United States
Zip Code
60637