The process of hormone and neurotransmitter release by exocytosis involves the calcium-stimulated attachment and fusion of secretory or synaptic vesicles to the plasma membrane of endocrine cells or neurons. This process is presumably directed by specific protein components of the secretory or synaptic vesicle, the cytoplasm, and the plasma membrane. The goal of this project is to determine the importance of specific vesicle membrane proteins in exocytosis and the biochemical details of their mechanism of action. The study will initially focus on synaptotagmin, a transmembrane protein that has a cytoplasmic domain homologous to the calcium and lipid-binding domain of protein kinase C, and synaptophysin, another transmembrane protein that has an extended, proline rich domain that may interact with cytoplasmic or plasma membrane components. A novel approach will be adopted involving the expression of mammalian cDNAs for these proteins in yeast, biochemical characterization of the properties of yeast secretory vesicles conferred by these proteins, and examination of the physiology of secretion in the transformed yeast cells. The structural features of the mammalian proteins essential for biochemical or physiological effects will be determined by site-directed mutagenesis. The basic understanding of the mechanisms underlying hormone release and neurotransmission gained in this study may lead to the development of treatments for disorders of neuromuscular transmission, central nervous system function, or endocrine function.
