PO Box 800735
Charlottesville, VA 22908
Calcium-dependent, membrane-binding proteins and mechanisms of exocytosis
Our research group is studying the subcellular mechanisms that underlie the release of hormones and neurotransmitters by exocytosis. In this process a secretory vesicle moves through the cytoplasmic matrix and attaches to the plasma membrane of the cell. Then the membranes of the vesicle and the cell fuse and the contents of the vesicle are released. Subsequently, the vesicle membrane is recovered by endocytosis and degraded or reused to form new secretory vesicles. Our interests have focused on proteins that may regulate or mediate individual steps in this process. In particular, we are studying a novel group of calcium-dependent, membrane-binding proteins that can promote membrane fusion in vitro. This group of proteins, collectively called the annexins, includes synexin, calpactin, lipocortin, and several other members that have been proposed to mediate calcium-dependent events on membrane surfaces. (For a historical review see: https://pdfs.semanticscholar.org/a221/27aab83c1e0a8aced19b2c635d65d88a061e.pdf ). More recently we have extended our studies to the synaptotagmins and the copines, proteins that interact with membranes through a domain homologous to the calcium- and lipid-binding domain of protein kinase C. In these studies we draw on techniques from biophysics, biochemistry, molecular biology and genetics. For example, we use fluorescence resonance energy transfer techniques to monitor protein-membrane interactions, membrane fusion, and protein self-assembly on membrane surfaces. We use site-directed mutagenesis and bacterial or yeast expression of recombinant proteins to assess the role of particular protein structural domains in promoting membrane fusion. We use cultured bovine chromaffin cells and in vitro kinase assays to assess the role of phosphorylation in regulating annexin function. We use yeast and nematode genetics to analyze the function of calcium-dependant, membrane-binding proteins in the secretory pathway. These fundamental studies on the mechanism of exocytosis may provide a basis for understanding the action of drugs that affect hormone or neurotransmitter release, and may also lead to the development of new pharmacological agents that influence these processes.