Investigation of secretory vesicle membrane sorting has been hindered principally due to a lack of information on the secretory membrane proteins themselves and the absence of any corresponding cDNAs. This proposal addresses both problems. The adrenal medullary chromaffin cell shares many of the characteristics of the sympathetic neuron; upon stimulation, both respond by fusing secretory vesicle membrane with the plasma membrane with consequent release of their stored contents. The vesicular membrane is then efficiently re-extracted and apparently re-cycled to serve in another round of secretion. Between 40 and 60 proteins are found in this membrane; few of these have been correlated with any known function and structural information is sparse. Given this relatively small number of proteins found in the granule membrane and the biochemically unique vantage that the granule occupies as a neural model system, it is clear that a systematic structural investigation of the granule membrane proteins by molecular genetic analysis will be of enormous benefit in understanding the biologically important processes of stimulus-secretion and exocytosis, vesicular membrane fusion, membrane trafficking, and bio-molecule uptake. We have begun a molecular genetic characterization of these membrane proteins by cloning portions of the genes encoding a subset of them; we are currently involved in identification of the clones, to be followed by the determination of the cloned nucleotide sequences, and inferrence of the encoded amino acid structures. Testable predictions of the protein structure with respect to the membrane will be made and the intracellular location of each will be determined by immunoelectron microscopy. This work will therefore immediately result in new structural information about these proteins. We will also possess their cognate cDNAs, permitting molecular genetic investigations of the membrane's biogenesis. The future goal of examining secretory membrane sorting at the molecular level will become feasible. The information obtained should largely apply to the entire sympatho-adrenal system.
