The research described in this proposal is designed to elucidate the mechanisms by which phospholipids are assembled into the membranes of subcellular organelles, and the role that specific phospholipids play in membrane structure and function. These goals will be accomplished by isolating and characterizing mutants of Chinese hamster ovary cells that are defective in the synthesis of phosphatidylserine and phosphatidylethanolamine. These mutants will be isolated using 5 bromeoxyuridine enrichment and auxotrophic selection, or replicaplating and auxotrophic screening, or temperature sensitive screening procedures. These methods will permit the isolation of mammalian cells defective in phosphatidylserine synthesis, translocation and decarboxylation. Mutants defective in lipid translocation will be used to elucidate the mechanism of this process and to determine if it is identical to that of phospholipid exchange proteins observed in vitro. If the process appears to be independent of phospholipid exchange protein activity the techniques of in vitro complementation will be used to characterize and purify the factors responsible for lipid translocation. Mutants defective in phospholipid synthesis will be manipulated to deplete cells of specific phospholipids in order to determine the effects upon specific organelle functions and cell physiology. From this line of investigation we will determine if the deletion of certain phospholipids pleiotropically effects all membrane systems or if it is confined to one organelle system or enzyme activity in particular. The development of mutants in phosphatidylethanolamine synthesis will also be complemented by the purification and characterization of phosphatidylserine decarboxylase from rat liver mitochondria. Strategies for purification of the decarboxylase include detergent solubilization and application of conventional protein purification methods, for the isolation of active enzyme, affinity labeling and purification of the inactive enzyme, and development of monoclonal antibodies and immunoaffinity purification. Collectively these studies will provide biochemical and genetic tools to study the regulation of membrane biogenesis in mammalian cells. The regulation of this process must ultimately be related to the control of cell growth and differentiation.
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