The goals of this project are to elucidate the biochemistry and molecular biology of the mechanisms by which eukaryotic cells assemble membranes. The focus of this proposal is upon the synthesis and translocation phosphatidylserine within eukaryotic cells. The synthesis of phosphatidylserine will be examined in isolated membranes and permeabilized cells. The coupling of phosphatidylserine synthesis to Ca2+ transport mechanisms that occurs within the endoplasmic reticulum will be investigated to determine how these processes are coupled and how the kinetic characteristics of these two reactions are related. The ATP dependent translocation of newly synthesized phosphatidylserine from its site of synthesis to the inner mitochondrial membrane will also be examined in detail. Permeabilized animal cells and subcellular fractions derived from these cells will be used to determine the physical elements of cell structure that are required to reconstitute the ATP dependent lipid translocation reactions that occur in intact cells. Both permeabilized cells and the derived subcellular fractions will be manipulated to biochemically dissect the events which occur in the inter- and intraorganelle movement of phosphatidylserine. This line of experimentation will address the topics of a) the pools of phosphatidylserine that are transferred from the endoplasmic reticulum to the mitochondria, b) the commonality between phosphatidylserine and protein import into the mitochondria, c) the kinetics of the transport process and the stoichiometry of ATP utilization, and d) the coupling of mitochondrial import of phosphatidylserine to the synthesis and export of phosphatidylethanolamine. The experimentation with animal -cells will be complemented by biochemical and genetic studies with yeast cells. The biochemical analysis of the yeast cell system will utilize intact and permeabilized cells in conjunction with isolated mitochondria to determine if ATP is required for either the interorganelle or intramitochondrial translocation of phosphatidyl serine. Genetic experiments will be conducted in parallel with these experiments for the purpose of isolating yeast mutants that are defective in the translocation and decarboxylation of phosphatidylserine. The mutant strains are identified from mutagenized populations of cells by analyzing the ability of permeabilized clonal populations to decarboxylate the lipid analog NBD phosphatidyl[1-14C]serine. The isolation of yeast mutants defective in phosphatidylserine translocation and decarboxylation will provide the genetic tools necessary for evaluating the effects of these mutations upon cell growth and membrane structure and function. These mutant strains will also provide the necessary genetic background required for the molecular cloning of the genes for phosphatidylserine translocation and decarboxylation and elucidation of their molecular structure. These studies will provide new information about the fundamental cellular processes of membrane assembly and organelle biogenesis.
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