The long-term goals of this project are to understand the role of the outer mitochondrial membrane in lipid synthesis. Three critical enzymes are located in the outer membrane and this proposal focuses on one of them, glycerophophate acyltransferase (GAT). Results obtained in the current funding period have provided evidence that the catalytic site faces the cytosol and that the enzyme is held in the membrane by two transmembrane segments with the connecting segment facing the cytosol. In this renewal project the PI plans to investigate the biosynthesis of GAT. Whether the first or second methionine deduced from the cDNA sequence is the initiator methionine will be determined and whether or not the signal sequence is cleaved. Antibodies will also be used to determine whether the mitochondrial protein import complexes, TIM and TOM, or the heat shock proteins hsp60 and hsp70 are involved. Regulation of GAT will also be investigated including examination of the role of phosphorylation, and the roles of ATP and citrate as allosteric modulators. It is also planned to purify and clone a putative outer membrane stimulator protein and examine the mode of stimulation of the enzyme by polymyxin B.
