Phospholipids have multiple functions in cells in forming the membrane bilayer which serves as the permeability barrier of cells and organelles, serves as the matrix for membrane associated processes, and provides the metabolites involved in signal transduction cascades which control cell growth, cell differentiation and responses to extracellular signals. This proposal will focus on a central step of phospholipid metabolism catalyzed by the CDP-diacylglycerol (-DAG) synthase (CDS1 gene product) which provides the precursor to the major phospholipids found in all organelles of eukaryotic cells including Saccharomyces cerevisiae, the simple eukaryotic organism in which these studies will be carried out. This CDP-DAG synthase activity has been found in the endoplasmic reticulum, the mitochondrial inner membrane, and the plasma membrane. Phospholipid synthesis in yeast is controlled by a complex composite of extracellular inositol-regulation of gene expression, cellular levels of substrates, intracellular effector molecules, the growth phase of the cell, and membrane phospholipid composition. 1) Using engineered strains overproducing the synthase, the enzyme will be isolated and characterized with respect to its physical, chemical, and enzymological properties in order to relate in vitro properties of the enzyme to its in vivo regulation and function. 2) Mutants in the CDS1 gene will be isolated that result in rapid loss of function in order to determine those processes that require a continual short term supply of CDP-DAG. Addition functional relationships of CDP-DAG to other yet unknown cellular processes will be uncovered by isolating second-site suppressor mutations of the primary mutations. Neither mutations of this type nor suppressor mutations have been previously reported in yeast phospholipid metabolism. 3) The regulation of CDS1 gene expression as well as gene product activity by the complex inositol regulatory system, the growth phase of cells, and the factors that regulate mitochondrial biogenesis and function will be investigated. 4) More definitive evidence for the apparent multi-organelle distribution of CDP-DAG synthase will be sought by coupling a better understanding of the biosynthesis and membrane assembly of the protein with a more thorough investigation of its subcellular location. The sum total of the results from these four specific aims will better define regulation of phospholipid metabolism in a multi-organelle eukaryotic organism and define the role this central step in phospholipid metabolism plays in cell physiology.
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