Recent studies indicate that the metabolism of diacylglycerol pyrophosphate (DGPP) is involved in a novel lipid signaling pathway. DGPP was first identified as the product of the phosphatidate (PA) kinase reaction in the plant Catharanthus roseus. DGPP has since been found in a variety of plants and in the yeast Saccharomyces cerevisiae. The amounts of DGPP in plants and in wild-type yeast are barely detectable and reminiscent of other lipid signaling molecules. Recent studies have shown that DGPP accumulates in plant tissues upon G protein activation through the stimulation of PA kinase activity and metabolic labeling studies have shown that DGPP is rapidly metabolized to PA, and then to diacylglycerol (DG). It has been suggested that the function of DGPP is to attenuate the signaling functions of PA, that DGPP is the precursor of the PA which serves as a signaling molecule, or that DGPP itself functions as a signaling molecule. DGPPase catalyzes the dephosphorylation of DGPP to yield PA and then catalyzes the dephosphorylation of PA to yield DG. The enzyme has been identified in a wide range of organisms including S. cerevisiae. The investigators hypothesize that the regulation of DGPPase regulates the cellular levels of DGPP, PA and DG. During the past grant period, the investigators purified and characterized two DGPPases from S. cerevisiae and isolated two genes (DPP1 and DPP2) encoding for the enzyme. The cloned genes will be characterized, and the investigators will examine the regulation of the DGPPases in response to inositol, growth mutants, and of elevated levels of DGPPase in strains that overexpress the enzymes. Enzymological and kinetic characterization of the purified DGPPases will be expanded. The regulation of the DGPPase activities by lipids and water-soluble intermediates of lipid metabolism will be examined. The investigators will also examine the DGPPase enzymes for covalent modification by phosphorylation. The proposed studies are expected to provide insight into the molecular and biochemical regulation of DGPPase and into the role DGPP plays in phospholipid metabolism and lipid signaling in S. cerevisiae. The results of these studies should be relevant to higher eukaryotic organisms.