In the yeast Saccharomyces cerevisiae, phosphatidate (PA) phosphatase plays an important role in the regulation of phospholipid biosynthesis by the primary (phosphatidylethanolamine methylation) and auxiliary (CDP-ethanolamine- and CDP-- choline-based) pathways. The enzyme also plays a role in the overall regulation controlling the proportional synthesis of phosholipids and triacylglycerols. In the past grant period we purified and characterized 45-kDa and 104-kDa forms of PA phosphatase. Our studies indicated that the expression of the two forms of PA phosphatase were regulated differentially by inositol and the enzyme activities were regulated differentially by phosphorylation. In this competitive renewal application we propose studies that are expected to provide insight into the regulation of PA phosphatase activity as well as the expression of the enzyme. The phosphorylation of PA phosphatase by cAMP-dependent protein kinase and the effects of phosphorylation on enzyme activity and kinetics will be examined using pure enzyme. The phosphorylation of PA phosphatase in vivo will be examined in wild-type and mutant cells defective in cAMP-dependent protein kinase activity. The effects of PA phosphatase phosphorylation on overall lipid biosynthesis will be examined. Systematic kinetic studies are proposed to examine the effect of lipid activators and inhibitors on the activity of pure PA phosphatase. Kinetic experiments will be performed using well-defined Triton X-100/phospholipid mixed micelles as an experimental system. The mixed micelle studies will be complemented with studies using pure enzymes reconstituted into unilamellar phospholipid vesicles. We will initiate a project to clone the structural genes for the 45-kDa and 104-kDa forms of PA phosphatase. We will use specific antibodies to the enzymes and lambda-gtll genomic expression and lambda-ZAP cDNA expression libraries. The cloned genes will be used as probes to study the regulation of the mRNA levels of the 45-kDa and 104-kDa forms of PA phosphatase in response to inositol and during the growth phase. The essential nature of PA phosphatase will also be addressed. The results of the proposed studies with S. cerevisiae should be relevant to higher eukaryotic organisms.
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