The phosphorylation of phosphatidylinositol by the reaction sequence phosphatidylinositol - phosphatidylinositol 4-phosphate - phosphatidylinositol 4, 5-bisphosphate is regulated in the yeast Saccharomyces cervisiae. Phosphatidylinositol 4, 5-bisphosphate, or its breakdown products diacylglycerol and inositol trisphosphate, play an essential role in S. cerevisiae cell proliferation. Phosphatidylinositol kinase is the first enzyme in the phosphorylation pathway of phosphatidylinositol. The regulation of this enzyme should therefore play a major role in phosphatidylinositol metabolism and cell growth in S. cerevisiae. Regulation of phosphatidylinositol kinase by cAMP-dependent protein kinase will be examined in vitro and in vivo. In vitro phosphorylation of phosphatidylinositol kinase and the effects of phosphorylation of enzyme activity and kinetics will be examined using pure phosphatidylinositol kinase. In vivo phosphorylation of phosphatidylinositol kinase will be examined in wild-type and mutants defective in cAMP-dependent protein kinase activity. The effects of phosphatidylinositol kinase phosphorylation on phosphatidylinositol metabolism and on overall phospholipid biosynthesis will be examined. Systematic studies are proposed to examine the effects of phospholipid composition and acyl moiety of the membrane on phosphatidylinositol kinase activity. The effects of the acyl composition of the substrate PI on activity will also be examined. These studies will be performed with pure enzyme reconstituted into unilamellar phospholipid vesicles. The reconstitution studies will be complemented with studies using a Triton X-100-phospholipid mixed micelle system. The regulation phosphatidylinositol kinase activity, subunit, and mRNA levels will be examined in response to inositol and the growth phase. We will also initiate a project to clone the structural gene for phosphatidylinositol kinase using specific antibodies to the enzyme and a lambda gt11 expression vector library. The essential nature of PI kinase will also be addressed. The results of the proposed studies should be relevant to higher eucaryotic organisms.
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