In the yeast Saccharomyces cerevisiae, CTP is a precursor of all membrane phospholipids synthesized by the phosphatidylethanolamine methylation (primary) and CDP-ethanolamine- and CDP-choline-based (auxiliary) pathways. This grant application addresses the hypothesis that cellular levels of CTP regulate the synthesis of phospholipids by the primary and auxiliary pathways by controlling the expression and/or activities of the enzymes in these pathways. The effects of CTP on phospholipid synthesis will be examined in vivo using S. cerevisiae strains expressing various levels of CTP. CTP levels will be varied through the expression of the URA7 gene, which encodes CTP synthetase. The mRNA abundance and protein levels of phospholipid biosynthetic enzymes will be examined in response to various levels of CTP. Biochemical regulation of enzyme activities in response to CTP will also be examined. Since cellular CTP levels must be governed by the expression and/or activity of CTP synthetase. This application will also address if CTP synthetase activity is regulated by factors known to regulated phospholipid synthesis. CTP synthetase will be purified by molecular genetic and/or biochemical approaches for the purpose of preparing anti-CTP synthetase antibodies for regulation studies. The purified enzyme will be characterized with respect to its basic enzymological and kinetic properties. The regulation of CTP synthetase by cAMP-dependent protein kinase phosphorylation will be examined in vivo and in vitro. The information that will be obtained from these studies should be of great use to, and complement the work of other investigators in the field of phospholipid metabolism as well as those working on nucleotide metabolism.
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