PHOSPHATIDYLCHOLINE BIOSYNTHESIS IN SACCHAROMYCES-CEREVISIAE - REGULATORY INSIGHTS FROM STUDIES EMPLOYING NULL AND CHIMERIC SN-1,2-DIACYLGLYCEROL CHOLINEPHOSPHOTRANSFERASE AND ETHANOLAMINEPHOSPHOTRANSFERASE

The Saccharomyces cerevisiae CPT1 and EPT1 genes encode distinct choline- and choline/ethanolamine-phosphotransferases, respectively. In vitro, each gene product accounts for 50% of the measurable cholinephosphotransferase activity. Strains containing null mutations in the CPT1 and EPT1 loci were used to investigate the function of each gene product in vivo. The CPT1 gene product was responsible for 95% of phosphatidylcholine (PC) synthesis via the CDP-choline pathway in vivo. The EPT1 gene product accounted for only 5% of PC synthesis in vivo. Chimeric CPT1/EPT1 enzymes with diacylglycerol and CDP-aminoalcohol specificities both similar and distinct from the parental enzymes were used to determine the specific segments of the CPT1/EPT1 gene products required to restore PC synthesis to cpt(-) cells in vivo. Only chimeras expressing the CDP-aminoalcohol specificity region of CPT1 were capable of PC synthesis via the CDP-choline pathway in vivo. Analysis of phospholipids extracted from wild type, cpt(-), and ept(-) cells labeled with P-32(i) indicated an intact CPT1 gene product was required for the pleiotropic regulation of phospholipid synthesis by inositol. Chimeric CPT1/EPT1 enzymes expressed in a cpt(-) background mapped the regulatory region of the CPT1 gene product required for the inositol-dependent regulation of phospholipid synthesis to the CDP-aminoalcohol binding domain of CPT1. Strains harboring dysfunctional cholinephosphotransferase enzymes also displayed decreased levels of choline uptake, suggesting that a feedback loop exists to coordinate choline uptake with ongoing PC biosynthesis. The data also implicate the CPT1 gene product in PC biosynthesis from an endogenous source of choline derived from turnover of PC via the phosphatidylserine-dependent route for PC synthesis.