Abstract
The Saccharomyces cerevisiae CPT1 and EPT1 genes are structural genes encoding sn-1,2-diacylglycerol choline phosphotransferase and sn-1,2-diacylglycerol choline/ethanolamine phosphotransferase, respectively. Incorporation of 32Pi into phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine in wild type and ept1 strains was decreased in the presence of exogenous inositol. In contrast, inositol did not affect 32Pi incorporation into phospholipid in cpt1 or cpt1ept1 strains. In membranes isolated from wild type and ept1 strains grown in the presence of inositol or inositol/choline, the CPT1-derived cholinephosphotransferase activities were reduced 40-50 and 65%, respectively. Inositol-dependent reductions in CPT1 derived choline-phosphotransferase activity correlated with transcript levels in both wild type and ept- backgrounds. The ethanolaminephosphotransferase activity of the EPT1 gene product in wild type cells was reduced 40% by exogenous inositol alone and 50% by inositol/choline. In the cpt1 strain, however, the ethanolaminephosphotransferase activity was unaffected by exogenous inositol or inositol/choline. The inositol-dependent reduction of ethanolaminephosphotransferase activity observed in wild type cells correlated with reduced levels of EPT1 transcripts; in the cpt1 strain, EPT1 transcript levels were not affected by inositol. These results indicate that 1) a functional CPT1 gene or gene product is required for inositol-dependent regulation of phospholipid synthesis; 2) the enzyme activities of both the CPT1 and EPT1 gene products are repressed by inositol and inositol/choline, and require an intact CPT1 gene; 3) inositol mediates its regulatory effects on phospholipid synthesis via a transcriptional mechanism.
Highlights
Phatidylethanolamine, and phosphatidylserine in wild 1).the contributions of the Kennedy pathway toovertype and eptl strains was decreased in the presence of all synthesis of PC and PE in yeast is not well understood
A number of yeast phospholipid ferase activity was unaffected by exogenous inositol or biosynthetic enzymes, including CDP-diacylglycerol synthase inositollcholine.Theinositol-dependent reduction of (71, PS synthase (81, PS decarboxylase (9, lo), the PE N-methethanolaminephosphotransferase activity observed in yltransferases (11,121, and choline kinase (13) are repressed in wild type cells correlated with reduced levels of EPTl cells grown in the presence of inositol
2-5 x lo6countdmidml of hybridization solution. Prehybridization and This increase was not observed in the double null mutant in hybridization of radiolabeled probes were performed at 42 "C in 50% which the synthesis of PC was compromised, suggesting formamide, 6 x SSC (0.9 M NaCl, 0.09 M trisodium citrate), 5 x Den- higher levels of PC in the eptl strain may result from an inhardt's (0.1%bovine serum albumin, 0.1%polyvinylpyrrolidone, 0.1% Ficoll), 0.5% SDS, and 100 pg/ml of salmon spermDNA (27).Following hybridization, membranes were washed for 15 min each with 2x SSC, creased availability for the CPTl gene product of the common substrate, sn-l,2-diacylglycerol
Summary
Phatidylethanolamine, and phosphatidylserine in wild 1).the contributions of the Kennedy pathway toovertype and eptl strains was decreased in the presence of all synthesis of PC and PE in yeast is not well understood. Prehybridization and This increase was not observed in the double null mutant in hybridization of radiolabeled probes were performed at 42 "C in 50% which the synthesis of PC was compromised, suggesting formamide, 6 x SSC (0.9 M NaCl, 0.09 M trisodium citrate), 5 x Den- higher levels of PC in the eptl strain may result from an inhardt's (0.1%bovine serum albumin, 0.1%polyvinylpyrrolidone, 0.1% Ficoll), 0.5% SDS, and 100 pg/ml of salmon spermDNA (27).Following hybridization, membranes were washed for 15 min each with 2x SSC, creased availability for the CPTl gene product of the common substrate, sn-l,2-diacylglycerol.
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