Abstract
The regulation of the Saccharomyces cerevisiae DPP1-encoded diacylglycerol pyrophosphate (DGPP) phosphatase by inositol supplementation and growth phase was examined. Addition of inositol to the growth medium resulted in a dose-dependent increase in the level of DGPP phosphatase activity in both exponential and stationary phase cells. Activity was greater in stationary phase cells when compared with exponential phase cells, and the inositol- and growth phase-dependent regulations of DGPP phosphatase were additive. Analyses of DGPP phosphatase mRNA and protein levels, and expression of beta-galactosidase activity driven by a P(DPP1)-lacZ reporter gene, indicated that a transcriptional mechanism was responsible for this regulation. Regulation of DGPP phosphatase by inositol and growth phase occurred in a manner that was opposite that of many phospholipid biosynthetic enzymes. Regulation of DGPP phosphatase expression by inositol supplementation, but not growth phase, was altered in opi1Delta, ino2Delta, and ino4Delta phospholipid synthesis regulatory mutants. CDP-diacylglycerol, a phospholipid pathway intermediate used for the synthesis of phosphatidylserine and phosphatidylinositol, inhibited DGPP phosphatase activity by a mixed mechanism that caused an increase in K(m) and a decrease in V(max). DGPP stimulated the activity of pure phosphatidylserine synthase by a mechanism that increased the affinity of the enzyme for its substrate CDP-diacylglycerol. Phospholipid composition analysis of a dpp1Delta mutant showed that DGPP phosphatase played a role in the regulation of phospholipid metabolism by inositol, as well as regulating the cellular levels of phosphatidylinositol.
Highlights
The regulation of the Saccharomyces cerevisiae DPP1encoded diacylglycerol pyrophosphate (DGPP) phosphatase by inositol supplementation and growth phase was examined
We examined the influence of growth phase on DGPP phosphatase, since it has a major influence on the expression of many phospholipid biosynthetic enzymes [1, 2, 4, 7, 17]
We discovered that the activity of DGPP phosphatase was inhibited by CDP-DG and that the activity of PS synthase was stimulated by DGPP
Summary
Growth medium supplies were purchased from Difco. Methods for yeast growth were performed as described previously [26, 27]. Escherichia coli strain DH5␣ was grown in LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl, pH 7.4) at 37 °C. For the overexpression of CHO1-encoded PS synthase, cells were grown to the exponential phase in complete synthetic medium containing 2% raffinose. Plasmid maintenance and amplifications were performed in E. coli strain DH5␣. Construction of Plasmids—Plasmid pJO2 contains the putative promoter of the DPP1 gene fused to the lacZ gene of E. coli. The plasmid was constructed by replacing the EcoRI fragment of plasmid pSD90 with the DPP1 promoter, which was obtained by PCR (primers, 5ЈGTGAAGAAGCAGGAATTCATAAAGGGACAACACGG-3Ј and 5ЈGTTTTAATAAACGAAACTGAATTCATTTTGGTCG-3Ј) using strain W303-1A genomic DNA as a template. The pJO2 plasmid was introduced into the indicated strains to examine the expression of the DPP1 gene by measuring -galactosidase activity
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