Phospholipid Metabolism and Membrane Function

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The yeast Saccharomyces cerevisiae responds to a variety of stress conditions (e.g., zinc depletion) by regulating the expression of several enzyme activities including those involved in phospholipid synthesis. Zinc is an essential nutrient required for the growth and metabolism of S. cerevisiae, and of higher eukaryotic cells. The work proposed in this application will address mechanisms by which the expression of key Dhospholipid biosynthetic enzyme activities is regulated in response to zinc depletion. We will examine the lypothesis that zinc depletion induces a cytosolic Mg2+-dependent and NEM-sensitive phosphatidate phosphatase that reduces phosphatidate concentration in the ER. This in turn triggers the Opilp-mediated repression of CHO1 (encodes phosphatidylserine synthase) and other UAS|No-containing genes. This question will be addressed using a phosphatase mutant that will be isolated by a reverse genetic approach requiring the purification of the cytosolic Mg2+-dependent phosphatidate phosphatase enzyme. The regulation of expression of this phosphatase gene by zinc depletion will be examined, along with the biochemical properties of the purified enzyme. We will examine the hypothesis that E/C/7-encoded ethanolamine kinase and CK/t-encoded choline kinase are induced in response to zinc depletion. Mechanisms responsible for the zinc-mediated regulation of the EKI1 and CKI1 genes will be examined, and the biological relevance of regulation for each gene will be examined using selective promoter mutations that affect their expression. The biological relevance of the Opilp-mediated repression of CHO1 and the Zaplp- mediated induction of PIS1 (encodes phosphatidylinositol synthase) will be explored with mutants that are unable to down-regulate phosphatidylserine synthase or up-regulate phosphatidylinositol synthase activities, respectively, in response to zinc depletion. We will examine the hypothesis that the function of the high- affinity plasma membrane zinc transporter Zrt1 p is regulated by changes in phospholipid composition brought about by phospholipid synthesis regulation by zinc depletion. Zinc transport function will be addressed with mutants defective in phospholipid synthesis, and with Zrtlp reconstituted into unilamellar vesicles with varying phospholipid composition.