Uniform TitleRegulation of the PIS1-encoded phosphatiduylinositol synthase by zinc, and purification and characterization of the N-ethylmaleimide-sensitive Mg2+ dependent phosphatidate phosphatase
NameHan, Seung-Hee (author), Carman, George (chair), Mattews, Karl (internal member), Tchikindas, Mikhail (internal member), Storch, Judith (outside member), Rutgers University, Graduate School - New Brunswick,
DescriptionIn the yeast Saccharomyces cerevisiae, the mineral zinc is essential for growth and metabolism. The effects of zinc depletion on the regulation of the PIS1-encoded phosphatidylinositol synthase were examined. Phosphatidylinositol synthase activity increased when zinc was depleted from the growth medium. Analysis of a zrt1D zrt2D mutant defective in plasma membrane zinc transport indicated that the cytoplasmic levels of zinc were responsible for the regulation of phosphatidylinositol synthase. PIS1 mRNA, its encoded protein Pis1p, and the b-galactosidase activity driven by the PPIS1-lacZ reporter gene were elevated in zinc-depleted cells. This indicated that the increase in phosphatidylinositol synthase activity was due to a transcriptional mechanism. The regulation of PIS1 gene expression by zinc depletion was mediated by the zinc-regulated transcription factor Zap1p. This work advances understanding of phospholipid synthesis regulation by zinc and the transcription control of the PIS1 gene.
Mg2+-dependent phosphatidate phosphatase catalyzes the dephosphorylation of phosphatidate to synthesize diacylglycerol and inorganic phosphate. There are at least two genes that encode Mg2+-dependent phosphatidate phosphatase enzymes in yeast. The PAH1 gene encodes an enzyme that is insensitive to N-ethylmaleimide whereas the gene that encodes the N-ethylmaleimide-sensitive enzyme has yet to be identified. The aim of this work was to purify and characterize the N-ethylmaleimide-sensitive Mg2+-dependent phosphatidate phosphatase. To purify the enzyme, a pah1D dpp1D lpp1D triple mutant that is devoid of all known phosphatidate phosphatase enzymes was used. The Mg2+-dependent phosphatidate phosphatase was extracted from the membrane fraction using 1 M NaCl. The salt extracted enzyme was then purified by chromatography with phosphocellulose, Mono Q and Superose 6. The enzyme was enriched 2,250 fold by this procedure. The basic enzymological properties of the purified Mg2+-dependent phosphatidate phosphatase were examined. The enzyme had a pH optimum from 7 to 7.5, and was dependent on Mg2+ ions for activity. The enzyme activity followed surface dilution kinetics using Triton X-100-phosphatidate mixed micelles. The enzyme was inhibited by N-ethylmaleimide, phenylglyoxal and propranolol. In addition, the enzyme activity was regulated by nucleotides, sphingolipids and phospholipids. These studies provide information on the mode of action of this important enzyme in lipid metabolism.
NoteIncludes bibliographical references (p. 139-152).
CollectionGraduate School - New Brunswick Electronic Theses and Dissertations
Organization NameRutgers, The State University of New Jersey
RightsThe author owns the copyright to this work.