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Regulation of the PIS1-encoded phosphatiduylinositol synthase by zinc, and purification and characterization of the N-ethylmaleimide-sensitive Mg2+ dependent phosphatidate phosphatase
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Title
Regulation of the PIS1-encoded phosphatiduylinositol synthase by zinc, and purification and characterization of the N-ethylmaleimide-sensitive Mg2+ dependent phosphatidate phosphatase
Name
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Han
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Seung-Hee
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1972-
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Seung-Hee Han
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author
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Carman
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George
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Advisory Committee
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George M. Carman
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chair
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Mattews
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Karl
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Advisory Committee
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Karl R. Mattews
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internal member
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Tchikindas
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Mikhail
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Advisory Committee
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Mikhail Tchikindas
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internal member
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Storch
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Judith
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Advisory Committee
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Judith Storch
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outside member
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Rutgers University
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degree grantor
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Graduate School - New Brunswick
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Text
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theses
OriginInfo
DateCreated
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2007
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2007
Language
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English
PhysicalDescription
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electronic
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application/pdf
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text/xml
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xv, 153 pages
Abstract
In 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.
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.
Note
(type = degree)
Ph.D.
Note
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Includes bibliographical references (p. 139-152).
Subject
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Topic
Food Science
Subject
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Topic
Saccharomyces cerevisiae
Subject
(ID = SUBJ3);
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Topic
Phospholipids
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Graduate School - New Brunswick Electronic Theses and Dissertations
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rucore19991600001
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http://hdl.rutgers.edu/1782.2/rucore10001600001.ETD.16071
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ETD_344
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Identifier
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doi:10.7282/T3HH6KGH
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ETD doctoral
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Open
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Seung-Hee Han
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Rutgers University. Graduate School - New Brunswick
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Non-exclusive ETD license
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Author Agreement License
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I hereby grant to the Rutgers University Libraries and to my school the non-exclusive right to archive, reproduce and distribute my thesis or dissertation, in whole or in part, and/or my abstract, in whole or in part, in and from an electronic format, subject to the release date subsequently stipulated in this submittal form and approved by my school. I represent and stipulate that the thesis or dissertation and its abstract are my original work, that they do not infringe or violate any rights of others, and that I make these grants as the sole owner of the rights to my thesis or dissertation and its abstract. I represent that I have obtained written permissions, when necessary, from the owner(s) of each third party copyrighted matter to be included in my thesis or dissertation and will supply copies of such upon request by my school. I acknowledge that RU ETD and my school will not distribute my thesis or dissertation or its abstract if, in their reasonable judgment, they believe all such rights have not been secured. I acknowledge that I retain ownership rights to the copyright of my work. I also retain the right to use all or part of this thesis or dissertation in future works, such as articles or books.
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