Regulation of Phospholipid Synthesis in Saccharomyces cerevisiae by Zinc
2004; Elsevier BV; Volume: 279; Issue: 21 Linguagem: Inglês
10.1074/jbc.m402047200
ISSN1083-351X
AutoresWendy M. Iwanyshyn, Gil‐Soo Han, George Carman,
Tópico(s)Fungal and yeast genetics research
ResumoZinc is an essential nutrient required for the growth and metabolism of eukaryotic cells. In this work, we examined the effects of zinc depletion on the regulation of phospholipid synthesis in the yeast Saccharomyces cerevisiae. Zinc depletion resulted in a decrease in the activity levels of the CDP-diacylglycerol pathway enzymes phosphatidylserine synthase, phosphatidylserine decarboxylase, phosphatidylethanolamine methyltransferase, and phospholipid methyltransferase. In contrast, the activity of phosphatidylinositol synthase was elevated in response to zinc depletion. The level of Aut7p, a marker for the induction of autophagy, was also elevated in zinc-depleted cells. For the CHO1-encoded phosphatidylserine synthase, the reduction in activity in response to zinc depletion was controlled at the level of transcription. This regulation was mediated through the UASINO element and by the transcription factors Ino2p, Ino4p, and Opi1p that are responsible for the inositol-mediated regulation of UASINO-containing genes involved in phospholipid synthesis. Analysis of the cellular composition of the major membrane phospholipids showed that zinc depletion resulted in a 66% decrease in phosphatidylethanolamine and a 29% increase in phosphatidylinositol. A zrt1Δ zrt2Δ mutant (defective in the plasma membrane zinc transporters Zrt1p and Zrt2p) grown in the presence of zinc exhibited a phospholipid composition similar to that of wild type cells depleted for zinc. These results indicated that a decrease in the cytoplasmic levels of zinc was responsible for the alterations in phospholipid composition. Zinc is an essential nutrient required for the growth and metabolism of eukaryotic cells. In this work, we examined the effects of zinc depletion on the regulation of phospholipid synthesis in the yeast Saccharomyces cerevisiae. Zinc depletion resulted in a decrease in the activity levels of the CDP-diacylglycerol pathway enzymes phosphatidylserine synthase, phosphatidylserine decarboxylase, phosphatidylethanolamine methyltransferase, and phospholipid methyltransferase. In contrast, the activity of phosphatidylinositol synthase was elevated in response to zinc depletion. The level of Aut7p, a marker for the induction of autophagy, was also elevated in zinc-depleted cells. For the CHO1-encoded phosphatidylserine synthase, the reduction in activity in response to zinc depletion was controlled at the level of transcription. This regulation was mediated through the UASINO element and by the transcription factors Ino2p, Ino4p, and Opi1p that are responsible for the inositol-mediated regulation of UASINO-containing genes involved in phospholipid synthesis. Analysis of the cellular composition of the major membrane phospholipids showed that zinc depletion resulted in a 66% decrease in phosphatidylethanolamine and a 29% increase in phosphatidylinositol. A zrt1Δ zrt2Δ mutant (defective in the plasma membrane zinc transporters Zrt1p and Zrt2p) grown in the presence of zinc exhibited a phospholipid composition similar to that of wild type cells depleted for zinc. These results indicated that a decrease in the cytoplasmic levels of zinc was responsible for the alterations in phospholipid composition. Phospholipids are amphipathic molecules that are major structural components of cellular membranes (1Stryer L. Biochemistry. 4th Ed. W.H. Freeman and Co., New York1995Google Scholar). In addition, phospholipids provide precursors for the synthesis of macromolecules (2Becker G.W. Lester R.L. J. Bacteriol. 1980; 142: 747-754Crossref PubMed Google Scholar, 3Menon A.K. Stevens V.L. J. Biol. 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The induction of DGPP phosphatase expression in zinc-depleted cells correlates with diminished levels of the minor vacuole membrane phospholipids DGPP and PA (59Han G.S. Johnston C.N. Carman G.M. J. Biol. Chem. 2004; 279: 5338-5345Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). In addition to the changes in DGPP and PA, zinc depletion results in a reduction in the level of PE and an increase in the level of PI in the vacuole membrane (59Han G.S. Johnston C.N. Carman G.M. J. Biol. Chem. 2004; 279: 5338-5345Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Analysis of dpp1Δ mutant cells depleted for zinc indicates that the alterations in the major vacuole membrane phospholipids are not dependent on the regulation of DPP1 expression (60Johnston C.N. The Effects of Zinc and the DPP1-encoded Diacylglycerol Pyrophosphate Phosphatase on the Vacuolar Phospholipid Composition in Saccharomyces cerevisiae. Rutgers University, New Brunswick, NJ2002Google Scholar). In this work, we showed that zinc depletion resulted in a decrease in the activities of CDP-DAG pathway enzymes and an increase in the activity of PI synthase. The level of Aut7p, a marker for the induction of autophagy, was also elevated in zinc-depleted cells. For the CHO1-encoded PS synthase, the enzyme that catalyzes the committed step in the CDP-DAG pathway, the reduction in activity was controlled at the level of transcription. This regulation was mediated through the UASINO element in the CHO1 promoter and by the transcription factors Ino2p, Ino4p, and Opi1p. Materials—All chemicals were reagent grade. Growth medium supplies were from Difco, and yeast nitrogen base lacking zinc sulfate was purchased from Bio 101. Restriction endonucleases, modifying enzymes, and NEBlot kit were purchased from New England Biolabs, Inc. RNA size markers were purchased from Promega. The Yeastmaker™ yeast transformation kit was obtained from Clontech. The plasmid DNA purification and DNA gel extraction kits were from Qiagen, Inc. ProbeQuant G-50 columns, polyvinylidene difluoride membranes, and the enhanced chemifluorescence Western blotting detection kit were purchased from Amersham Biosciences. The DNA size ladder used for agarose gel electrophoresis, Zeta Probe blotting membranes, protein assay reagents, electrophoretic reagents, immunochemical reagents, isopropyl-β-d-thiogalactoside, protein molecular mass standards for SDS-PAGE, and acrylamide solutions were purchased from Bio-Rad. S-adenosylmethionine, ampicillin, aprotinin, benzamidine, bovine serum albumin, leupeptin, O-nitrophenyl β-d-galactopyranoside, pepstatin, phenylmethylsulfonyl fluoride, and Triton X-100 were purchased from Sigma. Radiochemicals and scintillation counting supplies were purchased from PerkinElmer Life Sciences and National Diagnostics, respectively. Phospholipids were purchased from Avanti Polar Lipids. TLC plates were from EM science, and DE52 (DEAE-cellulose) was from Whatman. Liqui-Nox detergent was from Alconox, Inc. Strains, Plasmids, and Growth Conditions—The strains and plasmids used in this work are presented in Table I. Methods for the growth of yeast were performed as described previously (61Rose M.D. Winston F. Heiter P. Methods in Yeast Genetics: Laboratory Course Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1990Google Scholar, 62Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Cells were grown at 30 °C in YEPD medium (1% yeast extract, 2% peptone, 2% glucose) or in synthetic complete medium containing 2% glucose. For selection of cells bearing plasmids, appropriate nutrients were omitted from synthetic complete medium. Zinc-depleted medium was synthetic complete medium prepared with yeast nitrogen base lacking zinc sulfate. For zinc-depleted cultures, cells were first grown for 24 h in synthetic complete medium supplemented with 1.4 μm zinc sulfate. Standard synthetic growth medium contains 1.4 μm zinc sulfate. Saturated cultures were harvested, washed in deionized distilled water, diluted to 1 × 106 cells/ml in medium containing 0 or 1.4 μm zinc sulfate, and grown for 24 h. Cultures were then diluted to 1 × 106 cells/ml and grown again in medium containing 0 or 1.4 μm zinc sulfate. This growth regimen with medium lacking zinc was used to deplete internal stores of zinc. Cells in liquid media were grown to the exponential phase (1–2 × 107 cells/ml), and cell numbers were determined spectrophotometrically at an absorbance of 600 nm. Plasmids were maintained and amplified in Escherichia coli strain DH5α, which was grown in LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl, pH 7.4) at 37 °C. Ampicillin (100 μg/ml) was added to cultures of DH5α-carrying plasmids. For growth on plates, yeast and bacterial media were supplemented with 2 and 1.5% agar, respectively. To prevent zinc contamination, glassware was washed with Liqui-Nox, rinsed with 0.1 mm EDTA, and then rinsed several times with deionized distilled water.Table IStrains and plasmids used in this workStrain or plasmidRelevant characteristicsReference/SourceS. cerevisiaeDY1457MATα ade6 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-52Ref. 96Zhao H. Eide D.J. Mol. Cell. Biol. 1997; 17: 5044-5052Crossref PubMed Google ScholarZHY6MATaade6 can1-100oc his3 leu2 ura3 zap1Δ::TRP1Ref. 96Zhao H. Eide D.J. Mol. Cell. Biol. 1997; 17: 5044-5052Crossref PubMed Google ScholarZHY3MATα ade6 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-52 zrt1Δ::LEU2 zrt2Δ::HIS3Ref. 56Zhao H. Eide D. J. Biol. Chem. 1996; 271: 23203-23210Abstract Full Text Full Text PDF PubMed Scopus (294) Google ScholarCM101MATacan1-100 his3-11,15 leu2-3,112 trp1-1 ura3-52 zrt3Δ::KanrRef. 117MacDiarmid C.W. Gaither L.A. Eide D. EMBO J. 2000; 19: 2845-2855Crossref PubMed Google ScholarCM104MATα can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-52 zrc1Δ::HIS3 cot1Δ::URA3Ref. 117MacDiarmid C.W. Gaither L.A. Eide D. EMBO J. 2000; 19: 2845-2855Crossref PubMed Google ScholarW303-1AMATaade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1Ref. 135Thomas B. Rothstein R. Cell. 1989; 56: 619-630Abstract Full Text PDF PubMed Scopus (1278) Google ScholarSH303MATatrp1Δ63 his3Δ200 ura3-52 leu2Δ1 ino2Δ::TRP1S. A. HenrySH307MATα trp1Δ63 his3Δ200 ura3-52 leu2Δ1 ino4Δ::LEU2S. A. HenrySH304MATatrp1Δ63 his3Δ200 ura3-52 leu2Δ1 opi1Δ::LEU2S. A. HenrySEY6210MATα his3-Δ200 leu2-3,112 lys2-801 suc2-Δ9 trp1-Δ901 ura3-52Ref. 136Robinson J.S. Klionsky D.J. Banta L.M. Emr S.D. Mol. Cell. 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Chem. 2004; 279: 12081-12087Abstract Full Text Full Text PDF PubMed Scopus (0) Google ScholarpJH359-LEU2PINO1-lacZ reporter gene containing the INO1 promoter with LEU2Ref. 116Lopes J.M. Hirsch J.P. Chorgo P.A. Schulze K.L. Henry S.A. Nucleic Acids Res. 1991; 19: 1687-1693Crossref PubMed Google Scholar/ S. A. Henry Open table in a new tab DNA Manipulations, RNA Isolation, and Northern Blot Analysis— Plasmid and genomic DNA were prepared according to standard protocols (62Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Transformations of yeast (63Ito H. Yasuki F. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Crossref PubMed Google Scholar) and E. coli (62Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) were performed as described previously. 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Prehybridization, hybridization with the probes, and washes to remove nonspecific binding were carried out according to the manufacturer's instructions. Radioactive images were acquired by phosphorimaging. Preparation of Cell Extracts and Protein Determination—Cell extracts were prepared as described previously (67Klig L.S. Homann M.J. Carman G.M. Henry S.A. Bacteriol. 1985; 162: 1135-1141Crossref PubMed Google Scholar, 68Oshiro J. Han G.-S. Iwanyshyn W.M. Conover K. Carman G.M. J. Biol. Chem. 2003; 278: 31495-31503Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). Cells were suspended in 50 mm Tris maleate buffer (pH 7.0) containing 1 mm EDTA, 0.3 m sucrose, 10 mm 2-mercaptoethanol, 0.5 mm phenylmethylsulfonyl fluoride, 1 mm benzamidine, 5 μg/ml aprotinin, 5 μg/ml leupeptin, and 5 μg/ml pepstatin. Cells were disrupted by homogenization with chilled glass beads (0.5 mm diameter) using a Biospec Products Mini-Bead-Beater-8. 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