Regulation of the Saccharomyces cerevisiae EKI1-encoded Ethanolamine Kinase by Zinc Depletion
2006; Elsevier BV; Volume: 281; Issue: 19 Linguagem: Inglês
10.1074/jbc.m601612200
ISSN1083-351X
AutoresMichael C. Kersting, George Carman,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoEthanolamine kinase catalyzes the committed step in the synthesis of phosphatidylethanolamine via the CDP-ethanolamine branch of the Kennedy pathway. Regulation of the EKI1-encoded ethanolamine kinase by the essential nutrient zinc was examined in Saccharomyces cerevisiae. The level of ethanolamine kinase activity increased when zinc was depleted from the growth medium. This regulation correlated with increases in the CDP-ethanolamine pathway intermediates phosphoethanolamine and CDP-ethanolamine, and an increase in the methylated derivative of phosphatidylethanolamine, phosphatidylcholine. The β-galactosidase activity driven by the PEKI1-lacZ reporter gene was elevated in zinc-depleted cells, indicating that the increase in ethanolamine kinase activity was attributed to a transcriptional mechanism. The expression level of PEKI1-lacZ reporter gene activity in the zrt1Δzrt2Δ mutant (defective in plasma membrane zinc transport) cells grown with zinc was similar to the activity expressed in wild-type cells grown without zinc. This indicated that EKI1 expression was sensitive to intracellular zinc. The zinc-mediated regulation of EKI1 expression was attenuated in the zap1Δ mutant defective in the zinc-regulated transcription factor Zap1p. Direct interactions between Zap1p and putative zinc-responsive elements in the EKI1 promoter were demonstrated by electrophoretic mobility shift assays. Mutations of these elements to a nonconsensus sequence abolished Zap1p-DNA interactions. Taken together, this work demonstrated that the zinc-mediated regulation of ethanolamine kinase and the synthesis of phospholipids via the CDP-ethanolamine branch of the Kennedy pathway were controlled in part by Zap1p. Ethanolamine kinase catalyzes the committed step in the synthesis of phosphatidylethanolamine via the CDP-ethanolamine branch of the Kennedy pathway. Regulation of the EKI1-encoded ethanolamine kinase by the essential nutrient zinc was examined in Saccharomyces cerevisiae. The level of ethanolamine kinase activity increased when zinc was depleted from the growth medium. This regulation correlated with increases in the CDP-ethanolamine pathway intermediates phosphoethanolamine and CDP-ethanolamine, and an increase in the methylated derivative of phosphatidylethanolamine, phosphatidylcholine. The β-galactosidase activity driven by the PEKI1-lacZ reporter gene was elevated in zinc-depleted cells, indicating that the increase in ethanolamine kinase activity was attributed to a transcriptional mechanism. The expression level of PEKI1-lacZ reporter gene activity in the zrt1Δzrt2Δ mutant (defective in plasma membrane zinc transport) cells grown with zinc was similar to the activity expressed in wild-type cells grown without zinc. This indicated that EKI1 expression was sensitive to intracellular zinc. The zinc-mediated regulation of EKI1 expression was attenuated in the zap1Δ mutant defective in the zinc-regulated transcription factor Zap1p. Direct interactions between Zap1p and putative zinc-responsive elements in the EKI1 promoter were demonstrated by electrophoretic mobility shift assays. Mutations of these elements to a nonconsensus sequence abolished Zap1p-DNA interactions. Taken together, this work demonstrated that the zinc-mediated regulation of ethanolamine kinase and the synthesis of phospholipids via the CDP-ethanolamine branch of the Kennedy pathway were controlled in part by Zap1p. The yeast Saccharomyces cerevisiae responds to a variety of stress conditions (e.g. nutrient depletion) by regulating the expression of several enzyme activities including those involved in phospholipid synthesis (1Becker G.W. Lester R.L. J. Biol. Chem. 1977; 252: 8684-8691Abstract Full Text PDF PubMed Google Scholar, 2Griac P. Henry S.A. Nucleic Acids Res. 1999; 27: 2043-2050Crossref PubMed Scopus (19) Google Scholar, 3Homann M.J. Poole M.A. Gaynor P.M. Ho C.-T. Carman G.M. J. Bacteriol. 1987; 169: 533-539Crossref PubMed Google Scholar, 4Iwanyshyn W.M. Han G.S. Carman G.M. J. Biol. Chem. 2004; 279: 21976-21983Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 5Han G.-S. Johnston C.N. Chen X. Athenstaedt K. Daum G. Carman G.M. J. Biol. Chem. 2001; 276: 10126-10133Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 6Carman G.M. Henry S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (264) Google Scholar). 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Chem. 2005; 280: 29017-29024Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The induction of PI synthase activity correlates with an increase in PI content, whereas the repression of PS synthase and PS decarboxylase activities correlate with a decrease in PE content (4Iwanyshyn W.M. Han G.S. Carman G.M. J. Biol. Chem. 2004; 279: 21976-21983Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Although the activities of the phospholipid methyltransferases (that methylate PE to form PC) are repressed in zinc-depleted cells, this growth condition does not have a major effect on PC content (4Iwanyshyn W.M. Han G.S. Carman G.M. J. Biol. Chem. 2004; 279: 21976-21983Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). In this work, we examined the contribution of the CDP-ethanolamine branch of the Kennedy pathway for the reduction in PE content in response to zinc depletion. We focused on the regulation of the EKI1-encoded ethanolamine kinase, the enzyme that catalyzes the committed step in the CDP-ethanolamine pathway. Unexpectedly, we found that the expression of ethanolamine kinase activity, and the CDP-ethanolamine pathway was induced upon zinc depletion. In addition, this growth condition resulted in an increase in PC derived from PE synthesized via the CDP-ethanolamine pathway. The induction of ethanolamine kinase activity was attributed to a transcriptional mechanism that was mediated in part by the Zap1p transcription factor. Materials—All chemicals were reagent grade. Growth medium supplies were from Difco, and yeast nitrogen base lacking zinc sulfate was purchased from BIO 101. The Yeastmaker™ yeast transformation kit was obtained from Clontech. Oligonucleotides for electrophoretic mobility shift assays were prepared by Genosys Biotechnology, Inc. ProbeQuant G-50 columns were purchased from Amersham Biosciences. Protein molecular mass standards for SDS-PAGE, protein assay reagents, electrophoretic reagents, and acrylamide solutions were purchased from Bio-Rad. Ampicillin, aprotinin, benzamidine, bovine serum albumin, ethanolamine, phosphoethanolamine, CDP-ethanolamine, leupeptin, O-nitrophenylβ-d-galactopyranoside, pepstatin, phenylmethylsulfonyl fluoride, and IGEPAL CA-630 were purchased from Sigma. Phospholipids were purchased from Avanti Polar Lipids. Radiochemicals and scintillation counting supplies were purchased from PerkinElmer Life Sciences and National Diagnostics, respectively. Liqui-Nox detergent was from Alconox, Inc. Silica gel 60 thin layer chromatography plates were from EM Science. Strains, Plasmids, and Growth Conditions—The strains and plasmids used in this work are listed in Table 1. Transformation of yeast (44Ito H. Yasuki F. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Crossref PubMed Google Scholar, 45Schiestl R.H. Gietz R.D. Curr. Genet. 1989; 16: 339-346Crossref PubMed Scopus (1775) Google Scholar) and bacteria (46Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning, A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) were performed as described previously. Yeast cultures were grown in YEPD medium (1% yeast extract, 2% peptone, 2% glucose) or in synthetic complete medium (47Rose M.D. Winston F. Heiter P. Methods in Yeast Genetics: A Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1990Google Scholar) containing 2% glucose at 30 °C. The appropriate amino acids of synthetic complete medium were omitted for selection purposes. Zinc-free medium was synthetic complete medium (47Rose M.D. Winston F. Heiter P. Methods in Yeast Genetics: A Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1990Google Scholar) prepared with yeast nitrogen base lacking zinc sulfate. For zinc-depleted cultures, cells were first grown for 24 h in synthetic complete medium containing 1.5 μm zinc sulfate (equivalent to the concentration of zinc in standard synthetic growth media). Saturated cultures were diluted into zinc-free medium at an initial concentration of 1 × 106 cells/ml, and grown for 24 h. Cultures were then diluted to 1 × 106 cells/ml and grown in zinc-free medium containing 0 or 1.5 μm zinc sulfate. This growth routine was used to deplete internal stores of zinc (5Han G.-S. Johnston C.N. Chen X. Athenstaedt K. Daum G. Carman G.M. J. Biol. Chem. 2001; 276: 10126-10133Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Plasmid maintenance and amplification were performed in Escherichia coli strain DH5α. E. coli cells were 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 bacterial cultures that carried plasmids. For growth on plates, yeast and bacterial media were supplemented with 2% and 1.5% agar, respectively. Yeast cell numbers in liquid medium were determined spectrophotometrically at an absorbance of 600 nm. Exponential phase cells were harvested at a density of 1.5 × 107 cells/ml. Glassware were washed with Liqui-Nox, rinsed with 0.1 mm EDTA, and then rinsed several times with deionized distilled water to remove zinc contamination.TABLE 1Strains and plasmids used in this workStrain or plasmidGenotype or relevant characteristicsSource or Ref.E. coliDH5αF- ϕ80dlacZΔM15 Δ(lacZYA-argF)U169 deoR recA1 endA1 hdR17(rk- mk+) phoA supE44 l-thi-1 gyrA96 relA146Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning, A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google ScholarS. cerevisiaeW303-1AMATa ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-169Thomas B. Rothstein R. Cell. 1989; 56: 619-630Abstract Full Text PDF PubMed Scopus (1352) Google ScholarDY1457MATα ade6 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-5237Zhao H. Eide D.J. Mol. Cell. Biol. 1997; 17: 5044-5052Crossref PubMed Scopus (223) Google ScholarZHY6MATa ade6 can1-100oc his3 leu2 ura3 zap1Δ::TRP137Zhao H. Eide D.J. Mol. Cell. Biol. 1997; 17: 5044-5052Crossref PubMed Scopus (223) Google ScholarZHY3MATα ade6 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-52 zrt1Δ::LEU2 zrt2Δ::HIS327Zhao H. Eide D. J. Biol. Chem. 1996; 271: 23203-23210Abstract Full Text Full Text PDF PubMed Scopus (321) Google ScholarSH303MATa his3Δ200 leu2Δ1 trp1Δ63 ura3-52 ino2Δ::TRP1S. A. HenrySH307MATα his3Δ200 leu2Δ1 trp1Δ63 ura3-52 ino4Δ::LEU2S. A. HenrySH304MATa his3Δ200 leu2Δ1 trp1Δ63 ura3-52 opi1Δ::LEU2S. A. HenryPlasmidpKSK10PEKI1-lacZ reporter gene containing the EKI1 promoter with URA354 Open table in a new tab Preparation of Cell Extracts and Protein Determination—All steps were performed at 5 °C. Yeast cells were disrupted with glass beads with a Mini-BeadBeater-8 (Biospec Products) in 50 mm Tris-HCl buffer, pH 7.5, 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 (48Klig L.S. Homann M.J. Carman G.M. Henry S.A. J. Bacteriol. 1985; 162: 1135-1141Crossref PubMed Google Scholar). Glass beads and cell debris were removed by centrifugation at 1,500 × g for 10 min, and the supernatant was used as the cell extract. The concentration of protein in cell extracts was estimated by the method of Bradford (49Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217399) Google Scholar) using bovine serum albumin as the standard. Enzyme Assays—Ethanolamine kinase activity was measured for 40 min at 30 °C by following the phosphorylation of [1,2-14C]ethanolamine (20,000 cpm/nmol) with ATP. The reaction mixture contained 50 mm Tris-HCl buffer, pH 8.5, 5 mm ethanolamine, 10 mm ATP, 10 mm MgSO4, and enzyme protein (0.12 mg/ml) in a final volume of 25 μl. Reaction mixtures were separated by thin layer chromatography on potassium oxalate-impregnated silica gel plates using the solvent system of methanol/0.6% sodium chloride/29.2% ammonium hydroxide (10: 10:1) (50Elabbadi N. Ancelin M.L. Vial H.J. Biochem. J. 1997; 324: 435-445Crossref PubMed Scopus (60) Google Scholar). The position of the labeled phosphoethanolamine on chromatograms was visualized by phosphorimaging and compared with a phosphoethanolamine standard. The amount of labeled product was determined by scintillation counting. β-Galactosidase activity was determined by measuring the conversion of O-nitrophenyl β-d-galactopyranoside to O-nitrophenol (molar extinction coefficient of 3500 m–1 cm–1) by following the increase in absorbance at 410 nm on a recording spectrophotometer (51Craven G.R. Steers E. Jr-Anfinsen C.B. J. Biol. Chem. 1965; 240: 2468-2477Abstract Full Text PDF PubMed Google Scholar). The reaction mixture contained 100 mm sodium phosphate buffer, pH 7.0, 3 mm O-nitrophenyl β-d-galactopyranoside, 1 mm MgCl2, 100 mm 2-mercaptoethanol, and enzyme protein in a total volume of 0.1 ml. A unit of ethanolamine kinase activity was defined as the amount of enzyme that catalyzed the formation of 1 nmol of product/min. A unit of β-galactosidase activity was defined as the amount of enzyme that catalyzed the formation of 1 μmol product/min. All assays were performed in triplicate and were linear with time and protein concentration. Specific activity was defined as units per mg of protein. Labeling and Analysis of CDP-ethanolamine Pathway Intermediates and Phospholipids—Exponential phase cells were labeled for five to six generations with [1,2-14C]ethanolamine (0.5 μCi/ml). The CDP-ethanolamine pathway intermediates and phospholipids were extracted from whole cells by a chloroform/methanol/water extraction, followed by the separation of the aqueous and chloroform phases (52Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (43093) Google Scholar). The aqueous phase was neutralized, dried in vacuo, and the residue was dissolved in deionized water. Samples were subjected to centrifugation at 12,000 × g for 3 min to remove insoluble material. The 14C-labeled CDP-ethanolamine pathway intermediates were then separated by thin-layer chromatography on silica gel plates using the solvent system methanol/0.6% sodium chloride/ammonium hydroxide (10:10:1, v/v). 14C-labeled phospholipids, which were contained in the chloroform phase, were analyzed by thin-layer chromatography on silica gel plates using the solvent system chloroform/pyridine/88% formic acid/methanol/water (60:35:10:5:2, v/v). The positions of the labeled compounds on chromatograms were determined by phosphorimaging and compared with standards. The amount of each labeled compound was determined by liquid scintillation counting. Electrophoretic Mobility Shift Assays—The double-stranded oligonucleotides used in the electrophoretic mobility shift assays are presented in Table 2. They were prepared by annealing 25 μm complementary single-stranded oligonucleotides in a reaction mixture (0.1 ml) containing 10 mm Tris-HCl, pH 7.5, 100 mm NaCl, and 1 mm EDTA. The annealing reactions were incubated for 5 min at 100 °C in a heat block, and then kept in the heat block for another 2 h after it had been turned off. The annealed oligonucleotides (100 pmol), which had a 5′ overhanging end, were labeled with [α-32P]dTTP (400–800 Ci/nmol) and Klenow fragment (5 units) for 30 min at room temperature. Labeled oligonucleotides were separated from unincorporated nucleotides by gel filtration using ProbeQuant G-50 spin columns.TABLE 2Oligonucleotides used for electrophoretic mobility shift assaysElementAnnealed oligonucleotidesaUnderlined sequences are putative ZRE sites. The mutations (Mt and C) in ZRE1 and ZRE3 are shown in bold letters. The lower case letters indicate the nucleotides filled with the Klenow fragment.EKI1 ZRE15′-ATCATACTACCTTTCAGAATATCtaa-3′3′-tagTATGATGGAAAGTCTTATAGATT-5′EKI1 ZRE25′-ATTCGCTCTCCTTTAAGACAGAAAtaa-3′3′-taaGCGAGAGGAAATTCTGTCTTTATT-5′EKI1 ZRE35′-GTAAAAAAATATCGTTTGGGTTTTGGcta-3′3′-catTTTTTTATAGCAAACCCAAAACCGAT-5′EKI1 ZRE1 (Mt)5′-ATCATACTGTTGGGCAGAATATCtaa-3′3′-tagTATGACAACCCGTCTTATAGATT-5′EKI1 ZRE3 (Mt)5′-GTAAAAAAATATCGTTTGTTTTTTGGcta-3′3′-catTTTTTTATAGCAAACAAAAAACCGAT-5′ZRE1 Consensus (C)5′-ATCATACTACCTTGAAGGTTATCtaa-3′3′-tagTATGATGGAACTTCCAATAGATT-5′ZRE3 Consensus (C)5′-GTAAAAAAATACCTTGAAGGTTTTGGcta-3′3′-catTTTTTTATGGAAATTCCAAAACCGAT-5′a Underlined sequences are putative ZRE sites. The mutations (Mt and C) in ZRE1 and ZRE3 are shown in bold letters. The lower case letters indicate the nucleotides filled with the Klenow fragment. Open table in a new tab GST-Zap1p687–880 was expressed and purified from E. coli (43Han S.-H. Han G.-S. Iwanyshyn W.M. Carman G.M. J. Biol. Chem. 2005; 280: 29017-29024Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The indicated amounts of GST-Zap1p687–880 were incubated with 10 mm Tris-HCl (pH 8.0), 10 mm MgCl2, 50 mm KCl, 1 mm dithiothreitol, 0.025 mg/ml poly(dI-dC)·poly(dI-dC), 0.2 mg/ml bovine serum albumin, 0.04% IGEPAL CA-630, 10% glycerol, and 1 pmol of radiolabeled DNA probe (2.5 × 105 cpm/pmol) for 15 min at room temperature in a total volume of 10 μl. The reaction mixtures were resolved on 6% polyacrylamide gels (1.5-mm thickness) in 0.5× Tris-borate-EDTA buffer at 100 V for 45 min. Gels were dried onto blotting paper, and the radioactive signals were visualized by phosphorimaging analysis. Data Analysis—Statistical significance was determined by performing the Student's t test using SigmaPlot software. p values < 0.05 were taken as a significant difference. Effect of Zinc Depletion on Ethanolamine Kinase Activity and on the Incorporation of Ethanolamine into CDP-ethanolamine Pathway Intermediates and Phospholipids—The effect of zinc depletion on ethanolamine kinase activity was examined. For this and subsequent experiments, the growth medium lacked inositol supplementation to preclude the regulatory effects that inositol has on the regulation of phospholipid synthesis (6Carman G.M. Henry S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (264) Google Scholar, 7Carman G.M. Henry S.A. Annu. Rev. Biochem. 1989; 58: 635-669Crossref PubMed Google Scholar, 8Greenberg M.L. Lopes J.M. Microbiol. Rev. 1996; 60: 1-20Crossref PubMed Google Scholar, 9Henry S.A. Patton-Vogt J.L. Prog. Nucleic Acids Res. 1998; 61: 133-179Crossref PubMed Google Scholar, 53Paltauf F. Kohlwein S.D. Henry S.A. Jones E.W. Pringle J.R. Broach J.R. The Molecular and Cellular Biology of the Yeast Saccharomyces: Gene Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1992: 415-500Google Scholar). Depletion of zinc from the growth medium of wild-type cells caused a 2-fold increase in ethanolamine kinase activity when compared with cells grown in the presence of zinc (Fig. 2). To examine the effects of zinc depletion on the synthesis of PE via ethanolamine kinase and the CDP-ethanolamine branch of the Kennedy pathway, wild-type cells that were grown in the absence and
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