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Regulation of the PIS1-encoded Phosphatidylinositol Synthase in Saccharomyces cerevisiae by Zinc

2005; Elsevier BV; Volume: 280; Issue: 32 Linguagem: Inglês

10.1074/jbc.m505881200

ISSN

1083-351X

Autores

Seunghee Han, Gil‐Soo Han, Wendy M. Iwanyshyn, George Carman,

Tópico(s)

Plant Micronutrient Interactions and Effects

Resumo

In the yeast Saccharomyces cerevisiae, the mineral zinc is essential for growth and metabolism. Depletion of zinc from the growth medium of wild type cells results in changes in phospholipid metabolism, including an increase in phosphatidylinositol content (Iwanyshyn, W. M., Han, G.-S., and Carman, G. M. (2004) J. Biol. Chem. 279, 21976–21983). We examined the effects of zinc depletion on the regulation of the PIS1-encoded phosphatidylinositol synthase, the enzyme that catalyzes the formation of phosphatidylinositol from CDP-diacylglycerol and inositol. Phosphatidylinositol synthase activity increased when zinc was depleted from the growth medium. Analysis of a zrt1Δ zrt2Δ 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 β-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 the result of a transcriptional mechanism. The zinc-mediated induction of the PPIS1-lacZ reporter gene, Pis1p, and phosphatidylinositol synthase activity was lost in zap1Δ mutant cells. These data indicated that the regulation of PIS1 gene expression by zinc depletion was mediated by the zinc-regulated transcription factor Zap1p. Direct interaction between glutathione S-transferase (GST)-Zap1p687–880 and a putative upstream activating sequence (UAS) zinc-responsive element in the PIS1 promoter was demonstrated by electrophoretic mobility shift assays. Mutations in the UAS zinc-responsive element in the PIS1 promoter abolished the GST-Zap1p687–880-DNA interaction in vitro and abolished the zinc-mediated regulation of the PIS1 gene in vivo. This work advances understanding of phospholipid synthesis regulation by zinc and the transcription control of the PIS1 gene. In the yeast Saccharomyces cerevisiae, the mineral zinc is essential for growth and metabolism. Depletion of zinc from the growth medium of wild type cells results in changes in phospholipid metabolism, including an increase in phosphatidylinositol content (Iwanyshyn, W. M., Han, G.-S., and Carman, G. M. (2004) J. Biol. Chem. 279, 21976–21983). We examined the effects of zinc depletion on the regulation of the PIS1-encoded phosphatidylinositol synthase, the enzyme that catalyzes the formation of phosphatidylinositol from CDP-diacylglycerol and inositol. Phosphatidylinositol synthase activity increased when zinc was depleted from the growth medium. Analysis of a zrt1Δ zrt2Δ 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 β-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 the result of a transcriptional mechanism. The zinc-mediated induction of the PPIS1-lacZ reporter gene, Pis1p, and phosphatidylinositol synthase activity was lost in zap1Δ mutant cells. These data indicated that the regulation of PIS1 gene expression by zinc depletion was mediated by the zinc-regulated transcription factor Zap1p. Direct interaction between glutathione S-transferase (GST)-Zap1p687–880 and a putative upstream activating sequence (UAS) zinc-responsive element in the PIS1 promoter was demonstrated by electrophoretic mobility shift assays. Mutations in the UAS zinc-responsive element in the PIS1 promoter abolished the GST-Zap1p687–880-DNA interaction in vitro and abolished the zinc-mediated regulation of the PIS1 gene in vivo. This work advances understanding of phospholipid synthesis regulation by zinc and the transcription control of the PIS1 gene. Phosphatidylinositol (PI) 1The abbreviations used are: PI, phosphatidylinositol; GST, glutathione S-transferase; HA, hemagglutinin; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; UASINO, upstream activating sequence inositol-responsive element; UASZRE, UAS zinc-responsive element. 1The abbreviations used are: PI, phosphatidylinositol; GST, glutathione S-transferase; HA, hemagglutinin; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; UASINO, upstream activating sequence inositol-responsive element; UASZRE, UAS zinc-responsive element. is the third most abundant phospholipid in the cellular membranes of the yeast Saccharomyces cerevisiae (1Rattray J.B. Schibeci A. Kidby D.K. Bacteriol. Rev. 1975; 39: 197-231Crossref PubMed Google Scholar, 2Henry S.A. Strathern J.N. Jones E.W. Broach J.R. The Molecular Biology of the Yeast Saccharomyces: Metabolism and Gene Expression. 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Nucleic Acids Res. 2002; 30: 80-83Crossref PubMed Scopus (40) Google Scholar) enzyme catalyzes the formation of PI and CMP from CDP-diacylglycerol and inositol (25Paulus H. Kennedy E.P. J. Biol. Chem. 1960; 235: 1303-1311Abstract Full Text PDF PubMed Google Scholar) (Fig. 1). The regulation of PI synthase activity in vivo is largely governed by the availability of its substrates inositol and CDP-diacylglycerol (26Carman G.M. Zeimetz G.M. J. Biol. Chem. 1996; 271: 13293-13296Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 27Carman G.M. Henry S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (261) Google Scholar, 28Kelley M.J. Bailis A.M. Henry S.A. Carman G.M. J. Biol. Chem. 1988; 263: 18078-18085Abstract Full Text PDF PubMed Google Scholar). Cellular inositol levels are controlled by expression of the INO1 gene encoding inositol-3-phosphate synthase and by inositol supplementation (26Carman G.M. Zeimetz G.M. J. Biol. 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Science. 2004; 304: 1644-1647Crossref PubMed Scopus (364) Google Scholar) (Fig. 1). PS synthase catalyzes the committed step in the synthesis of PC via the CDP-diacylglycerol pathway (27Carman G.M. Henry S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (261) Google Scholar) (Fig. 1). Indeed, the coordinate regulation of the PI synthase and PS synthase enzymes is part of an overall mechanism by which the synthesis of PI is coordinately regulated with the synthesis of PC (3Paltauf 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, 27Carman G.M. Henry S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (261) Google Scholar, 30Carman G.M. Henry S.A. Annu. Rev. Biochem. 1989; 58: 635-669Crossref PubMed Google Scholar, 31Greenberg M.L. Lopes J.M. Microbiol. Rev. 1996; 60: 1-20Crossref PubMed Google Scholar, 32Henry S.A. Patton-Vogt J.L. Prog. Nucleic Acids Res. 1998; 61: 133-179Crossref PubMed Google Scholar, 33Daum G. Lees N.D. Bard M. Dickson R. Yeast. 1998; 14: 1471-1510Crossref PubMed Scopus (514) Google Scholar, 34Kersting M.C. Choi H.S. Carman G.M. J. Biol. Chem. 2004; 279: 35353-35359Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar).Zinc is an essential nutrient required for the growth and metabolism of S. cerevisiae and of higher eukaryotes (35Vallee B.L. Falchuk K.H. Physiol. Rev. 1993; 73: 79-118Crossref PubMed Google Scholar). It is a cofactor for hundreds of enzymes (e.g. alcohol dehydrogenase, carbonic anhydrase, proteases, RNA polymerases, superoxide dismutase) (35Vallee B.L. Falchuk K.H. Physiol. Rev. 1993; 73: 79-118Crossref PubMed Google Scholar) and a structural constituent of many proteins (e.g. transcription factors, chaperones, lipid-binding proteins) (36Schwabe J.W. Klug A. Nat. Struct. Biol. 1994; 1: 345-349Crossref PubMed Scopus (241) Google Scholar, 37Ellis C.D. Wang F. MacDiarmid C.W. Clark S. Lyons T. Eide D.J. J. Cell Biol. 2004; 166: 325-335Crossref PubMed Scopus (153) Google Scholar). Zinc deficiency in rats is associated with oxidative damage to DNA, lipids, and proteins (38Oteiza P.L. Olin K.L. Fraga C.G. Keen C.L. Proc. Soc. Exp. Biol. Med. 1996; 213: 85-91Crossref PubMed Scopus (121) Google Scholar), and in humans, it is manifested by defects in appetite, cognitive function, embryonic development, epithelial integrity, and immune function (39Walsh C.T. Sandstead H.H. Prasad A.S. Newberne P.M. Fraker P.J. Environ. Health Perspect. 1994; 102: 5-46Crossref PubMed Scopus (343) Google Scholar). Despite its essential nature, zinc is toxic to cells when accumulated in excess amounts (35Vallee B.L. Falchuk K.H. Physiol. Rev. 1993; 73: 79-118Crossref PubMed Google Scholar).Recent studies have revealed that the synthesis of phospholipids in S. cerevisiae is influenced by zinc deficiency (40Iwanyshyn 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 particular, PI synthase activity is elevated in zinc-depleted cells, whereas several enzyme activities (e.g. PS synthase, PS decarboxylase, PE methyltransferase, and phospholipid methyltransferase) in the CDP-diacylglycerol pathway for PC synthesis are reduced in response to zinc depletion (40Iwanyshyn 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). The regulation of these activities by zinc availability contributes to alterations in the cellular levels of the major membrane phospholipids PI (elevated) and PE (reduced) (40Iwanyshyn 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). For the PS synthase enzyme, the reduction in activity in response to zinc depletion is controlled at the level of transcription through the UASINO element in the CHO1 promoter and by the transcription factors Ino2p, Ino4p, and Opi1p (40Iwanyshyn 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 explored the mechanism by which PI synthase activity is regulated in response to zinc depletion. Our data indicated that this regulation occurred by a transcriptional mechanism that was mediated by the transcriptional activator Zap1p.EXPERIMENTAL PROCEDURESMaterials—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 the 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. Plasmid DNA purification and DNA gel extraction kits were from Qiagen, Inc. The QuikChange site-directed mutagenesis kit was from Stratagene. Oligonucleotides for PCRs and electrophoretic mobility shift assays were prepared by Genosys Biotechnology, Inc. ProbeQuant G-50 columns, polyvinylidene difluoride membranes, an enhanced chemifluorescence Western blotting detection kit, and glutathione-Sepharose 4 fast flow were purchased from GE Healthcare. DNA markers for agarose gel electrophoresis, protein molecular mass standards for SDS-PAGE, Zeta Probe blotting membranes, protein assay reagents, electrophoretic reagents, immunochemical reagents, isopropyl 1-thio-β-d-galactopyranoside, and acrylamide solutions were purchased from Bio-Rad. Ampicillin, aprotinin, benzamidine, bovine serum albumin, leupeptin, o-nitrophenyl β-d-galactopyranoside, pepstatin, phenylmethylsulfonyl fluoride, reduced glutathione, IGEPAL CA-630, and Triton X-100 were purchased from Sigma. Mouse monoclonal anti-HA antibodies (12CA5) and ImmunoPure goat anti-mouse IgG (H+L) antibodies were purchased from Roche Applied Science and Pierce, respectively. Radiochemicals and scintillation counting supplies were purchased from PerkinElmer Life Sciences and National Diagnostics, respectively. 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. Yeast cells were grown according to standard methods (41Rose M.D. Winston F. Heiter P. Methods in Yeast Genetics: A Laboratory Course Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1990Google Scholar, 42Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) at 30 °C in YEPD medium (1% yeast extract, 2% peptone, 2% glucose) or in synthetic complete medium containing 2% glucose. Appropriate nutrients were omitted from synthetic complete medium for the selection of cells bearing plasmids. 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 1.5 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 1.5 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 1.5 μm zinc sulfate. This growth routine with medium lacking zinc was used to deplete internal stores of zinc (43Han 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). Cells in liquid medium were grown to the exponential phase (1 × 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 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 contained plasmids. Yeast and bacterial media were supplemented with 2% and 1.5% agar, respectively, for growth on plates. Glassware were washed with Liqui-Nox, rinsed with 0.1 mm EDTA, and then rinsed several times with deionized distilled water to prevent zinc contamination.Table IStrains and plasmids used in this workStrain or plasmidRelevant characteristicsRef.S. cerevisiaeW303-1AMATa ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1(78Thomas B. Rothstein R. Cell. 1989; 56: 619-630Abstract Full Text PDF PubMed Scopus (1335) Google Scholar)DY1457MATα ade6 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-52(61Zhao H. Eide D.J. Mol. Cell. Biol. 1997; 17: 5044-5052Crossref PubMed Scopus (216) Google Scholar)ZHY6MATa ade6 can1-100oc his3 leu2 ura3 zap1Δ::TRP1(61Zhao H. Eide D.J. Mol. Cell. Biol. 1997; 17: 5044-5052Crossref PubMed Scopus (216) Google Scholar)ZHY3MATα ade6 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-52 zrt1Δ::LEU2 zrt2Δ::HIS3(56Zhao H. Eide D. J. Biol. Chem. 1996; 271: 23203-23210Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar)SH303MATa 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. HenryE. coliDH5αF - φ80dlacZΔM15 Δ(lacZYA-argF)U169 deoR, recA1 endA1 hdR17(rk- mk+) phoA supE44 l-thi-1 gyrA96 relA1(42Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar)PlasmidspWMI1HA-tagged PIS1 gene ligated into the XmaI/XbaI sites of pRS416This studypRS416Single copy E. coli/yeast shuttle vector containing URA3(79Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar)pPI514PIS1 gene on a multicopy plasmid with LEU2(21Nikawa J. Yamashita S. Eur. J. Biochem. 1984; 143: 251-256Crossref PubMed Scopus (59) Google Scholar)pGEX-687E. coli expression plasmid for recombinant GST-Zap1p687-880(65Bird A. Evans-Galea M.V. Blankman E. Zhao H. Luo H. Winge D.R. Eide D.J. J. Biol. Chem. 2000; 275: 16160-16166Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar)pMA109PPIS1-lacZ reporter plasmid containing the PIS1 promoter with URA3(60Anderson M.S. Lopes J.M. J. Biol. Chem. 1996; 271: 26596-26601Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar)pPZM1Derivative of pMA109 with mutations in UASZRE1This studypPZM2Derivative of pMA109 with mutations in UASZRE2This studypPZM3Derivative of pMA109 with mutations in UASZRE3This study Open table in a new tab DNA Manipulations and Amplification of DNA by PCR—Plasmid and genomic DNA preparation, restriction enzyme digestion, and DNA ligation were performed by standard methods (42Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Conditions for the amplification of DNA by PCR were optimized as described previously (44Innis M.A. Gelfand D.H. Innis M.A. Gelfand D.H. Sninsky J.J. White T.J. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego1990: 3-12Google Scholar). Transformation of yeast (45Ito H. Yasuki F. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Crossref PubMed Google Scholar) and E. coli (42Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) was performed using standard protocols.Construction of Plasmids—Plasmid pWMI1 contains a 2.2-kb DNA fragment for the PIS1 gene with sequences for an HA epitope tag inserted after the start codon. Genomic DNA prepared from strain W303-1A was used as a template to produce a 5′-fragment of PIS1HA (primers 5′-CCCCCCGGGCTAATGCATGAGCCAATAGAG-3′ and 5′-AGCGTAGTCTGGGACGTCGTATGGGTACATCTTGTACTATCACACTTTCCCTCTTAT-3′) and a 3′ fragment of PIS1HA (primers 5′-TACCCATACGACGTCCCAGACTACGCTAGTTCGAATTCAACACCAGAAAAGGTTACT-3′ and 5′-CGTCTAGAGTGCAAGTTGGAGAGAATCGCTTCCG-3′). The 5′- and 3′-fragments of PIS1HA were digested with XmaI/AatII and AatII/XbaI, respectively, and inserted into the XmaI/XbaI sites of pRS416 to generate the plasmid pWMI1. The Stratagene QuikChange site-directed mutagenesis kit was utilized according to the manufacturer's instructions to generate plasmids pPZM1–pPZM3. These plasmids were derivatives of pMA109 (PPIS1-lacZ) and contained mutations in UASZRE1, UASZRE2 and UASZRE3 of the PIS1 promoter. Plasmids pPZM1 (mutagenic primers 5′-TTTTTCTTCCTTTTCCCTAACAATTCCAATTGCTTCTCTTCTCTTCTCCTT-3′ and 5′-AAGGAGAAGAGAAGAGAAGCAATTGGAATTGTTAGGGAAAAGGAAGAAAAA-3′), pPZM2 (mutagenic primers 5′-TTTTAGCCATGGACACTTCTCAATTCCAATTTGTTGATGTCCATGGCTAAAA-3′ and 5′-TCAATGGCAGTTTTATCAACCAATTGGAATTGGAATTGAGAAGTGTCCATGGCTAAAA-3′), and pPZM3 (mutagenic primers 5′-ATATAAGTAAAACATAAAAACAATTCCAATTGGTATGGTTTATTTG CCGTC-3′ and 5′-GACGGCAAATAAACCATACCAATTGGAATTGTTTTTATGTTTTACTTATAT-3′) were constructed by amplification of plasmid pMA109 by PCR. Plasmid pMA109 was eliminated from the mutant plasmid reactions by digestion with DpnI. The mutant plasmids were amplified in E. coli, and the purified plasmids were sequenced to confirm the mutations in the PIS1 promoter.RNA Isolation and Northern Blot Analysis—Total RNA was isolated from cells (46Schmitt M.E. Brown T.A. Trumpower B.L. Nucleic Acids Res. 1990; 18: 3091-3092Crossref PubMed Scopus (1147) Google Scholar, 47Herrick D. Parker R. Jacobson A. Mol. Cell. Biol. 1990; 10: 2269-2284Crossref PubMed Scopus (316) Google Scholar), resolved by agarose gel electrophoresis (48Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, New York1993Google Scholar), and then transferred to Zeta Probe membranes by vacuum blotting. The PIS1 and CMD1 probes were labeled with [α-32P]dTTP using the NEBlot random primer labeling kit, and unincorporated nucleotides were removed using ProbeQuant G-50 columns. Prehybridization, hybridization with the probes, and washes to remove nonspecific binding were carried out according to the manufacturer's instructions. Images of the radiolabeled mRNAs were acquired by phosphorimaging analysis.Anti-PI Synthase Antibodies and Immunoblotting—The peptide sequence AALILADNDAKNANE (residues 201–215 at the C-terminal end of the deduced amino acid sequence of PIS1) was synthesized and used to raise antibodies in New Zealand White rabbits by standard procedures at Bio-Synthesis, Inc. The IgG fraction was isolated from the antiserum using protein A-Sepharose CL-4B (49Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar). SDS-PAGE (50Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206048) Google Scholar) using 10% slab gels and the transfer of proteins to polyvinylidene difluoride membranes (51Haid A. Suissa M. Methods Enzymol. 1983; 96: 192-205Crossref PubMed Scopus (231) Google Scholar) were performed as described previously. The membrane was probed with 12.5 μg/ml purified anti-PI synthase IgG fraction. Mouse monoclonal anti-HA antibodies were used at a dilution of 1:1,000. Goat anti-rabbit and anti-mouse IgG-alkaline phosphatase conjugates were used as secondary antibodies at a dilution of 1:5,000. The PI synthase protein (Pis1p) was detected using the enhanced chemifluorescence Western blotting detection kit, and the signals were acquired by FluorImaging. The relative density of the signal was analyzed using ImageQuant software. Immunoblot signals were in the linear range of detectability.Preparation of Cell Extracts and Protein Determination—Cell extracts were prepared as described previously (52Klig L.S. Homann M.J. Carman G.M. Henry S.A. J. Bacteriol. 1985; 162: 1135-1141Crossref PubMed 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. Samples were homogenized for 10 1-min bursts followed by a 2-min cooling between bursts at 4 °C. The cell extract (supernatant) was obtained by centrifugation of the homogenate at 1,500 × g for 10 min. The protein concentration was determined by the method of Bradford (53Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (213462) Google Scholar) using bovine serum albumin as the standard.Enzyme Assays—All assays were conducted in triplicate at 30 °C in a total volume of 0.1 ml. PI synthase activity was measured by following the incorporation of [2-3H]inositol (10,000 cpm/nmol) into PI as described previously (54Carman G.M. Fischl A.S. Methods Enzymol. 1992; 209: 305-312Crossref PubMed Scopus (40) Google Scholar). The assay mixture contained 50 mm Tris-HCl, pH 8.0, 2 mm MnCl2, 0.5 mm inositol, 0.2 mm CDP-diacylglycerol, 2.4 mm Triton X-100, and enzyme protein. β-Galactosidase activity was measured by following the formation of o-nitrophenyl from o-nitrophenyl β-d-galactopyranoside spectrophotometrically at a wavelength of 410 nm (55Craven G.R. Steers Jr., E. Anfinsen C.B. J. Biol. Chem. 1965; 240: 2468-2477Abstract Full Text PDF PubMed Google Scholar). The assay mixture contained 100 mm sodium phosphate, pH 7.0, 3 mm o-nitrophenyl β-d-galactopyranoside, 1 mm MgCl2, 100 mm 2-mercaptoethanol, and enzyme protein. All assays were linear with time and protein concentration. The average standard deviation of all assays was ± 5%. A unit of PI synthase activity was defined as the amount of enzyme that catalyzed the formation of 1 nmol of product/min, whereas a unit of β-galactosidase activity was defined in μmol/min. Specific activity was defined as units/mg of protein.Expression and Purification of GST-Zap1p687–880 from E. coli—The GST-Zap1p687–880 fusion protein was expressed in E. coli BL21(DE3)pLysS bearing plasmid pGEX-687. A 500-ml culture was grown to A600 ∼ 0.8 at 28 °C, and the expression of GST-Zap1p687–880 was induced for 1 h with 0.1 mm isopropyl 1-thio-β-d-galactopyranoside. The culture was harvested, and the resulting pellet was resuspended in 20 ml of phosphate-buffered saline (10 mm Na2HPO4, 1.8 mm KH2PO4, 140 mm NaCl, 2.7 mm KCl, pH 7.3). Cells were disrupted with a French press at 20,000 pounds/square inch, and unbroken cells and cell debris were removed by centrifugation at 12,000 × g for 30 min at 4 °C. The supernatant (cell lysate) was mixed for 1 h with 1 ml of a 50% slurry of glutathione-Sepharose with gentle shaking. The glutathione-Sepharose resin was then packed in a 10-ml Poly-Prep disposable column and was washed with 25 ml of phosphate-buffered saline. Proteins bound to the column were eluted (0.5-ml fractions) with 50 mm Tris-HCl, pH 8.0, buffer containing 10 mm reduced glutathione. SDS-PAGE analysis indicated that the 48-kDa GST-Zap1p687–880 fusion protein was purified to ∼90% of homogeneity. The

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