Regulation of the DPP1-encoded Diacylglycerol Pyrophosphate (DGPP) Phosphatase by Inositol and Growth Phase
2000; Elsevier BV; Volume: 275; Issue: 52 Linguagem: Inglês
10.1074/jbc.m008144200
ISSN1083-351X
AutoresJune Oshiro, Shanthi Rangaswamy, Xiaohong Chen, Gil‐Soo Han, Jeannette E. Quinn, George Carman,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoThe regulation of the Saccharomyces cerevisiae DPP1-encoded diacylglycerol pyrophosphate (DGPP) phosphatase by inositol supplementation and growth phase was examined. Addition of inositol to the growth medium resulted in a dose-dependent increase in the level of DGPP phosphatase activity in both exponential and stationary phase cells. Activity was greater in stationary phase cells when compared with exponential phase cells, and the inositol- and growth phase-dependent regulations of DGPP phosphatase were additive. Analyses of DGPP phosphatase mRNA and protein levels, and expression of β-galactosidase activity driven by a PDPP1-lacZreporter gene, indicated that a transcriptional mechanism was responsible for this regulation. Regulation of DGPP phosphatase by inositol and growth phase occurred in a manner that was opposite that of many phospholipid biosynthetic enzymes. Regulation of DGPP phosphatase expression by inositol supplementation, but not growth phase, was altered in opi1Δ, ino2Δ, andino4Δ phospholipid synthesis regulatory mutants. CDP-diacylglycerol, a phospholipid pathway intermediate used for the synthesis of phosphatidylserine and phosphatidylinositol, inhibited DGPP phosphatase activity by a mixed mechanism that caused an increase in Km and a decrease inVmax. DGPP stimulated the activity of pure phosphatidylserine synthase by a mechanism that increased the affinity of the enzyme for its substrate CDP-diacylglycerol. Phospholipid composition analysis of a dpp1Δ mutant showed that DGPP phosphatase played a role in the regulation of phospholipid metabolism by inositol, as well as regulating the cellular levels of phosphatidylinositol. The regulation of the Saccharomyces cerevisiae DPP1-encoded diacylglycerol pyrophosphate (DGPP) phosphatase by inositol supplementation and growth phase was examined. Addition of inositol to the growth medium resulted in a dose-dependent increase in the level of DGPP phosphatase activity in both exponential and stationary phase cells. Activity was greater in stationary phase cells when compared with exponential phase cells, and the inositol- and growth phase-dependent regulations of DGPP phosphatase were additive. Analyses of DGPP phosphatase mRNA and protein levels, and expression of β-galactosidase activity driven by a PDPP1-lacZreporter gene, indicated that a transcriptional mechanism was responsible for this regulation. Regulation of DGPP phosphatase by inositol and growth phase occurred in a manner that was opposite that of many phospholipid biosynthetic enzymes. Regulation of DGPP phosphatase expression by inositol supplementation, but not growth phase, was altered in opi1Δ, ino2Δ, andino4Δ phospholipid synthesis regulatory mutants. CDP-diacylglycerol, a phospholipid pathway intermediate used for the synthesis of phosphatidylserine and phosphatidylinositol, inhibited DGPP phosphatase activity by a mixed mechanism that caused an increase in Km and a decrease inVmax. DGPP stimulated the activity of pure phosphatidylserine synthase by a mechanism that increased the affinity of the enzyme for its substrate CDP-diacylglycerol. Phospholipid composition analysis of a dpp1Δ mutant showed that DGPP phosphatase played a role in the regulation of phospholipid metabolism by inositol, as well as regulating the cellular levels of phosphatidylinositol. phosphatidylcholine phosphatidylethanolamine phosphatidylinositol phosphatidylserine phosphatidate CDP-diacylglycerol diacylglycerol pyrophosphate diacylglycerol polymerase chain reaction kilobase(s) base pair The yeast Saccharomyces cerevisiae serves as a model eukaryote where the regulation of phospholipid synthesis can be studied (1Carman G.M. Henry S.A. Annu. Rev. Biochem. 1989; 58: 635-669Crossref PubMed Google Scholar, 2Paltauf 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, 3Carman G.M. Zeimetz G.M. J. Biol. Chem. 1996; 271: 13293-13296Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 4Carman G.M. Henry S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (262) Google Scholar). The major phospholipids found in the membranes of S. cerevisiae include PC,1PE, PI, and PS (1Carman G.M. Henry S.A. Annu. Rev. Biochem. 1989; 58: 635-669Crossref PubMed Google Scholar, 2Paltauf 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, 3Carman G.M. Zeimetz G.M. J. Biol. Chem. 1996; 271: 13293-13296Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 4Carman G.M. Henry S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (262) Google Scholar). Mitochondrial membranes also contain phosphatidylglycerol and cardiolipin (1Carman G.M. Henry S.A. Annu. Rev. Biochem. 1989; 58: 635-669Crossref PubMed Google Scholar, 2Paltauf 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, 3Carman G.M. Zeimetz G.M. J. Biol. Chem. 1996; 271: 13293-13296Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 4Carman G.M. Henry S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (262) Google Scholar). The synthesis of these phospholipids is a complex process that contains a number of branch points (Fig. 1). PS, PE, and PC are synthesized from PA by the CDP-DG pathway (Fig. 1). CDP-DG is also used for the synthesis of PI and cardiolipin. PE and PC are also synthesized by the CDP-ethanolamine and CDP-choline pathways, respectively (Fig. 1). The CDP-DG pathway is primarily used by wild-type cells for the synthesis of PE and PC when they are grown in the absence of ethanolamine or choline (1Carman G.M. Henry S.A. Annu. Rev. Biochem. 1989; 58: 635-669Crossref PubMed Google Scholar, 2Paltauf 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, 4Carman G.M. Henry S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (262) Google Scholar, 5Henry S.A. Strathern J.N. Jones E.W. Broach J.R. The Molecular Biology of the Yeast Saccharomyces: Metabolism and Gene Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982: 101-158Google Scholar, 6Carman G.M. Vance D.E. Phosphatidylcholine Metabolism. CRC Press, Inc., Baca Raton, FL1989: 165-183Google Scholar). The CDP-ethanolamine and CDP-choline pathways assume a critical role in phospholipid synthesis when enzymes in the CDP-DG pathway are defective (1Carman G.M. Henry S.A. Annu. Rev. Biochem. 1989; 58: 635-669Crossref PubMed Google Scholar, 2Paltauf 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, 4Carman G.M. Henry S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (262) Google Scholar, 7Greenberg M.L. Lopes J.M. Microbiol. Rev. 1996; 60: 1-20Crossref PubMed Google Scholar). Mutants in the CDP-DG pathway require choline for growth and synthesize PC by way of CDP-choline (8Atkinson K. Fogel S. Henry S.A. J. Biol. Chem. 1980; 255: 6653-6661Abstract Full Text PDF PubMed Google Scholar, 9Atkinson K.D. Jensen B. Kolat A.I. Storm E.M. Henry S.A. Fogel S. J. Bacteriol. 1980; 141: 558-564Crossref PubMed Google Scholar, 10Trotter P.J. Voelker D.R. J. Biol. Chem. 1995; 270: 6062-6070Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 11Trotter P.J. Pedretti J. Yates R. Voelker D.R. J. Biol. Chem. 1995; 270: 6071-6080Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 12Kodaki T. Yamashita S. J. Biol. Chem. 1987; 262: 15428-15435Abstract Full Text PDF PubMed Google Scholar, 13Kodaki T. Yamashita S. Eur. J. Biochem. 1989; 185: 243-251Crossref PubMed Scopus (88) Google Scholar, 14Summers E.F. Letts V.A. McGraw P. Henry S.A. Genetics. 1988; 120: 909-922Crossref PubMed Google Scholar, 15McGraw P. Henry S.A. Genetics. 1989; 122: 317-330Crossref PubMed Google Scholar). Mutants defective in the synthesis of PS (8Atkinson K. Fogel S. Henry S.A. J. Biol. Chem. 1980; 255: 6653-6661Abstract Full Text PDF PubMed Google Scholar, 9Atkinson K.D. Jensen B. Kolat A.I. Storm E.M. Henry S.A. Fogel S. J. Bacteriol. 1980; 141: 558-564Crossref PubMed Google Scholar) or PE (10Trotter P.J. Voelker D.R. J. Biol. Chem. 1995; 270: 6062-6070Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 11Trotter P.J. Pedretti J. Yates R. Voelker D.R. J. Biol. Chem. 1995; 270: 6071-6080Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar) can also synthesize PC if they are supplemented with ethanolamine. The ethanolamine is used for PE synthesis by way of CDP-ethanolamine. The PE is subsequently methylated to form PC (Fig. 1). The CDP-ethanolamine and CDP-choline pathways were once viewed as auxiliary or salvage pathways used by cells when the CDP-DG pathway was compromised (1Carman G.M. Henry S.A. Annu. Rev. Biochem. 1989; 58: 635-669Crossref PubMed Google Scholar, 2Paltauf 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). However, it is now known that these pathways contribute to the synthesis of PE and PC even when wild-type cells are grown in the absence of ethanolamine and choline, respectively (16Kim K. Kim K.-H. Storey M.K. Voelker D.R. Carman G.M. J. Biol. Chem. 1999; 274: 14857-14866Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 17Henry S.A. Patton-Vogt J.L. Prog. Nucleic Acids Res. 1998; 61: 133-179Crossref PubMed Google Scholar, 18Patton-Vogt J.L. Griac P. Sreenivas A. Bruno V. Dowd S. Swede M.J. Henry S.A. J. Biol. Chem. 1997; 272: 20873-20883Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). For example, the PC synthesized via the CDP-DG pathway is constantly hydrolyzed to free choline and PA by phospholipase D (18Patton-Vogt J.L. Griac P. Sreenivas A. Bruno V. Dowd S. Swede M.J. Henry S.A. J. Biol. Chem. 1997; 272: 20873-20883Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 19Xie Z.G. Fang M. Rivas M.P. Faulkner A.J. Sternweis P.C. Engebrecht J. Bankaitis V.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12346-12351Crossref PubMed Scopus (145) Google Scholar). Free choline is incorporated back into PC via the CDP-choline pathway, and PA is incorporated into phospholipids via reactions utilizing CDP-DG and DG (2Paltauf 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, 3Carman G.M. Zeimetz G.M. J. Biol. Chem. 1996; 271: 13293-13296Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 4Carman G.M. Henry S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (262) Google Scholar) (Fig. 1). Genetic, molecular, and biochemical studies have shown that the regulation of phospholipid synthesis is a complex and highly coordinated process (2Paltauf 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, 3Carman G.M. Zeimetz G.M. J. Biol. Chem. 1996; 271: 13293-13296Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 4Carman G.M. Henry S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (262) Google Scholar). The mechanisms that govern this regulation control the mRNA and protein levels of the biosynthetic enzymes, as well as their activity (1Carman G.M. Henry S.A. Annu. Rev. Biochem. 1989; 58: 635-669Crossref PubMed Google Scholar, 2Paltauf 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, 3Carman G.M. Zeimetz G.M. J. Biol. Chem. 1996; 271: 13293-13296Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 4Carman G.M. Henry S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (262) Google Scholar). The factors that regulate phospholipid synthesis in S. cerevisiae include water-soluble phospholipid precursors, nucleotides, lipids, and growth phase (1Carman G.M. Henry S.A. Annu. Rev. Biochem. 1989; 58: 635-669Crossref PubMed Google Scholar, 3Carman G.M. Zeimetz G.M. J. Biol. Chem. 1996; 271: 13293-13296Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 4Carman G.M. Henry S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (262) Google Scholar, 7Greenberg M.L. Lopes J.M. Microbiol. Rev. 1996; 60: 1-20Crossref PubMed Google Scholar, 17Henry S.A. Patton-Vogt J.L. Prog. Nucleic Acids Res. 1998; 61: 133-179Crossref PubMed Google Scholar). DGPP is a minor phospholipid recently identified in S. cerevisiae (20Wu W.-I. Liu Y. Riedel B. Wissing J.B. Fischl A.S. Carman G.M. J. Biol. Chem. 1996; 271: 1868-1876Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). It contains a pyrophosphate group attached to DG (21Wissing J.B. Behrbohm H. FEBS Lett. 1993; 315: 95-99Crossref PubMed Scopus (47) Google Scholar). DGPP is derived from PA via the reaction catalyzed by PA kinase (20Wu W.-I. Liu Y. Riedel B. Wissing J.B. Fischl A.S. Carman G.M. J. Biol. Chem. 1996; 271: 1868-1876Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). DGPP phosphatase catalyzes the removal of the β-phosphate from DGPP to yield PA and then removes the phosphate from PA to generate DG (20Wu W.-I. Liu Y. Riedel B. Wissing J.B. Fischl A.S. Carman G.M. J. Biol. Chem. 1996; 271: 1868-1876Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The function of DGPP has not been established in S. cerevisiae. However, phospholipid composition analysis of adpp1Δ mutant devoid of DGPP phosphatase activity (22Toke D.A. Bennett W.L. Dillon D.A. Chen X. Oshiro J. Ostrander D.B. Wu W.-I. Cremesti A. Voelker D.R. Fischl A.S. Carman G.M. J. Biol. Chem. 1998; 273: 3278-3284Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) has revealed that the DPP1 gene product plays a role in the regulation of phospholipid metabolism (23Toke D.A. Bennett W.L. Oshiro J. Wu W.I. Voelker D.R. Carman G.M. J. Biol. Chem. 1999; 273: 14331-14338Abstract Full Text Full Text PDF Scopus (103) Google Scholar). The dpp1Δ mutant exhibits a reduction in the cellular level of PI and an elevation in the levels of PA and DGPP (23Toke D.A. Bennett W.L. Oshiro J. Wu W.I. Voelker D.R. Carman G.M. J. Biol. Chem. 1999; 273: 14331-14338Abstract Full Text Full Text PDF Scopus (103) Google Scholar). PA plays a central role in phospholipid synthesis as the precursor of all phospholipids synthesized via the CDP-DG, CDP-ethanolamine, and CDP-choline pathways (Fig. 1). Moreover, of all the major phospholipids in S. cerevisiae, PI is the only one that is essential (1Carman G.M. Henry S.A. Annu. Rev. Biochem. 1989; 58: 635-669Crossref PubMed Google Scholar, 2Paltauf 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, 24Henry S.A. Atkinson K.D. Kolat A.J. Culbertson M.R. J. Bacteriol. 1977; 130: 472-484Crossref PubMed Google Scholar, 25Becker G.W. Lester R.L. J. Biol. Chem. 1977; 252: 8684-8691Abstract Full Text PDF PubMed Google Scholar). Because inositol (precursor of PI (Fig. 1)) has regulatory effects on the expression of many phospholipid biosynthetic enzymes (1Carman G.M. Henry S.A. Annu. Rev. Biochem. 1989; 58: 635-669Crossref PubMed Google Scholar, 2Paltauf 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, 4Carman G.M. Henry S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (262) Google Scholar, 7Greenberg M.L. Lopes J.M. Microbiol. Rev. 1996; 60: 1-20Crossref PubMed Google Scholar, 17Henry S.A. Patton-Vogt J.L. Prog. Nucleic Acids Res. 1998; 61: 133-179Crossref PubMed Google Scholar), we examined the effect of inositol on expression of theDPP1-encoded DGPP phosphatase. We also examined the influence of growth phase on DGPP phosphatase, since it has a major influence on the expression of many phospholipid biosynthetic enzymes (1Carman G.M. Henry S.A. Annu. Rev. Biochem. 1989; 58: 635-669Crossref PubMed Google Scholar, 2Paltauf 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, 4Carman G.M. Henry S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (262) Google Scholar, 7Greenberg M.L. Lopes J.M. Microbiol. Rev. 1996; 60: 1-20Crossref PubMed Google Scholar, 17Henry S.A. Patton-Vogt J.L. Prog. Nucleic Acids Res. 1998; 61: 133-179Crossref PubMed Google Scholar). We discovered that DGPP phosphatase expression was regulated by inositol and growth phase. However, this regulation occurred in an opposite manner to that of most phospholipid biosynthetic enzymes. We also discovered that the activity of DGPP phosphatase was inhibited by CDP-DG and that the activity of PS synthase was stimulated by DGPP. The work reported here increases the understanding of the long and short term regulation of DGPP phosphatase and the influence of the enzyme on phospholipid metabolism. All chemicals were reagent grade. Growth medium supplies were purchased from Difco. Radiochemicals were from PerkinElmer Life Sciences. Scintillation counting supplies were from National Diagnostics. Triton X-100, bovine serum albumin, aprotinin, benzamidine, leupeptin, pepstatin, phenylmethylsulfonyl fluoride, diethyl pyrocarbonate, inositol, choline, CDP, andO-nitrophenyl β-d-galactopyranoside were purchased from Sigma. Lipids were purchased from Avanti Polar Lipids. DE52 (DEAE-cellulose) was from Whatman. Protein assay reagent, Zeta ProbeTM membranes, electrophoresis reagents, and immunochemical reagents were purchased from Bio-Rad. The NEBlot kit, restriction endonucleases, modifying enzymes, and recombinant Vent DNA polymerase with 5′- and 3′-exonuclease activity were purchased from New England Biolabs. Primers for polymerase chain reaction were prepared commercially by Genosys Biotechnologies, Inc. Nitrocellulose membranes were purchased from Schleicher & Schuell. ProbeQuant G-50 columns, polyvinylidene difluoride membranes, protein A-Sepharose, and the ECF Western blotting chemifluorescent detection kit were purchased from Amersham Pharmacia Biotech. Silica Gel 60 thin layer chromatography plates were from EM Science. The strains and plasmids used in this work are listed in TableI. Methods for yeast growth were performed as described previously (26Rose M.D. Winston F. Heiter P. Methods in Yeast Genetics: A Laboratory Course Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1990Google Scholar, 27Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Yeast cultures were grown in YEPD medium (1% yeast extract, 2% peptone, 2% glucose) containing adenine (40 mg/liter) or in complete synthetic medium minus inositol (28Culbertson M.R. Henry S.A. Genetics. 1975; 80: 23-40Crossref PubMed Google Scholar) containing 2% glucose at 30 °C. The appropriate amino acid of complete synthetic medium was omitted for selection purposes.Escherichia coli strain DH5α 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. Media were supplemented with 2% agar for growth on plates. Yeast cell numbers in liquid media were determined spectrophotometrically at an absorbance of 600 nm. Exponential phase corresponded to a cell density of 1 × 107 cells/ml, whereas stationary phase was 1 × 108 cells/ml. Stationary phase cultures were harvested 24 h after an initial inoculation density of 1 × 106 cells/ml. For the overexpression of CHO1-encoded PS synthase, cells were grown to the exponential phase in complete synthetic medium containing 2% raffinose. Galactose (2%) was then added to the growth medium to induce the expression of PS synthase. Maximum induction (60-fold) was achieved after 2 h.Table IStrains and plasmids used in this workStrain or plasmidGenotype or relevant characteristicsSource or Ref.S. cerevisiae W303–1AMAT a leu2-3 112 trp1-1 can1-100 ura3-1 ade2-1 his2-11,1595Thomas B. Rothstein R. Cell. 1989; 56: 619-630Abstract Full Text PDF PubMed Scopus (1344) Google Scholar DTY1dpp1Δ∷TRP1/Kanrderivative of W303–1A22Toke D.A. Bennett W.L. Dillon D.A. Chen X. Oshiro J. Ostrander D.B. Wu W.-I. Cremesti A. Voelker D.R. Fischl A.S. Carman G.M. J. Biol. Chem. 1998; 273: 3278-3284Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar SH304MAT a trp1Δ63 his3Δ200 ura3–52 leu2Δ1 opi1Δ∷LEU2S. A. Henry SH303MAT a trp1Δ63 his3Δ200 ura3–52 leu2Δ1 ino2Δ∷TRP1S. A. Henry SH307MATα trp1Δ63, his3Δ200 ura3–52, leu2Δ1 ino4Δ∷LEU2S. A. HenryE. coliDH5αF−φ80dlacZΔM15 Δ(lacZYA-argF)U169 deoR, recA1 endA1 hsdR17(rk− mk+) phoA, supE44 λ− thi-1 gyrA96 relA127Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google ScholarPlasmidspDT1-DPP1DPP1 gene ligated into theSrfI site of pCRScript AMP SK(+)22Toke D.A. Bennett W.L. Dillon D.A. Chen X. Oshiro J. Ostrander D.B. Wu W.-I. Cremesti A. Voelker D.R. Fischl A.S. Carman G.M. J. Biol. Chem. 1998; 273: 3278-3284Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar pSD90PCRD1-lacZ reporter construct on a multicopy plasmid containing URA3, derivative of pMA109W. Dowhan pJO2PDPP1-lacZ reporter construct on a multicopy plasmid containing URA3, derivative of pSD90This work pJ699ZPINO1-lacZ reporter construct on a multicopy plasmid containing LEU2, derivative of pJH359S. A. Henry64Lopes J.M. Hirsch J.P. Chorgo P.A. Schulze K.L. Henry S.A. Nucleic Acids Res. 1991; 19: 1687-1693Crossref PubMed Scopus (100) Google Scholar pRS425Multicopy E. coli/yeast shuttle plasmid containing LEU233Christianson T.W. Sikorski R.S. Dante M. Shero J.H. Hieter P. Gene ( Amst. ). 1992; 110: 119-122Crossref PubMed Scopus (1433) Google Scholar YCpGPSSPGAL7-CHO1 on a centromere plasmid containing TRP132Hamamatsu S. Shibuya I. Takagi M. Ohta A. FEBS Lett. 1994; 348: 33-36Crossref PubMed Scopus (22) Google Scholar YEpGPSSPGAL7-CHO1 on a multicopy plasmid containing LEU2, derivative of YCpGPSSThis work Open table in a new tab Plasmid and genomic DNA preparation, restriction enzyme digestion, and DNA ligations were performed by standard methods (27Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Transformation of yeast (29Chen D.C. Yang B.C. Kuo T.T. Curr. Genet. 1992; 21: 83-84Crossref PubMed Scopus (586) Google Scholar) and E. coli (27Sambrook 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. Conditions for the amplification of DNA by PCR were optimized as described previously (30Innis 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, Inc., San Diego1990: 3-12Google Scholar). Plasmid maintenance and amplifications were performed in E. coli strain DH5α. Plasmid pJO2 contains the putative promoter of the DPP1 gene fused to the lacZ gene of E. coli. The plasmid was constructed by replacing theEcoRI fragment of plasmid pSD90 with the DPP1promoter, which was obtained by PCR (primers, 5′-GTGAAGAAGCAGGAATTCATAAAGGGACAACACGG-3′ and 5′-GTTTTAATAAACGAAACTGAATTCATTTTGGTCG-3′) using strain W303-1A genomic DNA as a template. The PCR primer used in the forward direction corresponds to −1156 bp to the start codon, and the primer used in the reverse direction corresponds to +25 bp to the start codon. Plasmid pSD90, derived from pMA109, contains the CRD1 promoter. Plasmid pMA109, derived from plasmid YEp357R, contains thePIS1 promoter (31Myers A.M. Tzagoloff A. Kinney D.M. Lusty C.J. Gene ( Amst. ). 1986; 45: 299-310Crossref PubMed Scopus (513) Google Scholar). The correct orientation of theDPP1 promoter in plasmid pJO2 was checked by digestion withPstI and by measurement of β-galactosidase activity. The PDPP1-lacZ construct does not contain any sequences of the DPP1 open reading frame. The pJO2 plasmid was introduced into the indicated strains to examine the expression of theDPP1 gene by measuring β-galactosidase activity. A 1.8-kb insert, containing the CHO1 gene fused to theGAL7 promoter, was released from plasmid YCpGPSS (32Hamamatsu S. Shibuya I. Takagi M. Ohta A. FEBS Lett. 1994; 348: 33-36Crossref PubMed Scopus (22) Google Scholar) by digestion with SalI/BamHI. This DNA fragment was ligated into the SalI/BamHI sites of pRS425, a multicopy E. coli/yeast shuttle vector containing theLEU2 gene (33Christianson T.W. Sikorski R.S. Dante M. Shero J.H. Hieter P. Gene ( Amst. ). 1992; 110: 119-122Crossref PubMed Scopus (1433) Google Scholar) to form plasmid YEpGPSS. This construct was transformed into W303-1A for the overexpression of theCHO1-encoded PS synthase. Total yeast RNA was isolated using the methods of Schmitt et al. (34Schmitt M.E. Brown T.A. Trumpower B.L. Nucleic Acids Res. 1990; 18: 3091-3092Crossref PubMed Scopus (1152) Google Scholar) and Herrick et al. (35Herrick D. Parker R. Jacobson A. Mol. Cell. Biol. 1990; 10: 2269-2284Crossref PubMed Scopus (320) Google Scholar). Equal amounts (25 μg) of total RNA from each sample were resolved on a 1.1% formaldehyde gel for 2.5 h at 100 V (36Ausubel 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, Inc., New York1993Google Scholar). The RNA samples were then transferred to a Zeta ProbeTM membrane by vacuum blotting. Pre-hybridization, hybridization with a specific probe, and washes to remove unbound probe were carried out according to the manufacturer's instructions. TheDPP1 probe was a 0.87-kb fragment isolated from pDT1-DPP1 byMfeI/BamHI digestion. A TCM1 probe was used as a constitutive standard and a loading control. This probe was generated from an PCR-amplified (primers, 5′-CTCACAGAAAGTACGAAGCACC-3′ and 5′-CAAGTCCTTCTTCAAAGTACC-3′) region of genomic DNA. TheDPP1 and TCM1 probes were labeled with [α-32P]dATP using the NEBlot random primer labeling kit. Unincorporated nucleotides were removed using ProbeQuant G-50 columns. Images of radiolabeled species were acquired by PhosphorImaging analysis. The peptide sequence SDVTLEEAVTHQRIPDE (residues 263–279 at the C-terminal end of the deduced protein sequence ofDPP1) was synthesized and conjugated to carrier protein at Bio-Synthesis, Inc. (Lewisville, TX). Antibodies were raised against the peptide in New Zealand White rabbits by standard procedures (37Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar) at Bio-Synthesis, Inc. The IgG fraction was isolated from antisera by protein A-Sepharose chromatography (37Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar). SDS-polyacrylamide gel electrophoresis (38Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207012) Google Scholar) using 12% slab gels and immunoblotting (39Haid A. Suissa M. Methods Enzymol. 1983; 96: 192-205Crossref PubMed Scopus (232) Google Scholar) using polyvinylidene difluoride membranes were performed as described previously. The anti-DGPP phosphatase antibodies were used at a dilution of 1:1000, and the DGPP phosphatase protein was detected using the ECF Western blotting chemifluorescent detection kit as described by the manufacturer. The DGPP phosphatase protein on immunoblots was acquired by FluoroImaging analysis. The relative density of the protein was analyzed using ImageQuant software. Immunoblot signals were in the linear range of detectability. Cells from all strains were disrupted with glass beads (40Klig L.S. Homann M.J. Carman G.M. Henry S.A. J. Bacteriol. 1985; 162: 1135-1141Crossref PubMed Google Scholar) in 50 mm Tris maleate buffer, pH 7.0, containing 1 mm Na2EDTA, 0.3m sucrose, 10 mm 2-mercaptoethanol, 0.5 mm phenylmethylsulfonyl fluoride, 1 mmbenzamidine, and 5 μg/ml each of aprotinin, leupeptin, and pepstatin. Glass beads and unbroken cells were removed by centrifugation at 1,500 × g for 10 min. The supernatant (cell extract) was used for enzyme assays and immunoblot analysis.DPP1-encode
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