FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2097074526> ?p ?o ?g. }

• W2097074526 endingPage "31227" @default.
• W2097074526 startingPage "31217" @default.
• W2097074526 abstract "The CHO1-encoded phosphatidylserine synthase (CDP-diacylglycerol:l-serine O-phosphatidyltransferase, EC 2.7.8.8) is one of the most highly regulated phospholipid biosynthetic enzymes in the yeast Saccharomyces cerevisiae. CHO1 expression is regulated by nutrient availability through a regulatory circuit involving a UASINO cis-acting element in the CHO1 promoter, the positive transcription factors Ino2p and Ino4p, and the transcriptional repressor Opi1p. In this work, we examined the post-transcriptional regulation of CHO1 by mRNA stability. CHO1 mRNA was stabilized in mutants defective in deadenylation (ccr4Δ), mRNA decapping (dcp1), and the 5′–3′-exonuclease (xrn1), indicating that the CHO1 transcript is primarily degraded through the general 5′–3′ mRNA decay pathway. In respiratory-sufficient cells, the CHO1 transcript was moderately stable with a half-life of 12 min. However, the CHO1 transcript was stabilized to a half-life of >45 min in respiratory-deficient (rho– and rhoo) cells, the cox4Δ mutant defective in the cytochrome c oxidase, and wild type cells treated with KCN (a cytochrome c oxidase inhibitor). The increased CHO1 mRNA stability in response to respiratory deficiency caused increases in CHO1 mRNA abundance, phosphatidylserine synthase protein and activity, and the synthesis of phosphatidylserine in vivo. Respiratory deficiency also caused increases in the activities of CDP-diacylglycerol synthase, phosphatidylserine decarboxylase, and the phospholipid methyltransferases. Phosphatidylinositol synthase and choline kinase activities were not affected by respiratory deficiency. This work advances our understanding of phosphatidylserine synthase regulation and underscores the importance of mitochondrial respiration to the regulation of phospholipid synthesis in S. cerevisiae. The CHO1-encoded phosphatidylserine synthase (CDP-diacylglycerol:l-serine O-phosphatidyltransferase, EC 2.7.8.8) is one of the most highly regulated phospholipid biosynthetic enzymes in the yeast Saccharomyces cerevisiae. CHO1 expression is regulated by nutrient availability through a regulatory circuit involving a UASINO cis-acting element in the CHO1 promoter, the positive transcription factors Ino2p and Ino4p, and the transcriptional repressor Opi1p. In this work, we examined the post-transcriptional regulation of CHO1 by mRNA stability. CHO1 mRNA was stabilized in mutants defective in deadenylation (ccr4Δ), mRNA decapping (dcp1), and the 5′–3′-exonuclease (xrn1), indicating that the CHO1 transcript is primarily degraded through the general 5′–3′ mRNA decay pathway. In respiratory-sufficient cells, the CHO1 transcript was moderately stable with a half-life of 12 min. However, the CHO1 transcript was stabilized to a half-life of >45 min in respiratory-deficient (rho– and rhoo) cells, the cox4Δ mutant defective in the cytochrome c oxidase, and wild type cells treated with KCN (a cytochrome c oxidase inhibitor). The increased CHO1 mRNA stability in response to respiratory deficiency caused increases in CHO1 mRNA abundance, phosphatidylserine synthase protein and activity, and the synthesis of phosphatidylserine in vivo. Respiratory deficiency also caused increases in the activities of CDP-diacylglycerol synthase, phosphatidylserine decarboxylase, and the phospholipid methyltransferases. Phosphatidylinositol synthase and choline kinase activities were not affected by respiratory deficiency. This work advances our understanding of phosphatidylserine synthase regulation and underscores the importance of mitochondrial respiration to the regulation of phospholipid synthesis in S. cerevisiae. The CHO1-encoded (1Letts V.A. Kim L.S. Bae-Lee M. Carman G.M. Henry S.A. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 7279-7283Crossref PubMed Scopus (105) Google Scholar, 2Nikawa J. Kim Y. Kodaki T. Yamashita S. Eur. J. Biochem. 1987; 167: 7-12Crossref PubMed Scopus (48) Google Scholar, 3Kiyono K. Kim K. Kushima Y. Hikiji T. Fukushima M. Shibuya I. Ohta A. J. Biochem. (Tokyo). 1987; 102: 1089-1100Crossref PubMed Scopus (58) Google Scholar) PS 2The abbreviations used are: PS, phosphatidylserine; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PI, phosphatidylinositol; PA, phosphatidate; CDP-DAG, CDP-diacylglycerol.2The abbreviations used are: PS, phosphatidylserine; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PI, phosphatidylinositol; PA, phosphatidate; CDP-DAG, CDP-diacylglycerol. synthase (CDP-diacylglycerol:l-serine O-phosphatidyltransferase, EC 2.7.8.8) 3The S. cerevisiae PS synthase enzyme should not be confused with the PS synthase enzyme from mammalian cells that catalyzes an exchange reaction between PE or PC with serine (94Vance J.E. Trends Biochem. Sci. 1998; 23: 423-428Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar).3The S. cerevisiae PS synthase enzyme should not be confused with the PS synthase enzyme from mammalian cells that catalyzes an exchange reaction between PE or PC with serine (94Vance J.E. Trends Biochem. Sci. 1998; 23: 423-428Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). is one of the most highly regulated enzymes of phospholipid synthesis in the yeast Saccharomyces cerevisiae (4Carman G.M. Kim G.M. J. Biol. Chem. 1996; 271: 13293-13296Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 5Carman G.M. Kim S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (262) Google Scholar, 6Yamashita S. Kim J. Biochim. Biophys. Acta. 1997; 1348: 228-235Crossref PubMed Scopus (43) Google Scholar). PS synthase is an integral membrane protein that is localized to the endoplasmic reticulum (7Natter K. Kim P. Faschinger A. Wolinski H. McCraith S. Fields S. Kohlwein S.D. Mol. Cell. Proteomics. 2005; 4: 662-672Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). It catalyzes the formation of PS by a Mn2+-dependent sequential reaction by displacing CMP from CDP-DAG with serine (8Bae-Lee M. Kim G.M. J. Biol. Chem. 1984; 259: 10857-10862Abstract Full Text PDF PubMed Google Scholar). The reaction product PS is a major structural component of yeast membranes accounting for 4–18% of total phospholipids depending on growth conditions (9Rattray J.B. Kim A. Kidby D.K. Bacteriol. Rev. 1975; 39: 197-231Crossref PubMed Google Scholar, 10Henry S.A. Strathern J.N. Jones E.W. Broach J.R. The Molecular Biology of the Yeast Saccharomyces. Metabolism and Gene Expression. 1982: 101-158, Cold Spring Harbor Laboratory Press, Cold Spring Harbor. NYGoogle Scholar, 11Paltauf F. Kim 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. 1992: 415-500, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar). PS also serves as the precursor for the synthesis of the most abundant membrane phospholipids PE (20–32%) and PC (35–55%) that are synthesized by the de novo CDP-DAG pathway (Fig. 1) (5Carman G.M. Kim S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (262) Google Scholar, 9Rattray J.B. Kim A. Kidby D.K. Bacteriol. Rev. 1975; 39: 197-231Crossref PubMed Google Scholar). cho1 mutants defective in PS synthase activity can still synthesize PE and PC if they are supplemented with ethanolamine or choline; indeed, cho1 mutants are ethanolamine/choline auxotrophs (12Atkinson K. Kim S. Henry S.A. J. Biol. Chem. 1980; 255: 6653-6661Abstract Full Text PDF PubMed Google Scholar, 13Atkinson K.D. Kim B. Kolat A.I. Storm E.M. Henry S.A. Fogel S. J. Bacteriol. 1980; 141: 558-564Crossref PubMed Google Scholar). Ethanolamine is used for PE synthesis via the CDP-ethanolamine branch of the Kennedy pathway (Fig. 1). The PE synthesized by the Kennedy pathway may be methylated to PC via the CDP-DAG pathway (Fig. 1). Choline is used for PC synthesis via the CDP-choline branch of the Kennedy pathway (Fig. 1). In wild type cells, both the CDP-DAG and Kennedy pathways contribute to the synthesis of PC regardless of whether choline is supplemented to the growth medium (14Morash S.C. Kim C.R. Hjelmstad R.H. Bell R.M. J. Biol. Chem. 1994; 269: 28769-28776Abstract Full Text PDF PubMed Google Scholar, 15McGee T.P. Kim H.B. Whitters E.A. Henry S.A. Bankaitis V.A. J. Cell Biol. 1994; 124: 273-287Crossref PubMed Scopus (152) Google Scholar, 16McMaster C.R. Kim R.M. J. Biol. Chem. 1994; 269: 28010-28016Abstract Full Text PDF PubMed Google Scholar, 17McDonough V.M. Kim R.J. Bruno M.E.C. Ozier-Kalogeropoulos O. Adeline M.-T. McMaster C.R. Bell R.M. Carman G.M. J. Biol. Chem. 1995; 270: 18774-18780Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 18Ostrander D.B. Kim D.J. Gorman J.A. Carman G.M. J. Biol. Chem. 1998; 273: 18992-19001Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 19Patton-Vogt J.L. Kim 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). If choline is not present in the growth medium, the choline required for the Kennedy pathway is derived from the phospholipase D-mediated turnover of PC synthesized by way of the CDP-DAG pathway (19Patton-Vogt J.L. Kim 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, 20Xie Z.G. Kim 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). PS synthase is regulated by biochemical and genetic mechanisms, both of which have an impact on the synthesis of PC via the CDP-DAG and Kennedy pathways (4Carman G.M. Kim G.M. J. Biol. Chem. 1996; 271: 13293-13296Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 5Carman G.M. Kim S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (262) Google Scholar, 6Yamashita S. Kim J. Biochim. Biophys. Acta. 1997; 1348: 228-235Crossref PubMed Scopus (43) Google Scholar, 21Chen M. Kim L.C. Lopes J.M. Biochim. Biophys. Acta. 2007; 1771: 310-321Crossref PubMed Scopus (66) Google Scholar). The activity of PS synthase is modulated (i.e. inhibited or activated) by membrane phospholipids (e.g. PA, phosphatidylglycerol, and cardiolipin) (22Hromy J.M. Kim G.M. J. Biol. Chem. 1986; 261: 15572-15576Abstract Full Text PDF PubMed Google Scholar, 23Bae-Lee M. Kim G.M. J. Biol. Chem. 1990; 265: 7221-7226Abstract Full Text PDF PubMed Google Scholar, 24Oshiro J. Kim S. Chen X. Han G.-S. Quinn J.E. Carman G.M. J. Biol. Chem. 2000; 275: 40887-40896Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) and is inhibited by inositol (25Kelley M.J. Kim A.M. Henry S.A. Carman G.M. J. Biol. Chem. 1988; 263: 18078-18085Abstract Full Text PDF PubMed Google Scholar) and by the nucleotide CTP (17McDonough V.M. Kim R.J. Bruno M.E.C. Ozier-Kalogeropoulos O. Adeline M.-T. McMaster C.R. Bell R.M. Carman G.M. J. Biol. Chem. 1995; 270: 18774-18780Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). In addition, the phosphorylation of the enzyme inhibits its activity, whereas dephosphorylation stimulates its activity (26Kinney A.J. Kim G.M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7962-7966Crossref PubMed Scopus (54) Google Scholar, 27Kinney A.J. Kim M. Singh Panghaal S. Kelley M.J. Gaynor P.M. Carman G.M. J. Bacteriol. 1990; 172: 1133-1136Crossref PubMed Google Scholar). In general, the inhibition of PS synthase activity favors PC synthesis via the Kennedy pathway (4Carman G.M. Kim G.M. J. Biol. Chem. 1996; 271: 13293-13296Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 5Carman G.M. Kim S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (262) Google Scholar). The biochemical regulation of PS synthase activity also governs the partitioning of the substrate CDP-DAG between PS and PI; the inhibition of PS synthase activity favors PI synthesis (Fig. 1) (4Carman G.M. Kim G.M. J. Biol. Chem. 1996; 271: 13293-13296Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). The expression of the PS synthase (CHO1) gene is regulated by the supplementation of water-soluble phospholipid precursors (e.g. inositol) (28Klig L.S. Kim M.J. Carman G.M. Henry S.A. J. Bacteriol. 1985; 162: 1135-1141Crossref PubMed Google Scholar, 29Poole M.A. Kim M.J. Bae-Lee M. Carman G.M. J. Bacteriol. 1986; 168: 668-672Crossref PubMed Google Scholar, 30Bailis A.M. Kim M.A. Carman G.M. Henry S.A. Mol. Cell. Biol. 1987; 7: 167-176Crossref PubMed Scopus (81) Google Scholar, 31Bailis A.M. Kim J.M. Kohlwein S.D. Henry S.A. Nucleic Acids Res. 1992; 20: 1411-1418Crossref PubMed Scopus (53) Google Scholar), zinc deprivation (32Iwanyshyn W.M. Kim G.S. Carman G.M. J. Biol. Chem. 2004; 279: 21976-21983Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), and by growth phase (33Homann M.J. Kim M.A. Gaynor P.M. Ho C.-T. Carman G.M. J. Bacteriol. 1987; 169: 533-539Crossref PubMed Google Scholar, 34Lamping E. Kim J. Paltauf F. Henry S.A. Kohlwein S.D. Genetics. 1995; 137: 55-65Crossref Google Scholar). CHO1 is maximally expressed in exponential phase cells when grown in the absence of inositol (28Klig L.S. Kim M.J. Carman G.M. Henry S.A. J. Bacteriol. 1985; 162: 1135-1141Crossref PubMed Google Scholar, 29Poole M.A. Kim M.J. Bae-Lee M. Carman G.M. J. Bacteriol. 1986; 168: 668-672Crossref PubMed Google Scholar, 30Bailis A.M. Kim M.A. Carman G.M. Henry S.A. Mol. Cell. Biol. 1987; 7: 167-176Crossref PubMed Scopus (81) Google Scholar, 31Bailis A.M. Kim J.M. Kohlwein S.D. Henry S.A. Nucleic Acids Res. 1992; 20: 1411-1418Crossref PubMed Scopus (53) Google Scholar) and grown in the presence of zinc (32Iwanyshyn W.M. Kim G.S. Carman G.M. J. Biol. Chem. 2004; 279: 21976-21983Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). CHO1 is repressed when inositol is supplemented to the growth medium (28Klig L.S. Kim M.J. Carman G.M. Henry S.A. J. Bacteriol. 1985; 162: 1135-1141Crossref PubMed Google Scholar, 29Poole M.A. Kim M.J. Bae-Lee M. Carman G.M. J. Bacteriol. 1986; 168: 668-672Crossref PubMed Google Scholar, 30Bailis A.M. Kim M.A. Carman G.M. Henry S.A. Mol. Cell. Biol. 1987; 7: 167-176Crossref PubMed Scopus (81) Google Scholar, 31Bailis A.M. Kim J.M. Kohlwein S.D. Henry S.A. Nucleic Acids Res. 1992; 20: 1411-1418Crossref PubMed Scopus (53) Google Scholar) or when zinc is depleted from the growth medium (32Iwanyshyn W.M. Kim G.S. Carman G.M. J. Biol. Chem. 2004; 279: 21976-21983Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). The zinc-mediated regulation of CHO1 occurs in the absence of inositol supplementation (32Iwanyshyn W.M. Kim G.S. Carman G.M. J. Biol. Chem. 2004; 279: 21976-21983Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Repression of CHO1 also occurs when cells enter the stationary phase of growth (33Homann M.J. Kim M.A. Gaynor P.M. Ho C.-T. Carman G.M. J. Bacteriol. 1987; 169: 533-539Crossref PubMed Google Scholar, 34Lamping E. Kim J. Paltauf F. Henry S.A. Kohlwein S.D. Genetics. 1995; 137: 55-65Crossref Google Scholar). These forms of regulation are dependent on the UASINO cis-acting element in the promoter of the CHO1 gene (21Chen M. Kim L.C. Lopes J.M. Biochim. Biophys. Acta. 2007; 1771: 310-321Crossref PubMed Scopus (66) Google Scholar). The derepression of CHO1 is mediated by a heterodimer complex of the positive transcription factors Ino2p and Ino4p that bind to a UASINO cis-acting element to drive transcription (5Carman G.M. Kim S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (262) Google Scholar, 21Chen M. Kim L.C. Lopes J.M. Biochim. Biophys. Acta. 2007; 1771: 310-321Crossref PubMed Scopus (66) Google Scholar, 35Greenberg M.L. Kim J.M. Microbiol. Rev. 1996; 60: 1-20Crossref PubMed Google Scholar, 36Henry S.A. Kim J.L. Prog. Nucleic Acids Res. 1998; 61: 133-179Crossref PubMed Google Scholar). Repression of CHO1 is mediated by the repressor Opi1p, which interacts with Ino2p to attenuate transcription (5Carman G.M. Kim S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (262) Google Scholar, 21Chen M. Kim L.C. Lopes J.M. Biochim. Biophys. Acta. 2007; 1771: 310-321Crossref PubMed Scopus (66) Google Scholar, 35Greenberg M.L. Kim J.M. Microbiol. Rev. 1996; 60: 1-20Crossref PubMed Google Scholar, 36Henry S.A. Kim J.L. Prog. Nucleic Acids Res. 1998; 61: 133-179Crossref PubMed Google Scholar). Opi1p repressor function is regulated by the cellular concentration of PA, which helps anchor the repressor to the nuclear/endoplasmic reticulum membrane apart from the Ino2p-Ino4p complex bound to the UASINO element (37Loewen C.J.R. Kim M.L. Jesch S.A. Delon C. Ktistakis N.T. Henry S.A. Levine T.P. Science. 2004; 304: 1644-1647Crossref PubMed Scopus (365) Google Scholar). PA concentration and Opi1p repressor function is mediated in part by the PAH1-encoded Mg2+-dependent PA phosphatase enzyme (38Santos-Rosa H. Kim J. Grimsey N. Peak-Chew S. Siniossoglou S. EMBO J. 2005; 24: 1931-1941Crossref PubMed Scopus (288) Google Scholar, 39Han G.-S. Kim W.-I. Carman G.M. J. Biol. Chem. 2006; 281: 9210-9218Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar, 40Carman G.M. Kim G.S. Trends Biochem. Sci. 2006; 31: 694-699Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). Data (41O'Hara L. Kim G.S. Peak-Chew S. Grimsey N. Carman G.M. Siniossoglou S. J. Biol. Chem. 2006; 281: 34537-34548Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar) are consistent with a model (40Carman G.M. Kim G.S. Trends Biochem. Sci. 2006; 31: 694-699Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar) whereby activation of Mg2+-dependent PA phosphatase activity results in a decrease in PA concentration followed by the translocation of Opi1p into the nucleus for interaction with Ino2p to repress CHO1 transcription. As discussed above for the biochemical regulation of PS synthase activity, the repression of CHO1 favors PI synthesis and the Kennedy pathway for PC synthesis (5Carman G.M. Kim S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (262) Google Scholar). Decay analysis of CHO1 mRNA in a cki1Δ eki1Δ mutant defective in the synthesis of phospholipids via the Kennedy pathway (Fig. 1) has revealed a novel mechanism by which CHO1 expression is regulated independent of the UASINO element in the CHO1 promoter (42Choi H.S. Kim A. Han G.-S. Carman G.M. J. Biol. Chem. 2004; 279: 12081-12087Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). In wild type cells, CHO1 mRNA is moderately stable with a half-life of 12 min when compared with other S. cerevisiae mRNAs that have half-lives ranging from 1 to 60 min (43Herrick D. Kim R. Jacobson A. Mol. Cell. Biol. 1990; 10: 2269-2284Crossref PubMed Scopus (320) Google Scholar). However, CHO1 mRNA is greatly stabilized with a half-life >45 min in the cki1Δ eki1Δ (KS106) mutant (42Choi H.S. Kim A. Han G.-S. Carman G.M. J. Biol. Chem. 2004; 279: 12081-12087Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). This results in increased levels of the PS synthase protein and its associated activity (42Choi H.S. Kim A. Han G.-S. Carman G.M. J. Biol. Chem. 2004; 279: 12081-12087Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). The objective of this work was to identify an intermediate or end product of the Kennedy pathway that was responsible for regulation of CHO1 mRNA stability. During the course of this work, we discovered that the stabilization of CHO1 mRNA was not mediated by components of the Kennedy pathway, but instead it was mediated by a defect in mitochondrial respiration. This work underscores the importance of respiration to the regulation of phospholipid synthesis and advances our understanding of PS synthase regulation in yeast. Materials—All chemicals were reagent grade. Growth medium supplies were purchased from Difco. The plasmid DNA purification and DNA gel extraction kits were from Qiagen. NEBlot kit, restriction endonucleases, recombinant Vent DNA polymerase, and modifying enzymes were purchased from New England Biolabs. RNA size markers were purchased from Promega. Yeast transformation kit was obtained from Clontech. Enhanced chemifluorescence Western blotting detection kit, polyvinylidene difluoride membranes, and ProbeQuant G-50 columns were purchased from GE Healthcare. BioRad was the supplier of Zeta Probe blotting membranes, protein assay reagents, electrophoretic reagents, acrylamide solutions, immunochemical reagents, the DNA size ladder used for agarose gel electrophoresis, and protein molecular mass standards for SDS-PAGE. AdoMet, ampicillin, aprotinin, benzamidine, bovine serum albumin, choline, CTP, leupeptin, N-ethylmaleimide, pepstatin, phenylmethylsulfonyl fluoride, l-serine, and Triton X-100 were purchased from Sigma. Phospholipids were purchased from Avanti Polar Lipids. TLC plates were from EM Science. Radiochemicals and scintillation counting supplies were from PerkinElmer Life Sciences and National Diagnostics, respectively. Thiolutin was a gift from Pfizer. Strains, Plasmids, and Growth Conditions—The bacterial and yeast strains used in this work are listed in Table 1. Methods for the growth of bacteria and yeast were described previously (44Rose M.D. Kim F. Heiter P. Methods in Yeast Genetics: A Laboratory Course Manual. 1990; (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY)Google Scholar, 45Sambrook J. Kim E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 1989; (2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY)Google Scholar). Cells were grown at 30 °C in complete synthetic medium without inositol (46Culbertson M.R. Kim S.A. Genetics. 1975; 80: 23-40Crossref PubMed Google Scholar) using either 2% glucose or 2% glycerol as the carbon source. For selection of cells bearing plasmids, appropriate nutrients were omitted from synthetic complete medium. Glucose-grown cells were also cultured in the presence of 1 mm hydrogen peroxide to induce oxidative stress (47Jamieson D.J. J. Bacteriol. 1992; 174: 6678-6681Crossref PubMed Google Scholar, 48Jamieson D.J. Kim S.L. Stephen D.W. Microbiology. 1994; 140: 3277-3283Crossref PubMed Scopus (118) Google Scholar). 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. The plasmids used in this work are listed in Table 2. Ampicillin (100 mg/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. Respiratory sufficiency was scored by growth on YPG (1% yeast extract, 2% peptone, 2% glycerol) and YPD (1% yeast extract, 2% peptone, 2% glucose) media plates (44Rose M.D. Kim F. Heiter P. Methods in Yeast Genetics: A Laboratory Course Manual. 1990; (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY)Google Scholar, 45Sambrook J. Kim E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 1989; (2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY)Google Scholar).TABLE 1Strains used in this workStrainRelevant characteristicsSource or Ref.E. coli DH5αF- ϕ80dlacZΔM15 Δ(lacZYA-argF)U169 deoR recA1 endA1 hsdR17(rk- mk+) phoA supE44 1- thi-1 gyrA96 relA145Sambrook J. Kim E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 1989; (2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY)Google ScholarS. cerevisiae yRP840MATa cup1::LEU2 (PM) his4-539 leu2-3,112 trp1 ura3-5295Hatfield L. Kim C.A. Stevens A. Parker R. Mol. Cell. Biol. 1996; 16: 5830-5838Crossref PubMed Scopus (145) Google Scholar yRP841MATa cup1::LEU2 (PM) leu2-3,112 lys2-201 trp1 ura3-5295Hatfield L. Kim C.A. Stevens A. Parker R. Mol. Cell. Biol. 1996; 16: 5830-5838Crossref PubMed Scopus (145) Google Scholar yRP1616ccr4Δ::NEO derivative of yRP84096Tucker M. Kim M.A. Staples R.R. Chen J. Denis C.L. Parker R. Cell. 2001; 104: 377-386Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar yRP1069dcp1::URA3 derivative of yRP84195Hatfield L. Kim C.A. Stevens A. Parker R. Mol. Cell. Biol. 1996; 16: 5830-5838Crossref PubMed Scopus (145) Google Scholar yRP884xrn1::URA3 derivative of yRP84095Hatfield L. Kim C.A. Stevens A. Parker R. Mol. Cell. Biol. 1996; 16: 5830-5838Crossref PubMed Scopus (145) Google Scholar W303-1BMATα ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-197Thomas B. Kim R. Cell. 1989; 56: 619-630Abstract Full Text PDF PubMed Scopus (1338) Google Scholar KS105cki1Δ::HIS3 derivative of W303-1B53Kim 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 KS101eki1Δ::TRP1 derivative of W303-1B [rhoo]53Kim 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 KS106cki1Δ::HIS3 eki1Δ::TRP1 derivative of W303-1B [rhoo]53Kim 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 HCY3ect1Δ::TRP1 derivative of W303-1BThis study HCY4ept1Δ::TRP1 derivative of W303-1BThis study HCY5eki1Δ::TRP1 derivative of strain W303-1BThis study HCY6rhoo derivative of HCY5This study HCY7cki1Δ::HIS3 eki1Δ::TRP1 derivative of W303-1BThis study HCY8rho- derivative of W303-1BThis study W303 [rhoo]MATa ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 [rhoo]M. Greenberg MGY100MATa ade1 oxi2 [rho-]M. Greenberg DL1MATα his3-11 15 leu2-3 12 ura3-251 328,37298van Loon A.P. Kim E. Grivell L.A. EMBO J. 1983; 2: 1765-1770Crossref PubMed Google Scholar WD1cox4Δ::LEU2 derivative of DL177Dowhan W. Kim C.R. Schatz G. EMBO J. 1985; 4: 179-184Crossref PubMed Scopus (74) Google Scholar Open table in a new tab TABLE 2Plasmids used in this workPlasmidRelevant characteristicsSource or Ref.pRS414Single copy vector containing TRP199Sikorski R.S. Kim P. Genetics. 1989; 122: 19-27Crossref PubMed Google ScholarpRS416Single copy vector containing URA399Sikorski R.S. Kim P. Genetics. 1989; 122: 19-27Crossref PubMed Google ScholarYEp352Multicopy E. coli/yeast shuttle vector containing URA3100Hill J.E. Kim A.M. Koerner T.J. Tzagoloff A. Yeast. 1986; 2: 163-167Crossref PubMed Scopus (1080) Google ScholarpAS103Plasmid containing a 1.2-kb fragment of the CHO1 gene17McDonough V.M. Kim R.J. Bruno M.E.C. Ozier-Kalogeropoulos O. Adeline M.-T. McMaster C.R. Bell R.M. Carman G.M. J. Biol. Chem. 1995; 270: 18774-18780Abstract Full Text Full Text PDF PubMed Scopus (54) Google ScholarpRIP1PGKPlasmid containing a 1.0-kb fragment of the PGK1 gene101He F. Kim S.W. Donahue J.L. Rosbash M. Jacobson A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7034-7038Crossref PubMed Scopus (219) Google ScholarpKSK1EKI1 gene derived from PCR ligated into the SrfI site of pCRScript™ AMP SK(+)53Kim 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 ScholarpKSK2TRP1 disruption cassette from pJA52 ligated into the BglII/BsaBI sites of plasmid pKSK153Kim 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 ScholarpHS9EKI1 gene from pKSK1 ligated into the PstI/SacI sites of YEp352This studypHS12EKI1 gene from pKSK1 ligated into the BamHI/SacI site of pRS416This study Open table in a new tab 4′,6-Diamidino-2-phenylindole Staining of Mitochondria—Mitochondrial DNA of S. cerevisiae cells was examined by 4′,6-diamidino-2-phenylindole staining (49Kaiser C. Kim S. Mitchell A. Methods in Yeast Genetics: A Laboratory Course Manual. 1994; (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY)Google Scholar) using a Nikon Eclipse E800 fluorescence microscope equipped with a Hamamatsu Orca digital camera. Images were captured in monochrome and processed using Improvision Openlab software. DNA Isolation and Manipulations—Plasmid and genomic DNA preparation, restriction enzyme digestion, and DNA ligations were performed according to standard protocols (45Sambrook J. Kim E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 1989; (2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY)Google Scholar). Transformations of yeast (50Ito H. Kim F. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Crossref PubMed Google Scholar) and E. coli (45Sambrook J. Kim E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 1989; (2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY)Google Scholar) were performed as described previously. Plasmid maintenance and amplifications were performed in E. coli strain DH5α. Conditions for the amplification of DNA by PCR were optimized as described previously (51Innis M.A. Kim D.H. Innis M.A. Gelfand D.H. Sninsky J.J. White T.J. PCR Protocols. A Guide to Methods and Applications. 1990: 3-12, Academic Press, Inc., San DiegoGoogle Scholar). Construction of eki1Δ, ect1Δ, ept1Δ, and cki1Δ eki1Δ Mutants—A new eki1Δ mutant (HCY5) was constructed in the W303-1B background by the one-step gene replacement technique (52Rothstein R. Methods Enzymol. 1991; 194: 281-301Crossref PubMed Scopus (1099) Google Scholar) with the eki1Δ::TRP1 disruption cassette as described by Kim et al. (53Kim 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). The ect1Δ (HCY3) an" @default.
• W2097074526 created "2016-06-24" @default.
• W2097074526 creator A5015880535 @default.
• W2097074526 creator A5089452267 @default.
• W2097074526 date "2007-10-01" @default.
• W2097074526 modified "2023-10-18" @default.
• W2097074526 title "Respiratory Deficiency Mediates the Regulation of CHO1-encoded Phosphatidylserine Synthase by mRNA Stability in Saccharomyces cerevisiae" @default.
• W2097074526 cites W1424883175 @default.
• W2097074526 cites W1459222285 @default.
• W2097074526 cites W1487585902 @default.
• W2097074526 cites W1491896962 @default.
• W2097074526 cites W1503292785 @default.
• W2097074526 cites W1508686037 @default.
• W2097074526 cites W1523851596 @default.
• W2097074526 cites W1547269929 @default.
• W2097074526 cites W1569151430 @default.
• W2097074526 cites W1597525158 @default.
• W2097074526 cites W1598131238 @default.
• W2097074526 cites W1601603816 @default.
• W2097074526 cites W1638738001 @default.
• W2097074526 cites W1663567163 @default.
• W2097074526 cites W1677751777 @default.
• W2097074526 cites W1681432328 @default.
• W2097074526 cites W1719011723 @default.
• W2097074526 cites W1798375282 @default.
• W2097074526 cites W1844084074 @default.
• W2097074526 cites W1847471860 @default.
• W2097074526 cites W1848942581 @default.
• W2097074526 cites W1859936003 @default.
• W2097074526 cites W1955545440 @default.
• W2097074526 cites W1966179958 @default.
• W2097074526 cites W1966869662 @default.
• W2097074526 cites W1968917541 @default.
• W2097074526 cites W1973782344 @default.
• W2097074526 cites W1975938982 @default.
• W2097074526 cites W1981388125 @default.
• W2097074526 cites W1983227864 @default.
• W2097074526 cites W1985435562 @default.
• W2097074526 cites W1989132347 @default.
• W2097074526 cites W1989849863 @default.
• W2097074526 cites W1990235046 @default.
• W2097074526 cites W1990625705 @default.
• W2097074526 cites W2004693594 @default.
• W2097074526 cites W2011764708 @default.
• W2097074526 cites W2020662133 @default.
• W2097074526 cites W2020725477 @default.
• W2097074526 cites W2031189145 @default.
• W2097074526 cites W2033538120 @default.
• W2097074526 cites W2039348210 @default.
• W2097074526 cites W2039407069 @default.
• W2097074526 cites W2043073327 @default.
• W2097074526 cites W2044480313 @default.
• W2097074526 cites W2052846823 @default.
• W2097074526 cites W2054046691 @default.
• W2097074526 cites W2055055808 @default.
• W2097074526 cites W2056928445 @default.
• W2097074526 cites W2060164471 @default.
• W2097074526 cites W2060859000 @default.
• W2097074526 cites W2062116865 @default.
• W2097074526 cites W2064825406 @default.
• W2097074526 cites W2066909405 @default.
• W2097074526 cites W2070425044 @default.
• W2097074526 cites W2074352392 @default.
• W2097074526 cites W2076510291 @default.
• W2097074526 cites W2076627305 @default.
• W2097074526 cites W2077731916 @default.
• W2097074526 cites W2079307234 @default.
• W2097074526 cites W2090375169 @default.
• W2097074526 cites W2095059919 @default.
• W2097074526 cites W2096068750 @default.
• W2097074526 cites W2100647001 @default.
• W2097074526 cites W2100837269 @default.
• W2097074526 cites W2102651270 @default.
• W2097074526 cites W2102971075 @default.
• W2097074526 cites W2110966981 @default.
• W2097074526 cites W2112727465 @default.
• W2097074526 cites W2115073295 @default.
• W2097074526 cites W2117403504 @default.
• W2097074526 cites W2118055097 @default.
• W2097074526 cites W2121770428 @default.
• W2097074526 cites W2126924371 @default.
• W2097074526 cites W2127570240 @default.
• W2097074526 cites W2127676205 @default.
• W2097074526 cites W2130994563 @default.
• W2097074526 cites W2139150415 @default.
• W2097074526 cites W2154430600 @default.
• W2097074526 cites W2158204713 @default.
• W2097074526 cites W2162295516 @default.
• W2097074526 cites W2168248533 @default.
• W2097074526 cites W2169333620 @default.
• W2097074526 cites W2171227040 @default.
• W2097074526 cites W2172293262 @default.
• W2097074526 cites W2300552860 @default.
• W2097074526 cites W2410585811 @default.
• W2097074526 cites W4293247451 @default.
• W2097074526 cites W941572373 @default.
• W2097074526 doi "https://doi.org/10.1074/jbc.m705098200" @default.
• W2097074526 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2150996" @default.

• next