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Reducing equivalents in the form of NADPH are essential for many enzymatic steps involved in the biosynthesis of cellular macromolecules. An adequate level of NADPH is also required to protect cells against oxidative stress. The major enzymatic source of NADPH in the cell is the reaction catalyzed by glucose-6-phosphate dehydrogenase, the first enzyme in the pentose phosphate pathway. Disruption of the ZWF1 gene, encoding glucose-6-phosphate dehydrogenase in the yeast Saccharomyces cerevisiae, results in methionine auxotrophy and increased sensitivity to oxidizing agents. It is assumed that both phenotypes are due to an NADPH deficiency in the zwf1Δ strain. We used a Met− phenotype displayed by the zwf1Δ strain to look for multicopy suppressors of this deletion. We found that overexpression of the ALD6 gene coding for cytosolic acetaldehyde dehydrogenase, which utilizes NADP+ as its cofactor, restores the Met+ phenotype of thezwf1Δ strain. Another multicopy suppressor identified in our screen, the ZMS1 gene encoding a putative transcription factor, regulates the level of ALD6 expression. A strain bearing a double ZWF1 ALD6 gene disruption is not viable. Thus, our results indicate the reaction catalyzed by Ald6p as an important source of reducing equivalents in the yeast cells. Reducing equivalents in the form of NADPH are essential for many enzymatic steps involved in the biosynthesis of cellular macromolecules. An adequate level of NADPH is also required to protect cells against oxidative stress. The major enzymatic source of NADPH in the cell is the reaction catalyzed by glucose-6-phosphate dehydrogenase, the first enzyme in the pentose phosphate pathway. Disruption of the ZWF1 gene, encoding glucose-6-phosphate dehydrogenase in the yeast Saccharomyces cerevisiae, results in methionine auxotrophy and increased sensitivity to oxidizing agents. It is assumed that both phenotypes are due to an NADPH deficiency in the zwf1Δ strain. We used a Met− phenotype displayed by the zwf1Δ strain to look for multicopy suppressors of this deletion. We found that overexpression of the ALD6 gene coding for cytosolic acetaldehyde dehydrogenase, which utilizes NADP+ as its cofactor, restores the Met+ phenotype of thezwf1Δ strain. Another multicopy suppressor identified in our screen, the ZMS1 gene encoding a putative transcription factor, regulates the level of ALD6 expression. A strain bearing a double ZWF1 ALD6 gene disruption is not viable. Thus, our results indicate the reaction catalyzed by Ald6p as an important source of reducing equivalents in the yeast cells. Glucose-6-phosphate dehydrogenase is a housekeeping enzyme, encoded in mammals by the G6PD 1The abbreviations used are: G6PDglucose-6-phosphate dehydrogenase5-FOA5-fluoroorotic acid1The abbreviations used are: G6PDglucose-6-phosphate dehydrogenase5-FOA5-fluoroorotic acid gene located on the X chromosome (1Vulliamy T. Mason P. Luzzatto L. Trends Genet. 1992; 8: 138-143Abstract Full Text PDF PubMed Scopus (117) Google Scholar). 2Online Mendelian Inheritance in Man (OMIMTM), McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins University (Baltimore, MD) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, MD), www.ncbi.nlm.nih.gov/omim.2Online Mendelian Inheritance in Man (OMIMTM), McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins University (Baltimore, MD) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, MD), www.ncbi.nlm.nih.gov/omim. It has important functions in intermediary metabolism since it catalyzes the first step in the pentose phosphate pathway, which supplies the cell with the reductive potential in the form of NADPH, required for a variety of biosynthetic pathways and redox reactions. Apart from generating NADPH, the pentose phosphate pathway contributes to the synthesis of ribose 5-phosphate, required for the biosynthesis of some amino acids, nucleotides, and coenzymes (3Stryer L. Biochemistry. 3rd Ed. W. H. Freeman and Co., San Francisco1989Google Scholar). Glucose-6-phosphate (G6PD) activity was reported in all organisms and cell types, and the enzyme structure is highly conserved. 3Molecular Modeling Data Base, www.ncbi.nlm.nih.gov/Structure.3Molecular Modeling Data Base, www.ncbi.nlm.nih.gov/Structure. Moreover, we found recently that human G6PD can functionally replace this enzyme inSaccharomyces cerevisiae. 4D. Grabowska, manuscript in preparation.4D. Grabowska, manuscript in preparation. G6PD deficiency is the most widespread human enzymopathy (5Beutler E. Stanbury J.B. Wyngaarden J.B. Fredrickson D.S. Goldstein J.L. Brown M.S. Fifth Ed. The Metabolic Basis of Inherited Disease. McGraw-Hill Book Co., New York1990Google Scholar);2still, a complete absence of G6PD activity has never been reported in mammals. Although it is possible to knock out the G6PD gene in the mammalian cell line, as it was shown in male mouse embryonic stem cells (6Pandolfi P.P. Sonati F. Rivi R. Mason P. Grosveld F. Luzzatto L. EMBO J. 1995; 14: 5209-5215Crossref PubMed Scopus (460) Google Scholar), it might be that at least residual G6PD activity is required at some step(s) in higher eucaryote development to such an extent that its total absence would be lethal. On the other hand G6PD-null mutants isolated in the unicellular organisms Escherichia coli andS. cerevisiae were both viable (7Fraenkel D.G. J. Bacteriol. 1968; 95: 1267-1271Crossref PubMed Google Scholar, 8Nogae I. Johnston M. Gene (Amst.). 1990; 96: 161-169Crossref PubMed Scopus (152) Google Scholar). Inactivation of theZWF1 gene encoding glucose-6-phosphate dehydrogenase in the yeast S. cerevisiae does not affect the cell growth in rich media supplemented with a variety of carbon sources, although it increases their sensitivity to oxidizing agents (8Nogae I. Johnston M. Gene (Amst.). 1990; 96: 161-169Crossref PubMed Scopus (152) Google Scholar) and leads to methionine auxotrophy (9Thomas D. Cherest H. Surdin-Kerjan Y. EMBO J. 1991; 10: 547-553Crossref PubMed Scopus (115) Google Scholar). It was suggested that the growth deficiencies are caused by an increased utilization of NADPH required for reductive assimilation of inorganic sulfur or for restoration of cellular pools of reduced glutathione and thioredoxin, which rapidly deplete under oxidative stress growth conditions (10Slekar K.H. Kosman D.J. Culotta V. J. Biol. Chem. 1996; 271: 28831-28836Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Due to surprisingly discrete phenotypes displayed by the zwf1-null mutant, the yeast S. cerevisiae was used as a model organism to clarify the contribution of the alternative routes of the NADPH synthesis, namely the reactions catalyzed by isocitrate dehydrogenases, to the overall pool of NADPH in the cell (11Minard K.I. Jennings G.T. Loftus T.M. Xuan D. McAlister-Henn L. J. Biol. Chem. 1998; 273: 31486-31493Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). There are three highly homologous but differentially compartmentalized isocitrate dehydrogenase isozymes in yeast encoded by the IDP1, IDP2,and IDP3 genes and localized in mitochondria, cytoplasm, and peroxisomes, respectively (12Haselbeck R.J. McAlister-Henn L. J. Biol. Chem. 1991; 266: 2339-2345Abstract Full Text PDF PubMed Google Scholar, 13Loftus T.M. Hall L.V. Anderson S.L. McAlister-Henn L. Biochemistry. 1994; 33: 9661-9667Crossref PubMed Scopus (51) Google Scholar, 14Henke B. Girzalsky W. Berteaux-Lecellier V. Erdmann R. J. Biol. Chem. 1998; 273: 3702-3711Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). The loss of either Idp1p and/or Idp2p activity produces no observable growth phenotype. The loss of Idp3p impairs growth on media containing unsaturated fatty acids as a carbon source, which indicates the role of this protein as a peroxisomal source of NADPH required for a double bond reduction (14Henke B. Girzalsky W. Berteaux-Lecellier V. Erdmann R. J. Biol. Chem. 1998; 273: 3702-3711Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar,15van Roermund C.W. Hettema E.H. Kal A.J. van den Berg M. Tabak H.F. Wanders R.J. EMBO J. 1998; 17: 677-687Crossref PubMed Scopus (121) Google Scholar). Combinations of double, triple, and quadruple deletions ofZWF1, IDP1, IDP2, and IDP3 indicated that onlyZWF1 and IDP2 have partially overlapping functions leading to the enhancement of the zwf1Δ idp2Δmutant phenotype in comparison with single zwf1Δ oridp2Δ knockouts (11Minard K.I. Jennings G.T. Loftus T.M. Xuan D. McAlister-Henn L. J. Biol. Chem. 1998; 273: 31486-31493Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). However, the ability of thezwf1Δ idp1Δ idp2Δ idp3Δ strain to grow under conditions including the use of glucose, glycerol, ethanol, or acetate as carbon sources indicates that the reactions catalyzed by G6PD and cytosolic isocitrate dehydrogenase, postulated to be the major contributors of biosynthetic reducing equivalents in eucaryotic cells, are apparently not essential under many growth conditions (11Minard K.I. Jennings G.T. Loftus T.M. Xuan D. McAlister-Henn L. J. Biol. Chem. 1998; 273: 31486-31493Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Here we present our evidence of another, as yet unidentified, activity contributing to the pool of NADPH in the yeast S. cerevisiaecells. Glucose-6-phosphate dehydrogenase is a housekeeping enzyme, encoded in mammals by the G6PD 1The abbreviations used are: G6PDglucose-6-phosphate dehydrogenase5-FOA5-fluoroorotic acid1The abbreviations used are: G6PDglucose-6-phosphate dehydrogenase5-FOA5-fluoroorotic acid gene located on the X chromosome (1Vulliamy T. Mason P. Luzzatto L. Trends Genet. 1992; 8: 138-143Abstract Full Text PDF PubMed Scopus (117) Google Scholar). 2Online Mendelian Inheritance in Man (OMIMTM), McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins University (Baltimore, MD) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, MD), www.ncbi.nlm.nih.gov/omim.2Online Mendelian Inheritance in Man (OMIMTM), McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins University (Baltimore, MD) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, MD), www.ncbi.nlm.nih.gov/omim. It has important functions in intermediary metabolism since it catalyzes the first step in the pentose phosphate pathway, which supplies the cell with the reductive potential in the form of NADPH, required for a variety of biosynthetic pathways and redox reactions. Apart from generating NADPH, the pentose phosphate pathway contributes to the synthesis of ribose 5-phosphate, required for the biosynthesis of some amino acids, nucleotides, and coenzymes (3Stryer L. Biochemistry. 3rd Ed. W. H. Freeman and Co., San Francisco1989Google Scholar). Glucose-6-phosphate (G6PD) activity was reported in all organisms and cell types, and the enzyme structure is highly conserved. 3Molecular Modeling Data Base, www.ncbi.nlm.nih.gov/Structure.3Molecular Modeling Data Base, www.ncbi.nlm.nih.gov/Structure. Moreover, we found recently that human G6PD can functionally replace this enzyme inSaccharomyces cerevisiae. 4D. Grabowska, manuscript in preparation.4D. Grabowska, manuscript in preparation. G6PD deficiency is the most widespread human enzymopathy (5Beutler E. Stanbury J.B. Wyngaarden J.B. Fredrickson D.S. Goldstein J.L. Brown M.S. Fifth Ed. The Metabolic Basis of Inherited Disease. McGraw-Hill Book Co., New York1990Google Scholar);2still, a complete absence of G6PD activity has never been reported in mammals. Although it is possible to knock out the G6PD gene in the mammalian cell line, as it was shown in male mouse embryonic stem cells (6Pandolfi P.P. Sonati F. Rivi R. Mason P. Grosveld F. Luzzatto L. EMBO J. 1995; 14: 5209-5215Crossref PubMed Scopus (460) Google Scholar), it might be that at least residual G6PD activity is required at some step(s) in higher eucaryote development to such an extent that its total absence would be lethal. On the other hand G6PD-null mutants isolated in the unicellular organisms Escherichia coli andS. cerevisiae were both viable (7Fraenkel D.G. J. Bacteriol. 1968; 95: 1267-1271Crossref PubMed Google Scholar, 8Nogae I. Johnston M. Gene (Amst.). 1990; 96: 161-169Crossref PubMed Scopus (152) Google Scholar). Inactivation of theZWF1 gene encoding glucose-6-phosphate dehydrogenase in the yeast S. cerevisiae does not affect the cell growth in rich media supplemented with a variety of carbon sources, although it increases their sensitivity to oxidizing agents (8Nogae I. Johnston M. Gene (Amst.). 1990; 96: 161-169Crossref PubMed Scopus (152) Google Scholar) and leads to methionine auxotrophy (9Thomas D. Cherest H. Surdin-Kerjan Y. EMBO J. 1991; 10: 547-553Crossref PubMed Scopus (115) Google Scholar). It was suggested that the growth deficiencies are caused by an increased utilization of NADPH required for reductive assimilation of inorganic sulfur or for restoration of cellular pools of reduced glutathione and thioredoxin, which rapidly deplete under oxidative stress growth conditions (10Slekar K.H. Kosman D.J. Culotta V. J. Biol. Chem. 1996; 271: 28831-28836Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Due to surprisingly discrete phenotypes displayed by the zwf1-null mutant, the yeast S. cerevisiae was used as a model organism to clarify the contribution of the alternative routes of the NADPH synthesis, namely the reactions catalyzed by isocitrate dehydrogenases, to the overall pool of NADPH in the cell (11Minard K.I. Jennings G.T. Loftus T.M. Xuan D. McAlister-Henn L. J. Biol. Chem. 1998; 273: 31486-31493Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). There are three highly homologous but differentially compartmentalized isocitrate dehydrogenase isozymes in yeast encoded by the IDP1, IDP2,and IDP3 genes and localized in mitochondria, cytoplasm, and peroxisomes, respectively (12Haselbeck R.J. McAlister-Henn L. J. Biol. Chem. 1991; 266: 2339-2345Abstract Full Text PDF PubMed Google Scholar, 13Loftus T.M. Hall L.V. Anderson S.L. McAlister-Henn L. Biochemistry. 1994; 33: 9661-9667Crossref PubMed Scopus (51) Google Scholar, 14Henke B. Girzalsky W. Berteaux-Lecellier V. Erdmann R. J. Biol. Chem. 1998; 273: 3702-3711Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). The loss of either Idp1p and/or Idp2p activity produces no observable growth phenotype. The loss of Idp3p impairs growth on media containing unsaturated fatty acids as a carbon source, which indicates the role of this protein as a peroxisomal source of NADPH required for a double bond reduction (14Henke B. Girzalsky W. Berteaux-Lecellier V. Erdmann R. J. Biol. Chem. 1998; 273: 3702-3711Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar,15van Roermund C.W. Hettema E.H. Kal A.J. van den Berg M. Tabak H.F. Wanders R.J. EMBO J. 1998; 17: 677-687Crossref PubMed Scopus (121) Google Scholar). Combinations of double, triple, and quadruple deletions ofZWF1, IDP1, IDP2, and IDP3 indicated that onlyZWF1 and IDP2 have partially overlapping functions leading to the enhancement of the zwf1Δ idp2Δmutant phenotype in comparison with single zwf1Δ oridp2Δ knockouts (11Minard K.I. Jennings G.T. Loftus T.M. Xuan D. McAlister-Henn L. J. Biol. Chem. 1998; 273: 31486-31493Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). However, the ability of thezwf1Δ idp1Δ idp2Δ idp3Δ strain to grow under conditions including the use of glucose, glycerol, ethanol, or acetate as carbon sources indicates that the reactions catalyzed by G6PD and cytosolic isocitrate dehydrogenase, postulated to be the major contributors of biosynthetic reducing equivalents in eucaryotic cells, are apparently not essential under many growth conditions (11Minard K.I. Jennings G.T. Loftus T.M. Xuan D. McAlister-Henn L. J. Biol. Chem. 1998; 273: 31486-31493Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Here we present our evidence of another, as yet unidentified, activity contributing to the pool of NADPH in the yeast S. cerevisiaecells. glucose-6-phosphate dehydrogenase 5-fluoroorotic acid glucose-6-phosphate dehydrogenase 5-fluoroorotic acid We thank Dr. Dominique Thomas for kindly providing the CD101–1A strain. We are grateful to Prof. Joanna Rytka for helpful advice and discussions. We also thank Dr. Marek Skoneczny for a critical reading of the manuscript.
Grabowska et al. (Tue,) studied this question.