Sources of NADPH in Yeast Vary with Carbon Source

Abstract

Production of NADPH in Saccharomyces cerevisiae cells grown on glucose has been attributed to glucose-6-phosphate dehydrogenase (Zwf1p) and a cytosolic aldehyde dehydrogenase (Ald6p) (Grabowska, D., and Chelstowska, A. (2003) J. Biol. Chem. 278, 13984 -13988). This was based on compensation by overexpression of Ald6p for phenotypes associated with ZWF1 gene disruption and on the apparent lethality resulting from co-disruption of ZWF1 and ALD6 genes. However, we have found that a zwf1⌬ald6⌬ mutant can be constructed by mating when tetrads are dissected on plates with a nonfermentable carbon source (lactate), a condition associated with expression of another enzymatic source of NADPH, cytosolic NADP ؉ -specific isocitrate dehydrogenase (Idp2p). We demonstrated previously that a zwf1⌬idp2⌬ mutant loses viability when shifted to medium with oleate or acetate as the carbon source, apparently because of the inadequate supply of NADPH for cellular antioxidant systems. In contrast, the zwf1⌬ald6⌬ mutant grows as well as the parental strain in similar shifts. In addition, the zwf1⌬ald6⌬ mutant grows slowly but does not lose viability when shifted to culture medium with glucose as the carbon source, and the mutant resumes growth when the glucose is exhausted from the medium. Measurements of NADP(H) levels revealed that NADPH may not be rapidly utilized in the zwf1⌬ald6⌬ mutant in glucose medium, perhaps because of a reduction in fatty acid synthesis associated with loss of Ald6p. In contrast, levels of NADP ؉ rise dramatically in the zwf1⌬idp2⌬ mutant in acetate medium, suggesting a decrease in production of NADPH reducing equivalents needed both for biosynthesis and for antioxidant functions.Reducing equivalents in the form of NADPH are required for numerous biosynthetic enzymatic reactions and antioxidant mechanisms involving glutathione and/or thioredoxin. The major cellular source of NADPH is thought to be the hexose monophosphate pathway. However, disruption of the Saccharomyces cerevisiae ZWF1 gene encoding glucose-6-phosphate dehydrogenase, the first and rate-limiting enzyme in that pathway, was found to produce relatively mild growth phenotypes including methionine auxotrophy and an increased sensitivity to exogenous oxidizing agents like hydrogen peroxide (1, 2). In studies designed to identify other crucial sources of NADPH in yeast, co-disruption of ZWF1 and the gene (IDP2) encoding cytosolic NADP ϩ -specific isocitrate dehydrogenase was found to produce a rapid loss in cell viability following shifts from medium containing glucose to medium containing either oleate or acetate as the carbon source (3, 4). This loss of viability correlated with an increase in levels of endogenous cellular oxidants. Fatty acid metabolism requires rapid flux through ␤-oxidation, a peroxisomal process in yeast that produces hydrogen peroxide in the first reaction of each cycle (5, 6), and acetate is a stringent carbon source that produces rapid flux through mitochondrial respiration, a process associated with the generation of dele...

Changes in disulfide bond content of proteins in a yeast strain lacking major sources of NADPH

“…Le Moan et al [55] also reported a substantial level of protein-thiol oxidation in glucose-grown yeast cells, and found that the presence of hydrogen peroxide increased the oxidation state of some of these proteins. We previously found that NADPH is required more for biosynthetic reactions than for resistance to oxidative stress in yeast cells growing on glucose [12]. With fermentative growth, macromolecular synthesis is rapid but respiratory rates are relatively slow.…”

“…In genetic studies using the yeast Saccharomyces cerevisiae [9][10][11][12], we determined that the major enzymatic sources of NADPH for antioxidant functions are two cytosolic enzymes: glucose-6-phosphate dehydrogenase (Zwf1p) which catalyzes the first step in the oxidative branch of the pentose phosphate pathway, and NADP + -specific isocitrate dehydrogenase isozyme 2 (Idp2p) which catalyzes the oxidative decarboxylation of isocitrate to form α -ketoglutarate. Others previously demonstrated that disruption of the yeast ZWF1 gene, which is constitutively expressed, produced only mild growth phenotypes including methionine auxotrophy and sensitivity to exogenous hydrogen peroxide in cells grown with glucose as the carbon source [13,14].…”

“…(c) No loss in viability was observed during similar media shifts of yeast mutants lacking cytosolic catalase and/or the major mitochondrial peroxidase [11], suggesting that these peroxidative enzymes do not provide crucial antioxidant functions under these conditions. (d) Direct measurements of cellular NADP + and NADPH levels [12] indicated a rapid cycling of NADPH in wild-type cells during such shifts (i.e., the ratio of NADP + /NADPH increased three-fold without a major change in total levels of cofactor). In the shifted idp2Δzwf1Δ mutant strain, levels of NADPH also decreased.…”

The ORF YNL274c (GOR1) codes for glyoxylate reductase in Saccharomyces cerevisiae

“…The NADPH needed for biosynthesis is produced by the oxidative branch of the pentose phosphate pathway and by aldehyde dehydrogenase, encoded by ALD6 (Grabowska and Chelstowska, 2003;Minard and McAlister-Henn, 2005). NADPH is also required for antioxidation involving glutathione and/or thioredoxin, especially during oxidative metabolism.…”

Degradation of the Gluconeogenic Enzymes Fructose-1,6-bisphosphatase and Malate Dehydrogenase Is Mediated by Distinct Proteolytic Pathways and Signaling Events

“…In addition to FBPase, the cytosolic enzyme malate dehydrogenase (MDH2) is subjected to rapid degradation when glucosestarved cells are replenished with fresh glucose (42,43). Mu-tagenesis studies have shown that the first 12 amino acids at the N terminus in MDH2 (⌬nMDH2) are essential for degradation.…”