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 zwf1Delta ald6Delta 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 zwf1Delta idp2Delta 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 zwf1Delta ald6Delta mutant grows as well as the parental strain in similar shifts. In addition, the zwf1Delta ald6Delta 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 zwf1Delta ald6Delta 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 zwf1Delta idp2Delta mutant in acetate medium, suggesting a decrease in production of NADPH reducing equivalents needed both for biosynthesis and for antioxidant functions.
Highlights
Tion, 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 deleterious reactive oxidative species [7, 8]
Construction of a zwf1⌬ald6⌬ Mutant—Because previous attempts to obtain a zwf1⌬ald6⌬ mutant strain using methods employing glucose as the carbon source were unsuccessful [12], we attempted to construct this strain by mating and sporulation on plates containing a nonfermentable carbon source
We have demonstrated that construction of a haploid zwf1⌬ald6⌬ mutant is feasible when haploid yeast strains containing the individual gene disruptions are mated, and tetrads from resulting diploids are sporulated on plates containing lactate as the carbon source
Summary
In studies designed to identify other crucial sources of NADPH in yeast, co-disruption of ZWF1 and the gene (IDP2) encoding cytosolic NADPϩ-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. In a search for alternative sources of NADPH in cells grown on glucose, Grabowska and Chelstowska [12] found the ALD6 gene encoding cytosolic NADPϩ-specific acetaldehyde dehydrogenase to be a multicopy suppressor of the MetϪ phenotype of a zwf1⌬ strain They reported that a zwf1⌬ald6⌬ mutant is not viable and concluded that Ald6p is essential for production of NADPH in the absence of Zwf1p. We describe construction of the zwf1⌬ald6⌬ mutant strain and evaluation of the roles of Zwf1p, Idp2p, and Ald6p as enzymatic sources of NADPH
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