Karyl I. Minard

Sources of NADPH in yeast vary with carbon source

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 zwf1Δald6Δ 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 zwf1Δidp2Δ 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 zwf1Δald6Δ mutant grows as well as the parental strain in similar shifts. In addition, the zwf1Δald6Δ 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 zwf1Δald6Δ 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 zwf1Δidp2Δ mutant in acetate medium, suggesting a decrease in production of NADPH reducing equivalents needed both for biosynthesis and for antioxidant functions.

Antioxidant function of cytosolic sources of NADPH in yeast.

The relative antioxidant functions of thiol-dependent mechanisms and of direct catalytic inactivation of H2O2 were examined using a collection of yeast mutants containing disruptions in single or multiple genes encoding two major enzymatic sources of NADPH [glucose-6-phosphate dehydrogenase (ZWF1) and cytosolic NADP+-specific isocitrate dehydrogenase (IDP2)] and in genes encoding two major cellular peroxidases [mitochondrial cytochrome c peroxidase (CCP1) and cytosolic catalase (CTT1)]. Both types of mechanisms were found to be important for growth in the presence of exogenous H2O2. In the absence of exogenous oxidants, however, loss of ZWF1 and IDP2, but not loss of CTT1 and CCP1, was found to be detrimental not only to growth but also to viability of cells shifted to rich medium containing oleate or acetate. The loss in viability correlates with increased levels of intracellular oxidants apparently produced during normal metabolism of these carbon sources. Acute effects in DeltaZWF1DeltaIDP2 mutants following shifts to these nonpermissive media include an increase in the number of cells demonstrating a transient decrease in growth rate and in cells containing apparent nuclear DNA strand breaks. Cumulative effects are reflected in phenotypes, including sensitivity to acetate medium and a reduction in mating efficiency, that become more pronounced with time following disruption of the ZWF1 and IDP2 genes. These results suggest that cellular mechanisms dependent on NADPH are crucial metabolic antioxidants.

Sources of NADPH and expression of mammalian NADP+-specific isocitrate dehydrogenases in Saccharomyces cerevisiae

To compare roles of specific enzymes in supply of NADPH for cellular biosynthesis, collections of yeast mutants were constructed by gene disruptions and matings. These mutants include haploid strains containing all possible combinations of deletions in yeast genes encoding three differentially compartmentalized isozymes of NADP+-specific isocitrate dehydrogenase and in the gene encoding glucose-6-phosphate dehydrogenase (Zwf1p). Growth phenotype analyses of the mutants indicate that either cytosolic NADP+-specific isocitrate dehydrogenase (Idp2p) or the hexose monophosphate shunt is essential for growth with fatty acids as carbon sources and for sporulation of diploid strains, a condition associated with high levels of fatty acid synthesis. No new biosynthetic roles were identified for mitochondrial (Idp1p) or peroxisomal (Idp3p) NADP+-specific isocitrate dehydrogenase isozymes. These and other results suggest that several major presumed sources of biosynthetic reducing equivalents are non-essential in yeast cells grown under many cultivation conditions. To develop an in vivo system for analysis of metabolic function, mammalian mitochondrial and cytosolic isozymes of NADP+-specific isocitrate dehydrogenase were expressed in yeast using promoters from the cognate yeast genes. The mammalian mitochondrial isozyme was found to be imported efficiently into yeast mitochondria when fused to the Idp1p targeting sequence and to substitute functionally for Idp1p for production of α-ketoglutarate. The mammalian cytosolic isozyme was found to partition between cytosolic and organellar compartments and to replace functionally Idp2p for production of α-ketoglutarate or for growth on fatty acids in a mutant lacking Zwf1p. The mammalian cytosolic isozyme also functionally substitutes for Idp3p allowing growth on petroselinic acid as a carbon source, suggesting partial localization to peroxisomes and provision of NADPH for β-oxidation of that fatty acid.

Dependence of Peroxisomal β-Oxidation on Cytosolic Sources of NADPH

Growth of Saccharomyces cerevisiaewith a fatty acid as carbon source was shown previously to require function of either glucose-6-phosphate dehydrogenase (ZWF1) or cytosolic NADP+-specific isocitrate dehydrogenase (IDP2), suggesting dependence of β-oxidation on a cytosolic source of NADPH. In this study, we find that ΔIDP2ΔZWF1 strains containing disruptions in genes encoding both enzymes exhibit a rapid loss of viability when transferred to medium containing oleate as the carbon source. This loss of viability is not observed following transfer of a ΔIDP3 strain lacking peroxisomal isocitrate dehydrogenase to medium with docosahexaenoate, a nonpermissive carbon source that requires function of IDP3 for β-oxidation. This suggests that the fatty acid− phenotype ofΔIDP2ΔZWF1 strains is not a simple defect in utilization. Instead, we propose that the common function shared by IDP2 and ZWF1 is maintenance of significant levels of NADPH for enzymatic removal of the hydrogen peroxide generated in the first step of peroxisomal β-oxidation in yeast and that inadequate levels of the reduced form of the cofactor can produce lethality. This proposal is supported by the finding that the sensitivity to exogenous hydrogen peroxide previously reported for ΔZWF1 mutant strains is less pronounced when analyses are conducted with a nonfermentable carbon source, a condition associated with elevated expression of IDP2. Under those conditions, similar slow growth phenotypes are observed forΔZWF1 and ΔIDP2 strains, and co-disruption of both genes dramatically exacerbates the H2O2 s phenotype. Collectively, these results suggest that IDP2, when expressed, and ZWF1 have critical overlapping functions in provision of reducing equivalents for defense against endogenous or exogenous sources of H2O2.

Dual compartmental localization and function of mammalian NADP+-specific isocitrate dehydrogenase in yeast

Isozymes of NADP+-specific isocitrate dehydrogenase (IDP) provide NADPH in cytosolic, mitochondrial, and peroxisomal compartments of eukaryotic cells. Analyses of purified IDP isozymes from yeast and from mouse suggest a general correspondence of pH optima for catalysis and pI values with pH values reported for resident cellular compartments. However, mouse IDP2, which partitions between cytosolic and peroxisomal compartments in mammalian cells, exhibits a broad pH optimum and an intermediate pI value. Mouse IDP2 was found to similarly colocalize in both cellular compartments when expressed in yeast at levels equivalent to those of endogenous yeast isozymes. The mouse enzyme can compensate for loss of yeast cytosolic IDP2 and of peroxisomal IDP3. Removal of the peroxisomal targeting signal of the mouse enzyme precludes both localization in peroxisomes and compensation for loss of yeast IDP3.

Effects of Excess Succinate and Retrograde Control of Metabolite Accumulation in Yeast Tricarboxylic Cycle Mutants

Cellular and mitochondrial metabolite levels were measured in yeast TCA cycle mutants (sdh2Δ or fum1Δ) lacking succinate dehydrogenase or fumarase activities. Cellular levels of succinate relative to parental strain levels were found to be elevated ∼8-fold in the sdh2Δ mutant and ∼4-fold in the fum1Δ mutant, and there was a preferential increase in mitochondrial levels in these mutant strains. The sdh2Δ and fum1Δ strains also exhibited 3–4-fold increases in expression of Cit2, the cytosolic form of citrate synthase that functions in the glyoxylate pathway. Co-disruption of the SFC1 gene encoding the mitochondrial succinate/fumarate transporter resulted in higher relative mitochondrial levels of succinate and in substantial reductions of Cit2 expression in sdh2Δsfc1Δ and fum1Δsfc1Δ strains as compared with sdh2Δ and fum1Δ strains, suggesting that aberrant transport of succinate out of mitochondria mediated by Sfc1 is related to the increased expression of Cit2 in sdh2Δ and fum1Δ strains. A defect (rtg1Δ) in the yeast retrograde response pathway, which controls expression of several mitochondrial proteins and Cit2, eliminated expression of Cit2 and reduced expression of NAD-specific isocitrate dehydrogenase (Idh) and aconitase (Aco1) in parental, sdh2Δ, and fum1Δ strains. Concomitantly, co-disruption of the RTG1 gene reduced the cellular levels of succinate in the sdh2Δ and fum1Δ strains, of fumarate in the fum1Δ strain, and citrate in an idhΔ strain. Thus, the retrograde response is necessary for maintenance of normal flux through the TCA and glyoxylate cycles in the parental strain and for metabolite accumulation in TCA cycle mutants.

Redox responses in yeast to acetate as the carbon source

Following a shift to medium with acetate as the carbon source, a parental yeast strain exhibited a transient moderate 20% reduction in total cellular [NAD(+)+NADH] but showed a approximately 10-fold increase in the ratio of [NAD(+)]:[NADH] after 36h. A mutant strain (idhDelta) lacking the tricarboxylic acid cycle enzyme isocitrate dehydrogenase had 50% higher cellular levels of [NAD(+)+NADH] relative to the parental strain but exhibited similar changes in cofactor concentrations following a shift to acetate medium, despite an inability to grow on that carbon source; essentially all of the cofactor was in the oxidized form within 36h. The salvage pathway for NAD(H) biosynthesis was found to be particularly important for viability during early transition of the parental strain to stationary phase in acetate medium. However, oxygen consumption was not affected, suggesting that the NAD(H) produced during this time may support other cellular functions. The idhDelta mutant exhibited increased flux through the salvage pathway in acetate medium but was dependent on the de novo pathway for viability. Long-term chronological lifespans of the parental and idhDelta strains were similar, but viability of the mutant strain was dependent on both pathways for NAD(H) biosynthesis.

Pnc1p supports increases in cellular NAD(H) levels in response to internal or external oxidative stress.

Following transfer from medium with fermentable glucose to medium with nonfermentable acetate as the carbon source, cellular levels of NAD(H) were found to increase approximately 2-fold in a parental yeast strain. Similar transfer of a mutant strain subject to endogenous oxidative stress under these conditions produced more dramatic increases in cellular levels of NAD(H), and elevations above parental levels were shown to be due to the nicotinimidase Pnc1p. Similar transient increases in NAD(H) levels observed in the parental strain following addition of exogenous hydrogen peroxide were also attributable to Pnc1p.

Disulfide Bond Formation in Yeast NAD+-Specific Isocitrate Dehydrogenase

The tricarboxylic acid cycle NAD+-specific isocitrate dehydrogenase (IDH) of Saccharomyces cerevisiae is an octameric enzyme composed of four heterodimers of regulatory IDH1 and catalytic IDH2 subunits. Recent structural analyses revealed the close proximity of Cys-150 residues from IDH2 in adjacent heterodimers, and features of the structure for the ligand-free enzyme suggested that formation of a disulfide bond between these residues might stabilize an inactive form of the enzyme. We constructed two mutant forms of IDH, one containing a C150S substitution in IDH2 and the other containing C56S/C242S substitutions in IDH2 leaving Cys-150 as the sole cysteine residue. Treatment of the affinity-purified enzymes with diamide resulted in the formation of disulfide bonds and in decreased activities for the wild-type and C56S/C242S enzymes. Both effects were reversible by the addition of dithiothreitol. Diamide had no effect on the C150S mutant enzyme, suggesting that Cys-150 is essential for the formation of a disulfide bond that inhibits IDH activity. Diamide-induced formation of the Cys-150 disulfide bond was also observed in vivo for yeast transformants expressing the wild-type or C56S/C242S enzymes but not for a transformant expressing the C150S enzyme. Finally, natural formation of the Cys-150 disulfide bond with a concomitant decrease in cellular IDH activity was observed during the stationary phase for the parental strain and for transformants expressing wild-type or C56S/C242S enzymes but not for a transformant expressing the C150S enzyme. A reduction in viability for the latter strain suggests that a decrease in IDH activity is important for metabolic changes in stationary phase cells.