Redox responses in yeast to acetate as the carbon source

During the diauxic shift, as cultures of Saccharomyces cerevisiae exhaust glucose as a carbon source and begin to utilize C2 metabolites (ethanol and acetate) as carbon/energy sources, expression of many mitochondrial proteins is increased to support more rapid flux through oxidative metabolic pathways [1,2]. In addition to changes in expression, allosteric regulation of key enzymes, e.g. mitochondrial NAD+-specific isocitrate dehydrogenase (IDH),1 ensures accelerated flux through the tricarboxylic acid (TCA) cycle as ATP production by glycolysis slows [3–5]. Yeast IDH is an octameric enzyme composed of four IDH1 and four IDH2 subunits [6–8]. Mutants lacking either IDH1 and/or IDH2 lack cellular enzyme activity and are unable to grow with acetate as a carbon source [9]. The acetate growth phenotype is shared with several other yeast mutants containing disruptions in genes encoding TCA cycle enzymes [10–12]. In recent studies [13], we found that levels of some TCA cycle intermediates, e.g. citrate and isocitrate, are substantially elevated in idhΔ mutants grown under permissive conditions (i.e. with glucose or galactose as the carbon source). In the current study, we have extended these studies to examine the effect of loss of IDH on cellular levels of NAD(H) ([NAD+] + [NADH]) and on the relative levels of oxidized to reduced cofactor ([NAD+]/[NADH]). Given that IDH is postulated to regulate rates of oxidative metabolism, we hypothesized that shifting cells lacking IDH from medium with glucose as the carbon source to medium with acetate would have dramatic effects particularly on the latter ratio. Also, for the parental strain, this medium shift recapitulates the growth transition to stationary phase [14], and there have been few rigorous analyses of changes in redox levels during this time. Previous measurements of pyridine nucleotide levels and ratios in yeast cells have primarily been conducted in the context of lifespan analyses. Lin et al. [15] demonstrated a requirement for respiration to support the extension of replicative lifespan (a measure of the times a single cell can divide) of yeast cells by caloric restriction. Respiration was reported to result in an increase in the cellular ratio of [NAD+]/[NADH] and subsequent stimulation of SIR2, an NAD+-dependent histone deacetylase implicated in regulation of lifespan [15,16]. However, Lin et al. [17] demonstrated a decrease in [NAD+] in older yeast cells, and Kaeberlein et al. [18] showed that severe caloric restriction extended lifespan by some mechanism that is independent of SIR2. Osorio et al. [19] reported a substantial decrease in cellular [NAD+] during the course of chronological lifespan analyses, which measure cellular survival during post-stationary phase growth. However, despite these examples of increasing interest in the relevance of cellular levels of the cofactor NAD(H) to aging, as well as to a number of health-related issues [20–23], there is a general dearth of knowledge about alterations in cellular redox conditions during changes in growth conditions as stated above and about the role of the TCA cycle in controlling the redox state of cells. We were also interested in determining the biosynthetic pathway(s) responsible for maintaining cellular NAD(H) under our experimental conditions. NAD(H) is synthesized from tryptophan by the cytosolic de novo pathway, catalyzed in yeast by enzymes designated BNA1–BNA6 [24]. NAD(H) can also be recycled from nicotinamide via the salvage pathway catalyzed by enzymes primarily localized in the nucleus [25,26]. The latter include PNC1, a nicotinamidase that converts nicotinamide to nicotinic acid, and NPT1, a nicotinate phosphoribosyltransferase. Codisruption of the BNA6 gene encoding quinolinate phosphoribosyl transferase and of the NPT1 gene was reported to be lethal [24], suggesting that the two pathways are the major cellular sources of net NAD(H). To examine whether the changes in NAD(H) levels and ratios we observed were due to de novo and/or salvage pathway function, we constructed and analyzed strains containing PNC1 or BNA6 gene disruptions in parental and idhΔ strains. This produced novel evidence for differential function of these pathways during entry into stationary phase. Finally, we examined the relevance of control of cellular redox levels to long-term viability using chronological lifespan analyses of stationary phase cultures.