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

Abstract

Cellular and mitochondrial metabolite levels were measured in yeast TCA cycle mutants (sdh2⌬ or fum1⌬) lacking succinate dehydrogenase or fumarase activities. Cellular levels of succinate relative to parental strain levels were found to be elevated ϳ8-fold in the sdh2⌬ mutant and ϳ4-fold in the fum1⌬ mutant, and there was a preferential increase in mitochondrial levels in these mutant strains. The sdh2⌬ and fum1⌬ strains also exhibited 3-4-fold increases in expression of Cit2, the cytosolic form of citrate synthase that functions in the glyoxylate pathway. Codisruption 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 sdh2⌬sfc1⌬ and fum1⌬sfc1⌬ strains as compared with sdh2⌬ and fum1⌬ strains, suggesting that aberrant transport of succinate out of mitochondria mediated by Sfc1 is related to the increased expression of Cit2 in sdh2⌬ and fum1⌬ strains. A defect (rtg1⌬) 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, sdh2⌬, and fum1⌬ strains. Concomitantly, co-disruption of the RTG1 gene reduced the cellular levels of succinate in the sdh2⌬ and fum1⌬ strains, of fumarate in the fum1⌬ strain, and citrate in an idh⌬ 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.Intermediates of the tricarboxylic (TCA) 2 cycle are believed to be present in relatively low concentrations in mitochondria of actively respiring eukaryotic cells due to rapid flux through the cycle and to alternative biosynthetic uses for many of the intermediates. Flux through the cycle thus requires anaplerotic enzymes (e.g. pyruvate carboxylase to produce oxaloacetate) or pathways (e.g. the glyoxylate pathway in yeast that produces succinate) to replenish these intermediates. Defects in a TCA cycle enzyme would be expected to result in increased mitochondrial and/or cellular concentrations of the substrate of that enzyme.We and others (1-4) have been analyzing effects of disruptions of genes encoding TCA cycle enzymes in the yeast Saccharomyces cerevisiae. These gene disruptions are not lethal because yeast cells can survive without mitochondrial functions. However, the disruptions have quite distinct effects depending on loss of particular enzymes (5). We initiated studies of changes in metabolite profiles associated with loss of yeast TCA cycle enzymes. In a previous report (2), we described that citrate, a relatively abundant metabolite in the parental strain, accumulates to very high cellular levels in a mutant (idh⌬) lacking both Idh1 and Idh2 subunits of NAD-specific isocitrate dehydrogenase and in a mutant (aco1⌬) lacking aconitase. In addition to sharing similar gene and...

Role of Mitochondrial Retrograde Pathway in Regulating Ethanol-Inducible Filamentous Growth in Yeast

“…The TCA cycle is compromised in cells experiencing mitochondrial defects, but flux through the pathway can be maintained by the action of the RTG pathway (Liu and Butow, 1999; Lin et al, 2011), which is a major function of the RTG pathway (Butow and Avadhani, 2004). Glutamate can suppress the requirement for the retrograde pathway in the TCA cycle by increasing metabolic flux (Liu and Butow, 1999).…”

“…How does the RTG pathway control ethanol-dependent filamentous growth without involving other major filamentation regulatory pathways? One possibility is that the RTG pathway is part of the sensing/signaling mechanism that controls the rate of flux through the TCA cycle (Liu and Butow, 1999; Lin et al, 2011). TCA cycle rate is dependent on carbon and nitrogen levels, which are key inducers of filamentous growth in yeast and other fungal species.…”

New Insights into the Nuclear Imaging Phenotypes of Cluster 1 Pheochromocytoma and Paraganglioma

“…This effect would be mediated by a succinate efflux for SDHx mutated cells. Analyses of compartmentalized levels of TCA cycle metabolites have revealed that yeast with sdh Δ mutations may aberrantly efflux succinate from the mitochondria to the cytosol [54]. Other studies have even reported that succinate could be excreted into the medium during cultivation of yeast sdh Δ mutants [55, 56].…”

Сучасні Можливості Енергопротекції При Критичних Станах

“…Вместе с тем при гипоксии дыхательная цепь митохондрий не может принять на себя водород от какого-либо иного субстрата, кроме янтарной кис-лоты, так как только при ее окислении водород по-ступает на наиболее близкий к кислороду участок дыхательной цепи, что является удобным и эко-номным в энергетическом плане для организма [8]. Феномен активного окисления сукцината получил название эффекта монополизации дыхательной цепи [9].…”