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 (sdh2 Delta or fum1 Delta) lacking succinate dehydrogenase or fumarase activities. Cellular levels of succinate relative to parental strain levels were found to be elevated similar to 8-fold in the sdh2 Delta mutant and similar to 4-fold in the fum1 Delta mutant, and there was a preferential increase in mitochondrial levels in these mutant strains. The sdh2 Delta and fum1 Delta 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 sdh2 Delta sfc1 Delta and fum1 Delta sfc1 Delta strains as compared with sdh2 Delta and fum1 Delta strains, suggesting that aberrant transport of succinate out of mitochondria mediated by Sfc1 is related to the increased expression of Cit2 in sdh2 Delta and fum1 Delta strains. A defect (rtg1 Delta) 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 Delta, and fum1 Delta strains. Concomitantly, co-disruption of the RTG1 gene reduced the cellular levels of succinate in the sdh2 Delta and fum1 Delta strains, of fumarate in the fum1 Delta strain, and citrate in an idh Delta 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.