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

An-Ping Lin,Sondra L Anderson,Karyl I Minard,Lee Mcalister-Henn

doi:10.1074/jbc.m111.266890

Abstract

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.

Highlights

Grant GM051265. 1 To whom correspondence should be addressed: 7703 Floyd Curl Dr, San cycle enzyme would be expected to result in increased mitochondrial and/or cellular concentrations of the substrate of that enzyme
We found that the high cellular levels of citrate in the idh⌬ and aco1⌬ mutants could be reduced by co-disruption of the gene encoding mitochondrial citrate synthase (CIT1)
Succinate Accumulation and Expression of Cit2—As described above, we previously reported an association between elevated cellular levels of citrate and protein expression patterns/growth phenotypes observed for yeast idh⌬ or aco1⌬ mutants (1, 2)

Summary

Introduction

Grant GM051265. 1 To whom correspondence should be addressed: 7703 Floyd Curl Dr, San cycle enzyme would be expected to result in increased mitochondrial and/or cellular concentrations of the substrate of that enzyme. Because protein levels are more likely to reflect metabolic changes, we conducted immunoblot analyses of a number of TCA cycle and related enzymes to determine, in particular, effects of elevated cellular concentrations of succinate or fumarate in sdh2⌬ and fum1⌬ mutants.

Results

Conclusion