Suppression of metabolic defects of yeast isocitrate dehydrogenase and aconitase mutants by loss of citrate synthase

An, Lin; Hakala, Kevin; Weintraub, Susan E; McAlister-Henn, Lee

doi:10.1016/j.abb.2008.03.005

Cited by 19 publications

(35 citation statements)

References 44 publications

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“…Metabolite Analyses-Methods for preparation of whole cell extracts and derivatization for metabolite analyses using gas chromatography/mass spectrometry were previously described in detail (2). Briefly, cells were cultured in YP galactose medium to A 600 nm Х 2.0, and growth was stopped by addition of 100 g/ml of cycloheximide and 40 mM sodium azide.…”

Section: Methodsmentioning

confidence: 99%

“…This extraction procedure and subsequent derivatization methods were empirically determined to permit reproducible and quantitative analyses of TCA cycle metabolites and related amino acids. Concentrations of denatured proteins in cell extracts were determined using a modified Bradford assay (22) following solubilization of the proteins as previously described (2).…”

Section: Methodsmentioning

confidence: 99%

“…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.…”

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confidence: 99%

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

Anderson

Minard

et al. 2011

Journal of Biological Chemistry

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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...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

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

Anderson

Minard

et al. 2011

Journal of Biological Chemistry

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“…Accumulation of intracellular citrate is deleterious in a number of ways, which include chelation of divalent cations and acidification of the intracellular milieu (24).…”

Section: Resultsmentioning

confidence: 99%

Deletion of Citrate Synthase Restores Growth of Sinorhizobium meliloti 1021 Aconitase Mutants

et al. 2009

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The symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti 1021 encodes only one predicted aconitase (AcnA) in its genome. AcnA has a significant degree of similarity with other bacterial aconitases that behave as dual proteins: enzymes and posttranscriptional regulators of gene expression. Similar to the case with these bacterial aconitases, AcnA activity was reversibly labile and was regained upon reconstitution with reduced iron. The aconitase promoter was active in root nodules. acnA mutants grew very poorly, had secondary mutations, and were quickly outgrown by pseudorevertants. The acnA gene was stably interrupted in a citrate synthase (gltA) null background, indicating that the intracellular accumulation of citrate may be deleterious for survival of strain 1021. No aconitase activity was detected in this mutant, suggesting that the acnA gene encodes the only functional aconitase of strain 1021. To uncover a function of AcnA beyond its catalytic role in the tricarboxylic acid cycle pathway, the gltA acnA double mutant was compared with the gltA single mutant for differences in motility, resistance to oxidative stress, nodulation, and growth on different substrates. However, no differences in any of these characteristics were found.Aconitases (EC 4.2.1.3) are key enzymes in the tricarboxylic acid (TCA) cycle in both prokaryotes and eukaryotes, catalyzing the reversible isomerization of citrate into isocitrate via the intermediate cis-aconitate. The active site contains a [4Fe-4S] cluster that is required for enzymatic activity.

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“…It was shown that ⌬icd and ⌬aco1 mutants of Saccharomyces cerevisiae share several growth phenotypes, for example, poor growth on glycerol, an inability to grow with acetate as the sole carbon source, or a propensity to generate petite segregants, which can be complemented or at least moderated by the codisruption of cit1 coding for mitochondrial citrate synthase (17,37,45).…”

Section: Vol 193 2011 Inactivation Of Citrate Synthase In Aconitasementioning

confidence: 99%

Deletion of the Aconitase Gene in Corynebacterium glutamicum Causes Strong Selection Pressure for Secondary Mutations Inactivating Citrate Synthase

et al. 2011

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The aconitase gene acn of Corynebacterium glutamicum is regulated by four transcriptional regulators, indicating that the synthesis of this enzyme is carefully controlled. To understand the causes for this elaborate regulation, the properties of the ⌬acn-1 deletion mutant were analyzed in detail. The mutant was glutamate auxotrophic in glucose minimal medium, showed a strong growth defect, and secreted large amounts of acetate. None of these phenotypes could be complemented by plasmid-encoded aconitase, suggesting the presence of a secondary mutation. In fact, a point mutation within the gltA gene encoding citrate synthase was identified that caused the instability of the protein and an almost complete lack of its enzymatic activity. Subsequently, 27 further, independent ⌬acn clones were isolated, and 15 of them were found to contain distinct mutations in gltA, causing the loss of citrate synthase activity. A similar result was observed for mutants lacking the isocitrate dehydrogenase gene icd. In this case, 8 of 24 ⌬icd clones contained additional mutations in gltA. Indirect evidence was obtained that elevated intracellular citrate concentrations could be the cause of this selection pressure. Accordingly, the careful control of aconitase synthesis might have evolved due to the necessity to avoid inhibitory cytoplasmic citrate levels on the one hand and to prevent the excessive synthesis of an oxygen-sensitive protein requiring both iron and sulfur on the other hand.For most organisms, the tricarboxylic acid (TCA) cycle, also known as the Krebs cycle, is a key metabolic pathway. In its catabolic function, acetyl-coenzyme A (CoA), a product of the degradation of sugars, fatty acids, amino acids, and other carbon sources, is oxidized to CO 2 , thereby generating reducing equivalents (NADH and reduced quinones) for respiration and ATP by substrate-level phosphorylation. In its anabolic function, the TCA cycle produces 2-oxoglutarate and oxaloacetate as precursors of the glutamate family and the aspartate family of amino acids, respectively, in addition to other important intermediates, such as succinyl-CoA. Moreover, in many bacteria, NADPH is produced for biosynthetic purposes by the isocitrate dehydrogenase reaction.The TCA cycle has been studied quite intensively in Corynebacterium glutamicum. This Gram-positive soil bacterium is used for large-scale industrial amino acid production, with the major products being the flavor enhancer L-glutamate (as monosodium salt) and the feed additive L-lysine, both of which are derived from intermediates of the TCA cycle. Many enzymes have been characterized, for example, citrate synthase (13), aconitase (4), isocitrate dehydrogenase (12), the 2-oxoglutarate dehydrogenase complex (25, 43, 59), fumarase (19), succinate dehydrogenase (34), malate-menaquinone oxidoreductase, and malate dehydrogenase (18,40,41). In addition, a complex regulatory network for the control of TCA cycle activity has been elucidated (for a review, see reference 7) that includes a variety of transcripti...

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Suppression of metabolic defects of yeast isocitrate dehydrogenase and aconitase mutants by loss of citrate synthase

Cited by 19 publications

References 44 publications

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

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

Deletion of Citrate Synthase Restores Growth of Sinorhizobium meliloti 1021 Aconitase Mutants

Deletion of the Aconitase Gene in Corynebacterium glutamicum Causes Strong Selection Pressure for Secondary Mutations Inactivating Citrate Synthase

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