Global Transcription Analysis of Krebs Tricarboxylic Acid Cycle Mutants Reveals an Alternating Pattern of Gene Expression and Effects on Hypoxic and Oxidative Genes

Abstract

To understand the many roles of the Krebs tricarboxylic acid (TCA) cycle in cell function, we used DNA microarrays to examine gene expression in response to TCA cycle dysfunction. mRNA was analyzed from yeast strains harboring defects in each of 15 genes that encode subunits of the eight TCA cycle enzymes. The expression of >400 genes changed at least threefold in response to TCA cycle dysfunction. Many genes displayed a common response to TCA cycle dysfunction indicative of a shift away from oxidative metabolism. Another set of genes displayed a pairwise, alternating pattern of expression in response to contiguous TCA cycle enzyme defects: expression was elevated in aconitase and isocitrate dehydrogenase mutants, diminished in α-ketoglutarate dehydrogenase and succinyl-CoA ligase mutants, elevated again in succinate dehydrogenase and fumarase mutants, and diminished again in malate dehydrogenase and citrate synthase mutants. This pattern correlated with previously defined TCA cycle growth–enhancing mutations and suggested a novel metabolic signaling pathway monitoring TCA cycle function. Expression of hypoxic/anaerobic genes was elevated in α-ketoglutarate dehydrogenase mutants, whereas expression of oxidative genes was diminished, consistent with a heme signaling defect caused by inadequate levels of the heme precursor, succinyl-CoA. These studies have revealed extensive responses to changes in TCA cycle function and have uncovered new and unexpected metabolic networks that are wired into the TCA cycle.

Metabolic pathway profiling of mitochondrial respiratory chain mutants in C. elegans

“…This result is consistent with observations of OXPHOS upregulation in previously published microarray analyses performed on human mitochondrial disease samples harboring a variety of mitochondrial DNA mutations [21]. Contrasting with this expression signature of primary respiratory chain dysfunction, downregulation of mitochondrial OXPHOS was seen upon transcriptional analysis of primary TCA cycle mutants in S. cerevisiae [22].…”

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

“…Growth phenotypes and protein expression in idhΔcit1 Δ and aco1 Δcit1 Δ mutants Yeast mutants lacking IDH or ACO1 have been reported to share some growth phenotypes, including an inability to grow with acetate as a carbon source [14] and a propensity for generation of petite segregants [10]. To better understand the phenotypes exhibited by IDH mutants and their suppression by CIT1 defects, we wished to first compare the properties of idhΔ and idhΔ cit1 Δ mutant strains with those of aco1 Δ and aco1 Δcit1 Δ mutant strains.…”

“…Diluted cell suspensions were plated onto YP glucose plates, and numbers of petite/total colonies were determined after 3-4 days of growth at 30°C. As previously described [9,10], the red pigment produced as a result of the ade2 mutation in these strains permits tabulation of the percentage of smaller white (petite) colonies relative to larger red (grande) colonies.…”

“…For derivatization, 10 µl samples of clarified supernatants or of standard metabolite mixtures (including TCA cycle intermediates plus aspartate and glutamate at concentrations ranging from 0-2.0 mM) were diluted with 10 µl of water containing labeled internal standards (1.0 mM [1,[5][6][7][8][9][10][11][12][13] Laboratories, respectively) and dried in a microfuge for 3 h using a Speed-Vac. The dry residues were dissolved in 50 µl of 20 mg/ml O-ethylhydroxylamine hydrochloride (Sigma-Aldrich) in pyridine, and reacted for 90 min at 30°C.…”

“…Concomitant loss of CIT1 reduces these frequencies to levels observed for the parental strain [9]. In addition, a global analysis of gene expression in yeast TCA cycle mutants [10] revealed a set of ~20 genes that exhibited elevated expression in idhΔ gene disruption mutants, depressed expression in cit1 Δ mutants, and intermediate expression in idhΔ cit1 Δ double mutant strains. Finally, a yeast mutant lacking IDH was found to exhibit reduced levels of mitochondrially-encoded proteins [11], which are hydrophobic protein components of the electron transport chain.…”