A Transcriptional Switch in the Expression of Yeast Tricarboxylic Acid Cycle Genes in Response to a Reduction or Loss of Respiratory Function

Abstract

The Hap2,3,4,5p transcription complex is required for expression of many mitochondrial proteins that function in electron transport and the tricarboxylic acid (TCA) cycle. We show that as the cells' respiratory function is reduced or eliminated, the expression of four TCA cycle genes, CIT1, ACO1, IDH1, and IDH2, switches from HAP control to control by three genes, RTG1, RTG2, and RTG3. The expression of four additional TCA cycle genes downstream of IDH1 and IDH2 is independent of the RTG genes. We have previously shown that the RTG genes control the retrograde pathway, defined as a change in the expression of a subset of nuclear genes, e.g., the glyoxylate cycle CIT2 gene, in response to changes in the functional state of mitochondria. We show that the cis-acting sequence controlling RTG-dependent expression of CIT1 includes an R box element, GTCAC, located 70 bp upstream of the Hap2,3,4,5p binding site in the CIT1 upstream activation sequence. The R box is a binding site for Rtg1p-Rtg3p, a heterodimeric, basic helix-loop-helix/leucine zipper transcription factor complex. We propose that in cells with compromised mitochondrial function, the RTG genes take control of the expression of genes leading to the synthesis of alpha-ketoglutarate to ensure that sufficient glutamate is available for biosynthetic processes and that increased flux of the glyoxylate cycle, via elevated CIT2 expression, provides a supply of metabolites entering the TCA cycle sufficient to support anabolic pathways. Glutamate is a potent repressor of RTG-dependent expression of genes encoding both mitochondrial and nonmitochondrial proteins, suggesting that it is a specific feedback regulator of the RTG system.

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

“…The RTG pathway results in elevated levels of CIT2 mRNA in yeast cells lacking mitochondrial DNA [34]. Also, most mitochondrial protein expression is under global control of the HAP2-5 transcription system but, as mitochondrial function becomes compromised, expression of CIT1, ACO1, and IDH1/2 genes was found to switch to control by the RTG pathway [35], suggesting a cellular need for regulation of glutamate synthesis under this condition. Another indication of a direct correlation is the shared phenotype of glutamate auxotrophy resulting from disruption of individual genes encoding RTG regulatory proteins [36,37], from disruption of ACO1 [38], and from co-disruption of genes encoding major cellular isozymes of citrate synthase [5] or of isocitrate dehydrogenase [25].…”

“…Thus, we find little correlation between levels of CIT2 and cellular levels of glutamate, suggesting either that CIT2 is not a reliable reporter for the RTG pathway or that the RTG pathway may be responsive to metabolites other than glutamate. However, since our studies have focused on CIT2 protein levels whereas previous analyses of the RTG response involved quantitative analyses of mRNA levels [33,35], it is important that future studies investigate different levels of the response. ACO1 and IDH have both been described to have bifunctional roles in yeast mitochondria.…”

RTG-dependent Mitochondria-to-Nucleus Signaling Is Regulated by MKS1 and Is Linked to Formation of Yeast Prion [URE3]

“…Rtg2p plays a pivotal role in the retrograde pathway, because it is both a sensor of the functional state of mitochondria and is required for the activation of RTG-dependent gene expression by promoting the cytoplasmic-to-nuclear translocation of Rtg1p and Rtg3p (Sekito et al., 2000). In addition, Rtg2p is required for the partial dephosphorylation of Rtg3p associated with its nuclear accumulation.The retrograde pathway senses the functional state of mitochondria via the level of glutamate (Liu and Butow, 1999;Sekito et al, 2000;Epstein et al, 2001). Because glutamate is a potent repressor of RTG-dependent gene expres- sion, the retrograde pathway is likely to be important for glutamate homeostasis.…”

“…Because glutamate is a potent repressor of RTG-dependent gene expres- sion, the retrograde pathway is likely to be important for glutamate homeostasis. In cells with compromised or dysfunctional mitochondria, the expression of CIT1, ACO1, IDH1, and IDH2 (genes encoding the enzymes catalyzing the first three steps in the TCA cycle that lead to the production of ␣-ketoglutarate, the direct precursor of glutamate) is controlled by the RTG genes (Liu and Butow, 1999); in cells with robust mitochondrial function, expression of these genes is under control of the HAP transcription complex (Forsburg and Guarente, 1989;Rosenkrantz et al, 1994). A connection between the retrograde pathway and nitrogen metabolism has recently been uncovered by the finding that the target of rapamycin (TOR) kinase pathway in yeast also involves the RTG genes (Komeili et al, 2000;Shamji et al, 2000).…”

“…Ureidosuccinic acid was added at a final centration of 200 g/ml. Rapamycin was dissolved in 10% Tween 20/90% ethanol and added at a final concentration of 0.2 g/ml.The ϩ and derivative of strain PSY142 (MAT␣ leu2 lys2 ura3) and their rtg2⌬ and rtg3⌬ derivatives have been previously described, as well as the derivatives of these strains containing a single chromosomal copy of a CIT2-lacZ reporter gene (Liu and Butow, 1999;Sekito et al, 2000). Similar derivatives of strain MLY42 (MAT␣ ura3 leu2), a ⌺1278b derivative, and CC34 (MAT␣ trp1 ade2 leu2 his3 ura2::HIS3) were constructed as done for strain PSY142.…”