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. 2012 Apr 27;287(18):14569-78.
doi: 10.1074/jbc.M111.302786. Epub 2012 Mar 6.

cAMP-induced mitochondrial compartment biogenesis: role of glutathione redox state

Edgar D Yoboue  1 , Eric Augier, Anne Galinier, Corinne Blancard, Benoît Pinson, Louis Casteilla, Michel Rigoulet, Anne Devin
Affiliations

cAMP-induced mitochondrial compartment biogenesis: role of glutathione redox state

Edgar D Yoboue et al. J Biol Chem. .

Abstract

Cell fate and proliferation are tightly linked to the regulation of the mitochondrial energy metabolism. Hence, mitochondrial biogenesis regulation, a complex process that requires a tight coordination in the expression of the nuclear and mitochondrial genomes, has a major impact on cell fate and is of high importance. Here, we studied the molecular mechanisms involved in the regulation of mitochondrial biogenesis through a nutrient-sensing pathway, the Ras-cAMP pathway. Activation of this pathway induces a decrease in the cellular phosphate potential that alleviates the redox pressure on the mitochondrial respiratory chain. One of the cellular consequences of this modulation of cellular phosphate potential is an increase in the cellular glutathione redox state. The redox state of the glutathione disulfide-glutathione couple is a well known important indicator of the cellular redox environment, which is itself tightly linked to mitochondrial activity, mitochondria being the main cellular producer of reactive oxygen species. The master regulator of mitochondrial biogenesis in yeast (i.e. the transcriptional co-activator Hap4p) is positively regulated by the cellular glutathione redox state. Using a strain that is unable to modulate its glutathione redox state (Δglr1), we pinpoint a positive feedback loop between this redox state and the control of mitochondrial biogenesis. This is the first time that control of mitochondrial biogenesis through glutathione redox state has been shown.

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Figures

SCHEME 1.
SCHEME 1.
Evolution of respiratory state values when the mitochondrial phosphorylating steady state changes (A) or does not change (B). If the in situ steady state gets closer to a non-phosphorylating state (state 4), the RSV decreases, whereas if the in situ steady state gets closer to a phosphorylating steady state, the RSV increases (A; amount of mitochondria constant). The “no changes” condition in the in situ mitochondrial steady state is associated with no changes in RSV (B; increase in the amount of mitochondria).
FIGURE 1.
FIGURE 1.
A, adenine nucleotide variations induced by cAMP. Adenine nucleotides were measured as described under “Experimental Procedures.” Results are means of at least three separate experiments ± S.D. (error bars). B, ATP/ADP and ATP/ADP·Pi ratio variations induced by cAMP. Pi was measured as described under “Experimental Procedures.” Results are means of at least three separate experiments ± S.D.
FIGURE 2.
FIGURE 2.
A, cellular cytochrome content variations induced by cAMP. Cytochrome content was determined as described under “Experimental Procedures.” Results are mean ± S.D. (error bars) of at least three measurements performed on three independent cell cultures. ●, cytochrome cc1; ♢, cytochrome b; ×, cytochrome aa3. B, mitochondrial enzymatic activities variations induced by cAMP. Enzymatic activities (i.e. citrate synthase (CS), cytochrome oxidase (COX), and glucose-6-phosphate dehydrogenase (G6PDH)) were measured as described under “Experimental Procedures.” Results are mean ± S.D. of at least three measurements performed on three independent cell cultures. ■, citrate synthase; ♢, cytochrome oxidase; ●, glucose-6-phosphate dehydrogenase. C, cellular cytochrome content, respiratory rates, and enzymatic activities in the presence of increasing concentrations of cAMP. Cytochromes, respiratory rates (JO2), and enzymatic activities were measured on yeast cells as described under “Experimental Procedures.”
FIGURE 3.
FIGURE 3.
Electron micrographs of cells upon cAMP treatment. Ultrastructural studies were performed as described under “Experimental Procedures.” The cells shown are representative of the average cell in the considered conditions. cAMP concentration (mm) is shown. Mitochondria are highlighted in white. Av, average number of mitochondria (assessed in about 20 cells/experimental condition).
FIGURE 4.
FIGURE 4.
A, activity of the transcription factors HAP2/3/4/5. The activity of the transcription factors HAP2/3/4/5 was assessed with a widely used reporter gene, pCYC1-lacZ(pLG669Z), as described under “Experimental Procedures.” Results are mean ± S.D. (error bars) of at least three measurements performed on three independent cell cultures. B, the amount of the co-activator Hap4p upon cAMP treatment. Western blot was performed as described under “Experimental Procedures.” The result is representative of at least four blots. Input was assessed using a commercial antibody directed against phosphoglycerate kinase (PGK1; Invitrogen).
FIGURE 5.
FIGURE 5.
Origin of HAP4p increase upon cAMP treatment. A, HAP4 mRNA was assessed as described under “Experimental Procedures.” The result is representative of three such experiments B, HAP4p turnover was assessed in the presence of cycloheximide (CHX) (0.4 mg/ml) for the indicated times (4 and 8 min). A residual amount of Hap4p is expressed as a percentage of the non-cycloheximide-treated condition (t = 0 min). Western blot was performed as described under “Experimental Procedures.” The result is representative of at least four blots. Input was assessed using a commercial antibody directed against phosphoglycerate kinase (PGK; Invitrogen).
FIGURE 6.
FIGURE 6.
A, glutathione redox state variations induced by cAMP. GSH and GSSG were measured as described under “Experimental Procedures.” Results are means of at least three separate experiments ± S.D. (error bars). B, relationship between the glutathione redox state and the cellular phosphate potential. Values for the cellular phosphate potential are from Fig. 1B.
FIGURE 7.
FIGURE 7.
A, respiratory rates in the wild type and Δglr1 strains. Cells were grown as described under “Experimental Procedures,” and respiratory rates were assessed in the absence or presence of 5 mm GSH, 5 mm ascorbate (ASC), and 5 μm lipoate (lipo). Error bars, S.D. B, the amount of the co-activator Hap4p in the wild type and Δglr1 strains. Western blot was performed as described under “Experimental Procedures.” The result is representative of at least four blots. Input was assessed using a commercial antibody directed against phosphoglycerate kinase (Pgk1p; Invitrogen).
FIGURE 8.
FIGURE 8.
Kinetics of Hap4p decrease reversion by reduced glutathione in the Δglr1 strain. Western blot was performed as described under “Experimental Procedures.” The blot is representative of three such experiments. Cells were grown as described under “Experimental Procedures.” When added, reduced glutathione was 5 mm. Input was assessed using a commercial antibody directed against phosphoglycerate kinase (Pgk1p; Invitrogen).
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