Evaluation of intramitochondrial ATP levels identifies G0/G1 switch gene 2 as a positive regulator of oxidative phosphorylation

Abstract

The oxidative phosphorylation (OXPHOS) system generates most of the ATP in respiring cells. ATP-depleting conditions, such as hypoxia, trigger responses that promote ATP production. However, how OXPHOS is regulated during hypoxia has yet to be elucidated. In this study, selective measurement of intramitochondrial ATP levels identified the hypoxia-inducible protein G0/G1 switch gene 2 (G0s2) as a positive regulator of OXPHOS. A mitochondria-targeted, FRET-based ATP biosensor enabled us to assess OXPHOS activity in living cells. Mitochondria-targeted, FRET-based ATP biosensor and ATP production assay in a semiintact cell system revealed that G0s2 increases mitochondrial ATP production. The expression of G0s2 was rapidly and transiently induced by hypoxic stimuli, and G0s2 interacts with OXPHOS complex V (FoF1-ATP synthase). Furthermore, physiological enhancement of G0s2 expression prevented cells from ATP depletion and induced a cellular tolerance for hypoxic stress. These results show that G0s2 positively regulates OXPHOS activity by interacting with FoF1-ATP synthase, which causes an increase in ATP production in response to hypoxic stress and protects cells from a critical energy crisis. These findings contribute to the understanding of a unique stress response to energy depletion. Additionally, this study shows the importance of assessing intramitochondrial ATP levels to evaluate OXPHOS activity in living cells.

Keywords: energy metabolism; live-cell imaging.

Fig. 1.

Establishment of a sensitive method to assess OXPHOS activity in living cells. (A) YFP/CFP emission ratio plots of (Upper) Mit-ATeam and (Lower) Cyto-ATeam fluorescence in cardiomyocytes. Various concentrations (0.001, 0.01, 0.1, 1, and 10 μg/mL) of oligomycin A or DMSO (control) were added at 5 min (arrowhead; n = 3). (B) Representative sequential YFP/CFP ratiometric pseudocolored images of (Upper) Mit-ATeam and (Lower) Cyto-ATeam in cardiomyocytes. Oligomycin A (0.01 μg/mL) was added at 5 min. (Scale bars: 20 μm.) (C) YFP/CFP emission ratio plots of Mit-ATeam and Cyto-ATeam fluorescence in cardiomyocytes (n = 10). (D) Representative sequential YFP/CFP ratiometric pseudocolored images of (Upper) Mit-ATeam and (Lower) Cyto-ATeam in cardiomyocytes. Cells were exposed to 1% hypoxia from the time point 30 min. All of the measurements were normalized to the YFP/CFP emission ratio at 0 min. Data are represented as the means ± SEMs. (Scale bars: 20 μm.)

Fig. 2.

G0s2, a hypoxia-inducible protein, affects intramitochondrial ATP concentration in cardiomyocytes. (A) Sequential YFP/CFP ratiometric pseudocolored images of Mit-ATeam fluorescence in cardiomyocytes expressing (Upper) shRNAs for LacZ (shLacZ) or (Lower) G0s2 (shG0s2). Oligomycin A (1 μg/mL) was added at the end of the time-lapse imaging to completely inhibit ATP synthesis. The indicated time represents the period after adenovirus infection. (B) Representative YFP/CFP ratiometric pseudocolored images of Mit-ATeam fluorescence in cardiomyocytes expressing the indicated adenovirus for 24 h. (Scale bar: A and B, 20 μm.) (C) The bar graph shows the mean YFP/CFP emission ratio of Mit-ATeam fluorescence in cardiomyocytes expressing shLacZ (n = 30), shG0s2 #1 (n = 30), shG0s2 #2 (n = 29), and shG0s2 #2 + G0s2 WT (n = 32) for 24 h. All of the measurements were normalized to the average of the control cells (shLacZ). ***P < 0.001. (D) Gene expression value plots of G0s2 (red line) and VEGF-α (Vegfa; black line) levels in cardiomyocytes under hypoxic conditions (1% O₂). Each value was compared with the level of Actb expression (n = 3). Values represent the means ± SEMs. (E) Immunoblotting of the G0s2 expression in cardiomyocytes under hypoxic conditions (1% O₂).

Fig. 3.

Overexpression of G0s2 before hypoxia rescues the decline of mitochondrial ATP production during hypoxia. (A) Sequential YFP/CFP ratiometric pseudocolored images of Mit-ATeam fluorescence in cardiomyocytes expressing (Upper) G0s2 WT or (Lower) LacZ during hypoxia and reoxygenation. (Scale bar: 20 μm.) (B) YFP/CFP emission ratio plots of Mit-ATeam fluorescence in cardiomyocytes expressing G0s2 WT (n = 20) or LacZ (n = 19) during hypoxia and reoxygenation. All of the measurements were normalized to the ratio at time 0 and compared between cardiomyocytes with G0s2 WT and LacZ at each time point. (C) The bar graph shows the cell viability of cardiomyocytes overexpressing G0s2 under hypoxic conditions. Cardiomyocytes expressing either LacZ or G0s2 WT were cultured under normoxic or hypoxic conditions for 18 h (n = 8). The asterisks denote statistical significance comparing G0s2 with LacZ. Data are represented as the means ± SEMs. n.s., not significant. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 4.

G0s2 interacts with the F_oF₁-ATP synthase in mitochondria. (A) Immunoprecipitation (IP) of G0s2-Flag in cardiomyocytes. Cell lysates from cardiomyocytes expressing G0s2-Flag or LacZ were immunoprecipitated with an anti-Flag antibody. (B) IP of endogenous G0s2 in cardiomyocytes. Endogenous G0s2 was induced by hypoxia and immunoprecipitated using an anti-G0s2 antibody. C, OXPHOS complex; F_oF₁, F_oF₁-ATP synthase. *IgG light chain. (C) IP of F_oF₁-ATP synthase in cardiomyocytes under normoxic or hypoxic conditions. Cell lysates from cardiomyocytes cultured under normoxia or hypoxia for 4 h were immunoprecipitated with an antibody against the whole F_oF₁-ATP synthase complex or a control IgG. *Nonspecific band. (D) IP of G0s2 mutants expressed in cardiomyocytes. Cell lysates were immunoprecipitated with an anti-Flag antibody. (E) Immunostained images of hypoxia-stimulated (4 h) cardiomyocytes with anti-G0s2 (green) and anti–F_oF₁-ATP synthase β-subunit (red) antibodies. (Scale bars: 20 μm.)

Fig. 5.

G0s2 enhances the mitochondrial ATP production rate. (A and B) MASC assay of (A) permeabilized HeLa cells expressing the indicated plasmids or (B) cardiomyocytes expressing the indicated adenovirus in the presence (dotted lines) or absence (solid lines) of 1 μg/mL oligomycin A (Oli A). Upper shows the ATP production plots, and Lower shows the mean ATP production rates between 0 and 10 min. (A) n = 12. (B) Solid lines, n = 12; dotted lines, n = 8. (C) MASC assay of permeabilized cardiomyocytes pretreated with hypoxia. Cells expressing the indicated adenovirus were pretreated with or without hypoxia for 4 h. After the pretreatment, the cells were permeabilized under room air conditions followed by MASC assay in the presence (dotted lines; n = 8) or absence (solid lines; n = 12) of 1 μg/mL Oli A. Upper shows the ATP production plot, and Lower shows the mean ATP production rate between 0 and 10 min. (D) The bar graph represents the cell viability of G0s2-depleted cardiomyocytes under hypoxic conditions. Cardiomyocytes expressing shLacZ or shG0s2 (#2) were cultured under normoxic or hypoxic conditions for 18 h. (E and F) The OCR in cardiomyocytes expressing shLacZ and shG0s2 (#2) under basal conditions and in response to the indicated mitochondrial inhibitors (n = 8). FCCP, carbonyl cyanide-4-(trifluoromethoxy)-phenylhydrazone. Data are represented as the means ± SEMs. n.s., not significant. **P < 0.01; ***P < 0.001.