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 (F o F 1 -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 F o F 1 -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.energy metabolism | live-cell imaging M aintaining cellular homeostasis and activities requires a stable energy supply. Most eukaryotic cells generate ATP as their energy currency mainly through the mitochondrial oxidative phosphorylation (OXPHOS) system. The OXPHOS system consists of five large protein complex units (i.e., complexes I-V), comprising more than 100 proteins. In this system, oxygen (O 2 ) is essential as the terminal electron acceptor for complex IV to finally produce the proton-motive force that drives the ATPgenerating molecular motor complex V (F o F 1 -ATP synthase).Hypoxia causes the depletion of intracellular ATP and triggers adaptive cellular responses to help maintain intracellular ATP levels and minimize any deleterious effects of energy depletion. Although the metabolic switch from mitochondrial respiration to anaerobic glycolysis is widely recognized (1-4), several recent reports have shown that hypoxic stimuli unexpectedly increase OXPHOS efficiency as well (5-7). In other words, cells have adaptive mechanisms to maintain intracellular ATP levels by enhancing OXPHOS, particularly in the early phase of hypoxia, in which the O 2 supply is limited but still remains. However, the mechanism by which OXPHOS is regulated during this early hypoxic phase is still not fully understood.Revealing the mechanism of this fine-tuned regulation of OXPHOS requires accurate and noninvasive measurements of OXPHOS activity. Although researchers have established methods to measure OXPHOS activity, precise measurement, especially in living cells, is still difficult. Measuring the intracellul...

Higd1a is a positive regulator of cytochrome c oxidase

“…FRET-based measurement of mitochondrial ATP concentration in cardiomyocytes was measured as previously described (8,33). Briefly, FRET signal was measured in cardiomyocytes infected with adenovirus encoding mit-AT1.03 with an Olympus IX-81 inverted fluorescence microscope (Olympus) using a PL APO 60×, 1.35 N.A., oil immersion objective lens (Olympus).…”

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Differential gene expression profiling of gastric intraepithelial neoplasia and early-stage adenocarcinoma

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