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...

It was found recently that a diabetes-associated protein in insulin-sensitive tissue (DAPIT) is associated with mitochondrial ATP synthase. Here, we report that the suppressed expression of DAPIT in DAPIT-knockdown HeLa cells causes loss of the population of ATP synthase in mitochondria. Consequently, DAPIT-knockdown cells show smaller mitochondrial ATP synthesis activity, slower growth in normal medium, and poorer viability in glucose-free medium than the control cells. The mRNA levels of ␣-and ␤-subunits of ATP synthase remain unchanged by DAPIT knockdown. These results indicate a critical role of DAPIT in maintaining the ATP synthase population in mitochondria and raise an intriguing possibility of active role of DAPIT in cellular energy metabolism.ATP synthase is a ubiquitous enzyme found in plasma membranes of bacteria, inner membranes of mitochondria, and chloroplast thylakoid membranes. It is a rotary motor enzyme that synthesizes ATP by proton flow along the gradient of electrochemical potential of proton across membranes (1-10). ATP synthase consists of two major portions: a membraneprotruding globular F 1 domain that acts as an ATPase when isolated, and a membrane-embedded F o domain that acts as a proton channel when F 1 is removed. In the typical bacterial ATP synthase, the subunit composition of F 1 is ␣ 3 ␤ 3 ␥␦⑀ and that of F o is ab 2 c n (n, different among species). The proton flow drives rotation of the oligomer ring of c-subunits (c-ring) that makes the ␥⑀ central stalk rotate with it, generating torque and conformational changes in the catalytic ␣ 3 ␤ 3 domain of F 1 to synthesize ATP. To prevent the dragged rotation, the second stalk, made of ␦ and b 2 , connects the stators in F 1 (␣ 3 ␤ 3 ) and F 0 (a-subunit). In addition to these core subunits, mitochondrial ATP synthase contains several other subunits, that is, d, e, f, g, A6L, F 6 , and mitochondrial ⑀, which is different from bacterial ⑀ (11). Further, it was found recently that two proteins with unknown function, DAPIT 3 (diabetes-associated protein in insulin-sensitive tissue) and MLQ (6.8-kDa proteolipid), are associated with mammalian mitochondrial ATP synthase (12,13).In this report, we focused on DAPIT, a small (58-amino acid), basic (pI ϭ 10.4) protein with a single putative transmembrane segment (14). DAPIT was first recognized as a gene product whose mRNA level increased during skeletal muscle growth in rats and decreased by treatment with streptozotocin, a drug that induces diabetes (15). DAPIT is associated with ATP synthase in a stoichiometric manner but is not found in ATP synthase purified in the absence of phospholipids (12). Also, DAPIT is prone to dissociation from ATP synthase in the presence of relatively strong detergents, but ATP synthase without DAPIT still retains the same ATP hydrolysis activity (13). Orthologs of DAPIT and MLQ are found in vertebrates and invertebrates but not in yeast and other fungi (12). From these observations, DAPIT has been assumed to have some minor roles that are dispensable for the c...

Background: IF1 inhibits ATPase activity of mitochondrial F o F 1 -ATP synthase. Results: Although IF1 alleviates ischemic injury, the cell can grow normally, manage to maintain ATP levels, and keep mitochondria morphology intact without IF1. Conclusion: IF1 helps ATP homeostasis, but activated glycolysis can cover deficiency of IF1. Significance: Integrated regulation of mitochondrial ATP synthesis is crucial for metabolic dynamism.

IF1 is an endogenous inhibitor protein of mitochondrial ATP synthase. It is evolutionarily conserved throughout all eukaryotes and it has been proposed to play crucial roles in prevention of the wasteful reverse reaction of ATP synthase, in the metabolic shift from oxidative phosphorylation to glycolysis, in the suppression of ROS (reactive oxygen species) generation, in mitochondria morphology and in haem biosynthesis in mitochondria, which leads to anaemia. Here, we report the phenotype of a mouse strain in which IF1 gene was destroyed. Unexpectedly, individuals of this IF1-KO (knockout) mouse strain grew and bred without defect. The general behaviours, blood test results and responses to starvation of the IF1-KO mice were apparently normal. There were no abnormalities in the tissue anatomy or the autophagy. Mitochondria of the IF1-KO mice were normal in morphology, in the content of ATP synthase molecules and in ATP synthesis activity. Thus, IF1 is not an essential protein for mice despite its ubiquitous presence in eukaryotes.

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