A sensitive, simple assay of mitochondrial ATP synthesis of cultured mammalian cells suitable for high-throughput analysis

Fujikawa, Makoto; Yoshida, Masasuke

doi:10.1016/j.bbrc.2010.09.089

Cited by 30 publications

(45 citation statements)

References 9 publications

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“…3A). Then, the ability of mitochondria to synthesize ATP was compared by MASC assay (see "Materials and Methods") (22). The results show that mitochondria in IF1-KD cells can synthesize ATP at nearly the same rate as control cells (Fig.…”

Section: Resultsmentioning

confidence: 96%

“…The sequence for IF1-KD was 5Ј-GAGCACAGAGTAGAGA ACAACTGGCAGCT-3Ј. Preparation and transduction of retroviruses were carried out as described (22).…”

Section: Methodsmentioning

confidence: 99%

“…The protein concentration of the lysate was also measured by BCA protein assay (Thermo Scientific). ATP synthesis was measured by MASC (Mitochondrial Activity of Storeptolysin O-permeabilized Cell) assay (22,23). Briefly, cells were passaged to a 96-well white plate and incubated overnight.…”

Section: Methodsmentioning

confidence: 99%

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Assessing Actual Contribution of IF1, Inhibitor of Mitochondrial FoF1, to ATP Homeostasis, Cell Growth, Mitochondrial Morphology, and Cell Viability

Fujikawa

Imamura

Nakamura

et al. 2012

Journal of Biological Chemistry

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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.

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Section: Resultsmentioning

confidence: 96%

“…The sequence for IF1-KD was 5Ј-GAGCACAGAGTAGAGA ACAACTGGCAGCT-3Ј. Preparation and transduction of retroviruses were carried out as described (22).…”

Section: Methodsmentioning

confidence: 99%

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Assessing Actual Contribution of IF1, Inhibitor of Mitochondrial FoF1, to ATP Homeostasis, Cell Growth, Mitochondrial Morphology, and Cell Viability

Fujikawa

Imamura

Nakamura

et al. 2012

Journal of Biological Chemistry

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“…To determine whether modulation of CcO activity by Higd1a affects ATP production, we performed the mitochondrial activity of streptolysin O permeabilized cells (MASC) assay, a sensitive means of measuring the mitochondrial ATP production rate in semi-intact cells (18). Indeed, Higd1a knockdown caused a significant decrease in the ATP production rate relative to the control (Fig.…”

Section: Higd1a Causes Structural Changes In Cco and Influences The Amentioning

confidence: 99%

Higd1a is a positive regulator of cytochrome c oxidase

Hayashi¹,

Asano²,

Shintani³

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

113

101

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Cytochrome c oxidase (CcO) is the only enzyme that uses oxygen to produce a proton gradient for ATP production during mitochondrial oxidative phosphorylation. Although CcO activity increases in response to hypoxia, the underlying regulatory mechanism remains elusive. By screening for hypoxia-inducible genes in cardiomyocytes, we identified hypoxia inducible domain family, member 1A (Higd1a) as a positive regulator of CcO. Recombinant Higd1a directly integrated into highly purified CcO and increased its activity. Resonance Raman analysis revealed that Higd1a caused structural changes around heme a, the active center that drives the proton pump. Using a mitochondria-targeted ATP biosensor, we showed that knockdown of endogenous Higd1a reduced oxygen consumption and subsequent mitochondrial ATP synthesis, leading to increased cell death in response to hypoxia; all of these phenotypes were rescued by exogenous Higd1a. These results suggest that Higd1a is a previously unidentified regulatory component of CcO, and represents a therapeutic target for diseases associated with reduced CcO activity.cytochrome c oxidase | oxidative phosphorylation | resonance Raman spectroscopy | ATP | oxygen

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“…4B). To resolve this issue and directly measure the rate of ATP production in mitochondria, we used a semiintact cell system called the mitochondrial activity of streptolysin O permeabilized cells (MASC) assay (25). In this assay, we permeabilized the plasma membrane to wash out any cytosolic components, such as creatine and glycolytic substrates, but left the mitochondria intact.…”

Section: G0s2 Binds To F O F 1 -Atp Synthase But Not Other Oxphos Promentioning

confidence: 99%

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

Kioka¹,

Kato²,

Fujikawa

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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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...

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A sensitive, simple assay of mitochondrial ATP synthesis of cultured mammalian cells suitable for high-throughput analysis

Cited by 30 publications

References 9 publications

Assessing Actual Contribution of IF1, Inhibitor of Mitochondrial FoF1, to ATP Homeostasis, Cell Growth, Mitochondrial Morphology, and Cell Viability

Assessing Actual Contribution of IF1, Inhibitor of Mitochondrial FoF1, to ATP Homeostasis, Cell Growth, Mitochondrial Morphology, and Cell Viability

Higd1a is a positive regulator of cytochrome c oxidase

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

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