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. 2012 May 25;287(22):18781-7.
doi: 10.1074/jbc.M112.345793. Epub 2012 Apr 9.

Assessing actual contribution of IF1, inhibitor of mitochondrial FoF1, to ATP homeostasis, cell growth, mitochondrial morphology, and cell viability

Makoto Fujikawa  1 , Hiromi Imamura, Junji Nakamura, Masasuke Yoshida
Affiliations

Assessing actual contribution of IF1, inhibitor of mitochondrial FoF1, to ATP homeostasis, cell growth, mitochondrial morphology, and cell viability

Makoto Fujikawa et al. J Biol Chem. .

Abstract

F(o)F(1)-ATP synthase (F(o)F(1)) synthesizes ATP in mitochondria coupled with proton flow driven by the proton motive force (pmf) across membranes. It has been known that isolated IF1, an evolutionarily well conserved mitochondrial protein, can inhibit the ATP hydrolysis activity of F(o)F(1). Here, we generated HeLa cells with permanent IF1 knockdown (IF1-KD cells) and compared their energy metabolism with control cells. Under optimum growth conditions, IF1-KD cells have lower cellular ATP levels and generate a higher pmf and more reactive oxygen species. Nonetheless, IF1-KD cells and control cells show the same rates of cell growth, glucose consumption, and mitochondrial ATP synthesis. Furthermore, contrary to previous reports, the morphology of mitochondria in IF1-KD cells appears to be normal. When cells encounter sudden dissipation of pmf, the cytoplasmic ATP level in IF1-KD cells drops immediately (~1 min), whereas it remains unchanged in the control cells, indicating occurrence of futile ATP hydrolysis by F(o)F(1) in the absence of IF1. The lowered ATP level in IF1-KD cells then recovers gradually (~10 min) to the original level by consuming more glucose than control cells. The viability of IF1-KD cells and control cells is the same in the absence of pmf. Thus, IF1 contributes to ATP homeostasis, but its deficiency does not affect the growth and survival of HeLa cells. Only when cells are exposed to chemical ischemia (no glycolysis and no respiration) or high concentrations of reactive oxygen species does IF1 exhibit its ability to alleviate cell injury.

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Figures

FIGURE 1.
FIGURE 1.
A, the amount of expressed IF1 in mitochondria. B, the amount of assembled FoF1 in mitochondria. Mitochondrial fractions from control and IF1-KD cells were subjected to SDS-PAGE (A) and stained with antibodies against α-, β-, and ϵ-subunits of FoF1 and with anti-IF1 antibody or blue-native PAGE (B) and stained with anti-β-subunit antibody. Arrowhead indicates the position of dimer of FoF1.
FIGURE 2.
FIGURE 2.
A, the amounts of ATP contained in IF1-KD and the control cells. The amounts of ATP relative to the amount of total proteins are shown. B, mitochondrial membrane potential (Δψ) estimated from TMRE fluorescence intensities averaged from 400 cells (p < 0.01). C, ROS levels in IF1-KD (n = 24) and control (n = 16) cells estimated from fluorescence intensities of dichlorofluorescein (DCF) (p < 0.01). Experimental details are as described under “Materials and Methods.” The error bars indicate S.D.
FIGURE 3.
FIGURE 3.
A, cell growth of IF1-KD and control cells. Cells were grown in optimum growth conditions (DMEM). Relative cell numbers are plotted. B, ATP synthesis activity of mitochondria in IF1-KD and control cells measured by MASC assay. Synthesized ATP was monitored with luciferase. Oligomycin (oligo) was added for the negative controls. C, typical electron microscopic images of IF1-KD and control cells. The scale bars are 500 nm. D, typical images of IF1-KD and control cells stained with TMRE. The scale bars are 50 μm. Experimental details are as described under “Materials and Methods.”
FIGURE 4.
FIGURE 4.
A–C, change of cytoplasmic [ATP] upon loss of pmf by addition of CCCP. [ATP] was monitored by an ATP-sensing probe, ATeam, in which the yellow fluorescence domain (YFP) comes closer to the cyan fluorescence domain (CFP) when the middle domain binds ATP, causing FRET. Left panels, typical FRET images of cells, representing by ratio of YFP/cyan fluorescence domain (1.6 (blue) to 2.2 (red)); right panels, changes of FRET values averaged from randomly chosen cells. Cell numbers of control and IF1-KD cells used for averaging were 17 and 23 (A), 18 and 13 (B), and 9 and 17 (C). The error bars indicate S.D. Diamonds, IF1-KD cells; squares, control cells. At time zero, CCCP (A; 50 μm), CCCP+oligomycin (B; 10 μg/ml), or CCCP+2-deoxyglucose (C; 10 mm) was added. The scale bar in A is 20 μm.
FIGURE 5.
FIGURE 5.
Shown are glucose consumption (A) and viability of cells (B) cultured in normal DMEM for 48 h in the absence or presence of 15 μm CCCP. Experimental details are as described under “Materials and Methods.”
FIGURE 6.
FIGURE 6.
A, viability of IF1-KD and control cells in 10 mm 2-deoxyglucose and 5 mm KCN (chemical ischemia). Cells were exposed to chemical ischemia for 0, 10, 30, and 120 min, and living cells were counted (n = 6). The double asterisk indicates that the p value is <0.01. B, viability of IF1-KD and control cells under ROS-producing conditions. Cells were cultured in 0, 1, and 5 mm paraquat for 2 days, and living cells were counted (n = 6). The living cell numbers in the absence of paraquat are set at 100%.
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