Effect of CsA on ATPase activity in wild-type and CyPD-null mouse liver mitochondria. Freshly prepared liver mitochondria from wild-type (WT, A) and CyPD-null mitochondria (KO, B) were suspended in buffer A containing 10 mm KH₂PO₄ and treated with the indicated concentrations of CsA. Aliquots were withdrawn to determine (i) the amount of ATPase present by immunoblotting with anti-F₁ α/β Ab (30 μg of protein in lanes 1 and 15 μg of protein in lanes 2, upper part of both panels) and (ii) ATP hydrolysis rate in the presence of 10 μm alamethicin (lower part of both panels). Oligomycin sensitivity was higher than 85%, and no difference through the samples was observed. Reported values are mean ± S.E. (error bars) of three independent experiments. In both panels, asterisks denote p < 0.05 relative to the 100% value measured in wild-type mitochondria.

CsA stimulates both ATP hydrolysis and synthesis, and displaces CyPD from MgATP-SMP. A, MgATP-SMP suspended in buffer A supplemented with 10 mm KH₂PO₄ were treated for 15 min at room temperature with the indicated concentrations of CsA. Aliquots were then used to determine the rate of oligomycin-sensitive ATP hydrolysis at 37 °C, which was always higher than 85% of the total. The rate in the absence of CsA was taken as 100%, and reported values are mean ± S.E. (error bars) of three independent experiments. B, MgATP-SMP were treated with CsA as in panel A, and ATP synthesis was determined at 30 °C with an ATPlite 1step luminescence detection system as described (30). The rate in the absence of CsA was taken as 100%, and reported values are mean ± S.E. (error bars) of three independent experiments. C, MgATP-SMP were treated with CsA as in panel A and subjected to SDS-PAGE followed by immunodetection using Ab against CyPD and the α/β subunits or F₁ (left), IF1 (center), or ANT (right). Peak areas of immunodetected bands were quantified as in Fig. 2A. The ratio between the peak area of CyPD and that of each of the other proteins was determined, 100% being the ratio observed in the absence of CsA. The results are representative of three independent experiments.

CsA treatment increases the ATPase activity of both intact mitochondria and digitonin extracts, where it displaces CyPD. A, freshly prepared mitochondria suspended in buffer A containing 10 mm KH₂PO₄ were treated with the indicated concentrations of CsA, and aliquots were withdrawn to determine spectrophotometrically the maximal rate of oligomycin-sensitive ATP hydrolysis (which was always higher than 85%) in the presence of 10 μm alamethicin. Values are mean ± S.E. (error bars) of three independent experiments. B, identical aliquots of mitochondria were incubated in phosphate-free buffer C (open bars) or in PBS (closed bars) with the indicated concentrations of CsA and supplemented with 1% (w/v) digitonin followed by determination of the ATPase activity. Oligomycin sensitivity was higher than 85%, and no difference through the samples was observed. Values are mean ± S.E. (error bars) of three independent experiments. C, digitonin extracts obtained using PBS buffer as in panel B were subjected to immunoprecipitation with anti-complex V antibodies followed by SDS-PAGE and immunodetection with antibodies against CyPD, F₁ α/β, IF1, and CyP. Each immunodetected band was analyzed by densitometry with the ImageQuant software, and the ratio between the peak area of CyPD and that of the corresponding α/β subunits in the absence of CsA was taken as 100% (graph to the right; experiments are representative of three independent determinations).

CyPD interacts with mitochondrial ATP synthase through the OSCP and b and d subunits. A, freshly prepared bovine heart mitochondria were solubilized in buffer B with 2% (w/v) DDM. ATP synthase (double arrow) was separated by one-dimensional BNE (1D BNE), stained by Coomassie Blue G (lane 1), and identified by in-gel ATPase assay (lane 2). The excised bands with enzymatic activity were subjected to two-dimensional SDS-PAGE (2D SDS-PAGE) and silver-stained (lane 3) or blotted on nitrocellulose for immunodetection using polyclonal Ab against F₁ α/β subunits and monoclonal Ab against CyPD (lane 4). B, mitochondria were suspended in PBS buffer, treated with 1% (w/v) DDM, and immunoprecipitated with anti-complex V Ab followed by SDS-PAGE (lane 1) and immunodetection using anti-α/β or CyPD Ab (lane 2). No signal was detected when affinity-purified IgG from preimmune rabbit or mouse serum were used (results not shown). C, mitochondria suspended in PBS buffer were treated with vehicle or with the indicated concentrations of DTBP at room temperature for 15 min prior to immunoprecipitation with anti-complex V Ab. The immunoprecipitates were incubated for 30 min at 37 °C in the absence or presence of 150 mm DTT as indicated and separated by SDS-PAGE in the absence of reducing agents. Immunodetection was performed with Ab against OSCP or b or d subunits or against CyPD as indicated. Each reported blot is representative of at least two independent experiments.

P_i and CsA modulate CyPD association to ATP synthase in intact mitochondria. A, freshly prepared mitochondria were suspended in phosphate-free saline (lanes 1 and 3) or in PBS (lanes 2 and 4), treated with 1% (w/v) DDM (lanes 1 and 2) or 1% (w/v) digitonin (lanes 3 and 4), and immunoprecipitated with anti-complex V Ab followed by SDS-PAGE and immunoblotting with anti-α/β or CyPD Ab (representative results of one experiment out of three are shown). Each immunodetected band was analyzed by densitometry with the ImageQuant software, and the ratio between the peak area of CyPD and that of the corresponding α/β subunits was measured and expressed relative to the ratio obtained in the absence of P_i, which was taken as 100% (graph on the right, values are mean ± S.E. (error bars) of three independent experiments). B, mitochondria were suspended in phosphate-free buffer A (see “Experimental Procedures”) alone or supplemented with 1 or 10 mm KH₂PO₄. ATP synthase oligomers were extracted with 1% (w/v) digitonin in phosphate-free buffer B, separated by one-dimensional BNE and two-dimensional SDS-PAGE, and blotted onto nitrocellulose followed by immunodetection with anti-α/β or CyPD Ab; CyPD association was quantified as in panel A (reported values are the mean ± S.E. (error bars) of three independent experiments). C, mitochondria in buffer A supplemented with 1 mm KH₂PO₄ were treated with vehicle or with 0.8 μm CsA for 15 min at room temperature, centrifuged at 17,000 × g for 15 min at 4 °C, solubilized in buffer B containing 2% (w/v) DDM or 1% (w/v) digitonin, and subjected to one-dimensional BNE. DDM extracts (yielding monomers of ATP synthase) and digitonin extracts (yielding oligomers of ATP synthase) were identified by in-gel ATPase assay, excised, subjected to two-dimensional SDS-PAGE, and blotted onto nitrocellulose followed by immunodetection with anti-α/β or CyPD Ab. Lanes 1 and 2, extraction with DDM; lanes 3 and 4, extraction with digitonin (representative results of one experiment out of three are shown). The peak area of each immunodetected band was analyzed by densitometry with the ImageQuant software, and the ratio between the peak area of CyPD and that of the corresponding α/β subunits was measured. Data were then expressed relative to the ratio obtained in lane 1, which was taken as 100% (graph on the right, where numbers refer to the lanes of the same panel; values are mean ± S.E. (error bars) of three independent experiments).