Figure 4. Distinct functional roles of the three tightly bound CLs.

(A–C) ADP/ATP exchange: The efflux of matrix ATP was detected with isolated mitochondria as NADPH formation (A340; absorbance at 340 nm) occurring coupled with an in vitro glycolysis reaction which contained glucose, hexokinase, and glucose-6-phosphate dehydrogenase. The reaction was initiated by adding ADP. The measurement was performed in the presence of 5 mM malate and 5 mM pyruvate (+Mal/Pyr). Where indicated, WT mitochondria were treated with 5 μM CATR prior to the efflux reaction. (A) The linear part of the initial velocity for the ATP efflux was plotted and curve fitting performed by nonlinear regression (mean with SEM, n = 6, biological replicates). Plots of aac2Δ and WT are repeated in all panels. (B) The initial velocity following the addition of 33 μM ADP was presented as scatter plots (mean with SEM, n = 6, biological replicates). (C) Fitted Km and Vmax values were obtained using the Michaelis–Menten equation (mean). The range of determined values is shown in brackets (n.d., not detected). (D) Oxygen consumption rate (OCR) in isolated mitochondria with sequential injections of the respiratory substrate (NADH or succinate), ADP, oligomycin, and CCCP was measured using Seahorse XF96e FluxAnalyzer. (E, F) ADP-stimulated respiration and respiratory control ratio (RCR) of WT and Aac2 mutant mitochondria under NADH (E) and succinate (F) were plotted. RCR was obtained by dividing OCR of ADP-stimulated respiration by that of basal respiration (see also Fig. S5). WT mitochondria was treated with 50 μM CATR before the measurement. Mean with SEM, n = 21–35, 3–5 biological replicates with 5–7 technical replicates. Significant differences obtained by two-way ANOVA followed by Tukey’s multiple comparisons test are shown as * for comparison with WT and † for comparison between pocket mutants; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Source data are available online for this figure.