Cardiolipin is essential for organization of complexes III and IV into a supercomplex in intact yeast mitochondria

Abstract

Digitonin extracts of mitochondria from cardiolipin-containing (wild type) and cardiolipin-lacking (crd1Delta mutant) Saccharomyces cerevisiae subjected to colorless native polyacrylamide gel electrophoresis in the presence of 0.003% digitonin displayed a supercomplex composed of homodimers of complexes III and IV in the former case but only the individual homodimers in the latter case. To avoid treatment with any detergent or dye, we compared organization of the respiratory chain in intact mitochondria from wild type and cardiolipin-lacking cells by using a functional analysis developed previously for the study of the organization of the respiratory chain of S. cerevisiae (Boumans, H., Grivell, L. A., and Berden, J. A. (1998) J. Biol. Chem. 273, 4872-4877). Dependence of the kinetics of NADH oxidation via complexes III, IV, and cytochrome c on the concentration of the complex III-specific inhibitor antimycin A was studied. A linear relationship between respiratory activity and saturation of complex III with antimycin A was obtained for wild type mitochondria consistent with single functional unit kinetics of the respiratory chain. Under the same conditions, cardiolipin-lacking mitochondria displayed a hyperbolic relationship indicating cytochrome c pool behavior. No release of cytochrome c from cardiolipin-lacking mitochondria or mitoplasts under our standard experimental conditions was detected. Identical cytochrome c pool behavior was observed for both wild type and cardiolipin-lacking mitochondria in the presence of a chaotropic agent, which disrupts the interaction between respiratory complexes. The results demonstrate that cardiolipin is essential for association of complexes III and IV into a supercomplex in intact yeast mitochondria.

Fig. 1. Lack of supercomplex formation in crd1Δ cells as detected by CN-PAGE

DL1 (CRD1, lanes 1 and 3) and YZD5 (crd1Δ, lanes 2 and 4) were grown in YPEG at 30 °C and harvested in the exponential phase of growth. Mitochondria were isolated, solubilized by digitonin, and displayed (75 μg of protein/lane) by CN-PAGE, as described under “Experimental Procedures.” Digitonin (0.003%) was added to the gel. The specificity of the antibodies used (Cox3p, lanes 1 and 2; Cobp, lanes 3 and 4) in each blot is indicated. The mobility of standard proteins of the indicated weight average M_r is on the left.

Fig. 2. Titration of the Q₂H₂:cytochrome c oxidoreductase (complex III) activity with antimycin A in mitochondria from wild type and crd1Δ cells

Mitochondria were isolated from both wild type (DL1, CRD1, ●) and cardiolipin-lacking cells (YZD5, crd1Δ, ■) grown at 30 °C in YPEG to exponential phase. Q₂H₂:cytrochrome c oxidoreductase activity of isolated mitochondria was determined with different concentrations of antimycin A, as described under “Experimental Procedures.” V/Vo represents the complex III activity at each antimycin A concentration relative to the activity with no antimycin A. The x-axis represents the fractional saturation by antimycin A with complete inhibition set to 1.

Fig. 3. Titration of NADH oxidation with antimycin A

Respiration of isolated mitochondria from wild type (DL1, CRD1) (A) and cardiolipin-lacking cells (YZD5, crd1Δ) (B) was measured in standard buffer (0.6 M sorbitol, 25 mM potassium P_i (pH 7.0), 1 mM EDTA, 1 mM MgCl₂, 0.5 mM NADH) (●) and in the standard buffer supplemented with 50 mM trichloroacetic acid (pH 7.0) (■) in the presence of different concentrations of antimycin A. Respiratory activities of both types of mitochondria ranged from 320 to 350 nmol of O₂/mg/min for the preparations used in this figure.

Fig. 4. Analysis of cytochrome c release from mitochondria and mitoplasts of wild type and crd1Δ cells

Mitochondria (A) and mitoplasts (B) isolated from both wild type and crd1Δ cells were incubated in the respiration buffer (0.6 M sorbitol, 25 mM potassium P_i, 1 mM EDTA, 1 mM MgCl₂ (pH 7.0), 0.5 mM NADH and methanol (4 μl/3 ml)) for the indicated time. Samples were processed as described under “Experimental Procedures.” SDS-PAGE and Western blotting were used to detect the presence of cytochrome c in the supernatants (released cytochrome c) and pellets (mitochondrial- or mitoplast-bound cytochrome c).