Fig. 1. Saccharomyces cerevisiae complex IV is quantitatively associated with complex III. The amounts of complex III in free dimeric form (III₂) or associated with one (III₂IV₁) or two complex IV monomers (III₂IV₂) vary with growth conditions. (A) BN-PAGE of mitochondria from strain W303-1A grown on glucose for 24 h, and two-dimensional resolution by SDS–PAGE. The subunits of complexes III and IV were assigned after N-terminal protein sequencing. (B) Growth on lactate increases the amount of III₂IV₂ complex, and decreases the fraction of free complex III. (C) Prolonged growth on glucose reduces the amounts of the III₂IV₂ complex, and increases the fraction of free complex III. V_Dim and V_Mon are dimeric and monomeric ATP synthase (complex V), respectively.

Fig. 2. Functional association of complexes III and IV in digitonin-solubilized yeast mitochondria. Detergent dependence (A) of ubiquinol cytochrome c reductase (mol cytochrome c reduced/mol complex III/s), (B) of cytochrome c oxidase (mol cytochrome c oxidized/mol complex IV/s) and (C) of ubiquinol oxidase (complexes III+IV; mol DBH oxidized/mol complex III/s) after addition of 5 µM yeast cytochrome c. Arrows indicate the solubilization range. A decrease in ubiquinol oxidase rates after solubilization by DDM indicates a separation of complexes III and IV. No decay of rates and no dissociation of complex III–IV occur after solubilization by digitonin (cf. text).

Fig. 3. Complex I from bovine heart mitochondria is associated with complexes III and IV. (A) BN-PAGE of bovine heart mitochondria after solubilization by digitonin. Most complex I and complex III was found assembled into two major supercomplexes a and b, and two minor supercomplexes c and d. The 200 kDa mass differences indicate the presence of varying copy numbers of monomeric complex IV. (B) Solubilization by DDM was used as a reference for quantitative solubilization of all OXPHOS complexes.

Fig. 4. Bovine complex I has binding sites for complex III and complex IV, and ATP synthase can be isolated as a dimer. (A) Complexes a–d and dimeric ATP synthase (V_Dim) separated by BN-PAGE (Figure 3A; 4 g/g digitonin) were dissociated by 2D BN-PAGE using addition of DDM to the cathode buffer. Direct interaction of complexes I and III was apparent from the dissociation of complex a (I₁III₂) into monomeric complex I and dimeric complex III. Complexes b–d comprised complex IV in addition (silver staining required for c and d). Dimeric complex V dissociated into the monomeric form V_Mon. (B) The line of complex b from 2D BN-PAGE (brackets in Figure 4A) was resolved by 3D SDS–PAGE and Coomassie Blue stained. The stoichiometry within this complex (I₁III₂IV₁) was determined by densitometric analysis using purified complexes I, III and IV for calibration. (C) 2D BN-PAGE similar to (A), but using addition of Triton X-100 to the cathode buffer dissociated complex b (I₁III₂IV₁) in a different way (two additional spots larger than complex I; less complex IV dissociated). (D) The line of complex b from 2D BN-PAGE (brackets in Figure 4C) was resolved by 3D SDS–PAGE. The additional spots were identified as undisociated I₁III₂IV₁ complex and as I₁IV₁ complex. COXI and COXII, subunits of cytochrome c oxidase (complex IV).

Fig. 5. Identification of dimeric complex IV, direct complex III–IV interactions, and complex e (I₁III₂IV₄) comprising four copies of complex IV. (A) BN-PAGE of bovine heart mitochondria using various Triton X-100/protein ratios for solubilization. Low Triton X-100(1.0 g/g) selectively solubilized complexes V and II. High Triton X-100 (2.4 g/g) solubilized all OXPHOS complexes quantitatively, but retained supercomplexes a (I₁III₂) and c (I₁III₂IV₂). Intermediate Triton X-100 (1.4 g/g) retained supercomplexes a–e (I₁III₂IV_X) comprising0–4 copies of complex IV. (B) 2D BN-PAGE of the boxed sector from (A). Arrowheads mark the lines of dissociated complexes a–e. The identification of dimeric complex IV in lines c–e, but not in b (only one copy of complex IV), indicates the presence of complex IV dimers within the supercomplexes. (C) BN-PAGE of bovine heart mitochondria using high (1.6 g/g) and low (0.6 g/g) DDM/protein ratios for solubilization. Low DDM solubilizes all OXPHOS complexes quantitatively, but retains ∼20% of total complex V in dimeric form (V_Dim). (D) 2D BN-PAGE of the lane from (C) using low DDM. Arrowheads mark the lines of complexes that were retained after first dimension BN-PAGE but dissociated by 2D BN-PAGE.

Fig. 6. Functional association of complexes I and III in digitonin-solubilized bovine heart mitochondria. (A) Dergent dependence of NADH ubiquinone reductase (mol NADH oxidized/mol complex I/s). Arrows indicate the solubilization range. (B) Detergent dependence of ubiquinol cytochrome c reductase (mol cytochrome c reduced/mol complex III/s). (C) Detergent dependence of NADH cytochrome c reductase (complexes I+III; mol cytochrome c/mol complex I/s). A decrease in NADH cytochrome c reductase rates after solubilization by DDM indicates a separation of complexes I and III. The absence of a decay of rates after complete solubilization by digitonin indicates a retained association of complexes I and III (cf. text).

Fig. 7. Model for of a network of mammalian respiratory chain complexes. This model is based on the identification of direct interactions of complexes and on the overall 1:3:6 stoichiometry of complex I:III:IV by Hatefi (1985). It postulates two copies of a large building block comprising complexes I, III and IV, and one smaller building block without complex I. Comparison of the different solubilization by DDM and Triton X-100 indicated that these building blocks can interact to form a network of respiratory chain complexes.