FIG. 1.

Respiratory supercomplexes and ATP synthase dimers in N. crassa wild-type mitochondria. (A and B) Digitonin-solubilized crude mitochondria were analyzed by BN-PAGE (A) and CN-PAGE (B) in the first dimension. In both panels, results are shown for in-gel activity of COX (upper panels) and subsequent 2D SDS-PAGE (silver stained) to resolve the subunits of all OXPHOS complexes and their supercomplexes (middle panels) and 2D BN-PAGE (Coomassie stained) with 0.02% DDM in the cathode buffer to dissociate the OXPHOS supercomplexes into their individual complexes (lower panels). For mass calibration, digitonin-solubilized bovine heart mitochondria were used: individual complexes I to V (130 to 1,000 kDa) and supercomplexes a to e (I₁III₂IV_0-4; 1,500 to 2,300 kDa). Additionally, 31 subunits of all five OXPHOS complexes separated in 2D BN-SDS-PAGE (A) were verified by MALDI-MS, which are marked in detail in Fig. 2 for the sake of clarity. The OXPHOS supercomplexes were assigned according to their subunit compositions and apparent molecular masses. Besides ATP synthase monomers and dimers (V₁ and V₂), the individual respiratory complexes I to IV as well as the respiratory supercomplexes I_xIII_yIV_z, I₁III₂, I₁IV₁, III₂IV₂, III₂IV₁, and IV₂ are indicated. Additionally, a subunit of complex IV (spot 43; Table 1) in the 2D SDS-PAGE (A) as well as the separated complex IV monomers, complex III dimers, and complex I monomers, which constitute the supercomplexes IV₂, III₂IV₁, III₂IV₂, I₁IV₁, and I_xIII_yIV_z, in the 2D BN-PAGE (A and B) are marked by arrowheads. Note that supercomplexes I₁IV₁ and III₂IV₂ have very similar apparent masses. Similarly, the mobility of dimeric complex IV (IV₂) is only slightly higher than that of complex III.