Fig. 5.

BN-PAGE and SDS-PAGE analyses of mitochondrial proteins. Experiments were performed with mitochondria isolated from strains WT+pØ, taz1Δ+ pØ, taz1Δ+pTAZ1 and taz1Δ+pODC1 grown as described in Fig. 2B. (A) BN-PAGE analyses of ATP synthase. The left panel shows a BN-gel of mitochondrial proteins (50 µg) dissolved with 2 g of digitonin per g of proteins, where ATP synthase is revealed by its ATPase activity as dimers (V2), monomers (V1) or free F₁ particles (F₁). In the right panel, ATP synthase was analyzed in samples (50 µg) obtained after treating the mitochondria with increasing concentrations of digitonin, from 0.5 to 3.0 g per g of protein. After their electrophoretic separation and transfer onto a nitrocellulose membrane, the proteins were probed with antibodies against the γ-F₁ subunit (ATP3) of ATP synthase. (B) BN-PAGE analysis of CIV and CIII. Mitochondrial proteins were extracted with 10 g digitonin per g of protein, separated by BN-PAGE (100 µg per lane), transferred onto a nitrocellulose membrane, and probed with antibodies against the COX2 subunit of CIV or the cytochrome b subunit of CIII. (C) BN-PAGE and SDS-PAGE analyses of CII. On the left panel, mitochondrial proteins were extracted with digitonin (10 g/g), separated by BN-PAGE, and assayed for in-gel complex II activity; in the right panel, 100 µg of total protein extracts were separated by SDS-PAGE, transferred onto a nitrocellulose membrane and probed antibodies against SDH2 and ADE13. (D) SDS-PAGE analyses. 100 µg of total mitochondrial proteins were separated by SDS-PAGE, transferred onto a nitrocellulose membrane and probed with antibodies against the indicated proteins. The right panel shows a quantification which as been done using ImageJ. Levels of COX2, ATPα-F₁ and cytochrome c are related to the mitochondrial protein Por1p. The data are all relative to WT.