Figure 2.
Respiratory Chain Supercomplexes Are Disassembled during Cristae Remodeling
(A) RCR of mitochondria energized with 5 mM/2.5 mM glutamate/malate (GLU/MAL) or 10 mM succinate (SUCC) treated for the indicated times with cBID. Data represent average ± SEM of five independent experiments. Inset, RCR values normalized to t = 0.
(B) Experiments were as in (A), except that mitochondria were incubated with the indicated mutants of cBID for 15 min. Data represent average ± SEM of four independent experiments. The asterisk denotes p < 0.05 in a paired sample Student’s t test versus untreated.
(C) BNGE of mitochondrial OXPHOS protein from DKO MEFs transduced as indicated and, after 2 days, metabolically labeled for 2 hr and lysed after 24 hr. Equal amounts of protein (100 μg) were separated by BNGE, and radioactivity was detected in the fixed and dried gels for 1 week. RCC and RCS of the respiratory chain are indicated.
(D) Densitometric analysis of the autoradiographic RCS/complex V signal ratio. Data represent average ± SEM of three independent experiments performed as in (E). The asterisk denotes p < 0.05 in a paired sample Student’s t test versus untreated.
(E) 2D BN/SDS PAGE analysis of mitochondrial OXPHOS proteins from DKO MEFs transduced as indicated, metabolically labeled for 2 hr, and lysed after 24 hr. Equal amount (50 μg) of proteins were separated in native condition, and then the lanes were excised and proteins separated by a second dimension SDS-PAGE. The gels were dried and the signal was detected following 1 week of exposure. RCC and RCS of the respiratory chain as well as the single-labeled proteins are indicated. Boxes and circles indicate RCS and cytochrome b, respectively.
(F) Densitometric analysis of the ratio of autoradiographic signal between supercomplex (boxed) and complex assembled cytochrome b (circled). Data represent average ± SEM of three independent experiments. The asterisk denotes p < 0.05 in a paired sample Student’s t test versus untreated.
(G) BNGE analysis of OXPHOS proteins in mitochondria from DKO MEFs transduced as indicated. Equal amounts (100 μg) of proteins were separated in native conditions, transferred onto PVDF membranes, and probed with the indicated antibodies. RCC and RCS are indicated.
(H) RCR of DKO mitochondria energized with 5 mM/2.5 mM GLU/MAL or 10 mM SUCC treated for 15 min with the indicated cBID mutants. Data represent average ± SEM of four independent experiments. The asterisk denotes p < 0.05 in a paired sample Student’s t test versus untreated.
See also Figure S2.