FIGURE 3.

Supramolecular association of ETC III and IV into III2IV2 supercomplex is hampered in taz1Δ, which could be reversed by YME1 deletion. A, representative BN-PAGE gel that was transferred to nitrocellulose membrane and probed with an antibody against Cox2. Briefly, mitochondria were isolated and solubilized with dodecylmaltoside from the indicated strains and separated by BN-PAGE in the presence of Coomassie Blue dye in the gel as described under “Materials and Methods.” Proteins were then transferred to a PVDF membrane that was probed with antisera against Cox2 to ascertain the formation of III2IV2 and III2IV ETC supercomplexes. Cox2 is one of the subunits of ETC IV; hence, antiserum against it is effective in assessing the efficiency of complex IV to associate with III to form higher order supercomplexes. TAZ1 (lane 2) deletion increases the accumulation of free IV monomer with a concomitant reduction in the formation of III2IV2 supercomplex. Interestingly, deletion of YME1 in taz1Δ yeast was able to restore III2IV2 supercomplex formation, preventing the accumulation of IV monomer. B, to assess the level of Cox2, equal amounts of mitochondrial extracts were separated under denaturing conditions (SDS-PAGE), and an antibody against Cox2 was used to determine the levels of Cox2 by Western blot. PI, phosphatidylinositol; PS, phosphatidylserine; PC, phosphatidylcholine.