Fig 4.

Rcf2, like Rcf1, associates with the cytochrome bc₁-COX supercomplex. (A and B) Rcf1_His and Rcf2_His were expressed in their respective null mutant strains (Δrcf1+Rcf1_His and Δrcf2+Rcf2_His), and mitochondria were isolated and, together with wild-type control mitochondria analyzed in parallel, were solubilized in digitonin and subjected to Ni-NTA chromatography. Following SDS-PAGE of purified material, the gel was subjected to silver staining (A) or Western blotting and immunodecoration (B). (A) The identity of subunits verified by immunoblotting of parallel samples is indicated. (B) The levels of Rcf2_His and Rcf1_His were directly compared using an antibody against the His epitope (indicated by α-His). The presence of Rcf1 in the Rcf2_His purified material was analyzed in a parallel sample using antibodies directed against the Rcf1 protein. Samples were further analyzed as for Fig. 1B. (C) Rcf1_His (top) and Rcf2_His (bottom) were purified under digitonin solubilization conditions from the indicated isolated mitochondria. Δrcf1,2 indicates Δrcf1 Δrcf2 mitochondria. Samples were further analyzed as described for Fig. 1B, and Cox3 and Core1 were immunodecorated. The recovery of Rcf1_His and Rcf2_His was monitored using an antibody directed against the His epitope.