Rcf1 can independently associate with Cox3 and the COX and cytochrome bc₁ complexes. (A and B) Mitochondria isolated from wild-type (WT) cells and Δrcf1 cells harboring Rcf1_His and control wild-type mitochondria were solubilized in 2% digitonin (A) or 0.25% Triton X-100 (B), subjected to Ni-NTA purification, and further analyzed as described for Fig. 1B. (C) In organello protein synthesis in the presence of [³⁵S]methionine was performed in mitochondria isolated from wild-type control cells or from Rcf1_His-containing cells. Following Triton X-100 solubilization, Ni-NTA purification was performed, followed by SDS-PAGE and autoradiography. (D) Rcf1_His was purified from Δrcf1 or Δcox18 mitochondria under digitonin solubilization conditions and further analyzed as described for Fig. 1B.

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.

Physical association with and supporting activity of the COX enzyme are a conserved feature of Hig1 proteins. (A) Ni-NTA purification of Rcf1_His and Rcf1ΔC_His following solubilization with Triton X-100. See Fig. 1B for further details. (B) COX enzyme activity measurements in mitochondria isolated from wild-type and Δrcf1 Δrcf2 strains harboring Rcf1_His or the C. elegans Hig1 homolog M05D6.5_His, as described for Fig. 3B. (C) Purification of Rcf1_His and M05D6.5_His from the Δrcf1 Δrcf2 strain performed under Triton X-100 solubilization conditions and analyzed by SDS-PAGE and silver staining (top) or in parallel by immunodecoration for Cox2, Cox6, and Cox12 subunits (bottom). The positions of COX subunits, Rcf1_His, and M05D6.5_His (and a smaller proteolytic fragment of this protein, f-M05D6.5_His) on the silver-stained gel, verified by immunodecoration, are indicated.

Rcf1, an inner membrane protein, associates with the cytochrome bc₁-COX supercomplex. (A) Wild-type mitochondria were solubilized with digitonin (1%) and subjected to BN-PAGE electrophoresis followed by a second-dimension SDS-PAGE. The resulting Western blot was immunodecorated with antibodies directed against subunits of the COX (Cox5 and Cox2), cytochrome bc₁ (Core2), and F₁F_o-ATP synthase (Atp4) complexes, AAC, and Rcf1. The positions of the dimeric and monomeric ATP synthase (V_dim and V_mon), cytochrome bc₁-COX supercomplex (III-IV), and 150- and 200-kDa markers are indicated. (B) Mitochondria were isolated from strains harboring either His-tagged Aac2, subunit e (Su e), F₁β, or cytochrome c₁ (Cytc₁) proteins or from control wild-type cells (i.e., lacking a His-tagged protein), solubilized with digitonin (0.5%), and subjected to Ni-NTA purification, Western blotting, and immunodecoration. “Total” indicates 5% of the solubilized mitochondria; “Bound” indicates the Ni-NTA-eluted material. (C) Depiction of the N_out-C_out orientation of S. cerevisiae (S.c.) Rcf1 and C. elegans (C.e.) M05D6.5 (Hig1 homolog) in the inner membrane (IM), with N and C tails exposed to the intermembrane space (IMS). (D) Isolated wild-type mitochondria (0.1 mg/ml) were extracted by alkaline treatment using 0.1 M carbonate buffer at pH 11.5. Control mitochondria (pH 7.5) were resuspended in 0.6 M sorbitol, 20 mM HEPES-KOH, pH 7.5. Following extraction, samples were subjected to ultracentrifugation, and the resulting membrane pellet and supernatant (after TCA precipitation) were analyzed by SDS-PAGE, Western blotting, and immunodecoration for Rcf1 and control proteins. (E) Proteinase K (20 μg/ml) treatment was performed on intact mitochondria (Mitoch.) or mitochondria which had been subjected to hypotonic swelling (Mitoplast) or Triton X-100 detergent lysis (T X-100) on ice. Samples were subjected to SDS-PAGE and Western blotting. Cytochrome c peroxidase (Ccpo), a soluble IMS marker protein, displays inherent protease stability and hence is not degraded by the proteinase K treatment in the presence of Triton X-100. D-LD, d-lactate dehydrogenase, an IM protein with a large C-terminal domain exposed to the IMS. (F) Wild-type mitochondria were hypotonically swollen, subjected to treatment with V8 protease (20 μg/ml), and further processed as described for panel B. OxaI and the fragment (f) of OxaI resulting from V8 degradation of the N-tail of OxaI exposed to the IMS are indicated.

RCF1 and RCF2 genetically interact and together are required to support the COX enzyme activity. (A) Serial 10-fold dilutions of wild-type (WT), Δrcf1, Δrcf2, or Δrcf1 Δrcf2 cells were spotted on YP plates containing glucose (YPD) or glycerol (YPG) and grown at 30°C. (B) The COX and cytochrome bc₁ relative specific enzyme activities were determined and expressed as a percentage of the value for the wild-type control. The averages of three replicate measurements are given. (C) Serial 10-fold dilutions of wild-type (WT) cells and Δrcf1 Δrcf2 cells expressing Rcf1_His or Rcf2_His or not (−) were spotted on YP plates containing glucose (YPD) or glycerol supplemented with 0.1% galactose (YPG + 0.1% Gal) and grown at 30°C.

The presence of Rcf1 and Rcf2 is required to secure the correct assembly of the COX complex and its association with the cytochrome bc₁ complex. (A) Steady-state levels of the OXPHOS subunits in the mitochondria (50 μg) isolated from strains with the indicated genotypes. Δrcf1,2 indicates Δrcf1 Δrcf2 mitochondria. Tim44 was used as a loading control. Rip1, Rieske Fe-S protein. (B) Mitochondria isolated from WT and Δrcf1 Δrcf2 strains were solubilized in Triton X-100 and analyzed by sucrose gradient centrifugation, as described in Materials and Methods. The fractions corresponding to the bottom and top of the gradient are indicated. Following SDS-PAGE and Western blotting, immunodecoration was performed against the indicated COX subunits and Hsp60, a control marker protein. The positions of the control marker complexes monomeric ATP synthase (500 kDa) and cytochrome b₂ (220 kDa) are indicated. (C) Mitochondria isolated from strains with the indicated genotypes were solubilized with digitonin (2%) and subjected to BN-PAGE analysis, Western blotting, and immunodecoration with Rip1, Cox12, and Cox13 antisera.

Rcf1 can be chemically cross-linked to AAC proteins. (A) Chemical cross-linking using MBS was performed on mitochondria isolated from wild-type (WT) [rho⁺] and [rho⁰] strains and the Δrcf1 strain. Following SDS-PAGE and Western blotting, immunodecoration with Rcf1 antiserum was performed. As the Rcf1 antibody recognizes some additional protein bands, the Δrcf1 mitochondria were analyzed in parallel to distinguish the Rcf1-specific adducts observed in WT mitochondria. ***, **, and *, 45-, 33-, and 28-kDa Rcf1-containing adducts, respectively. (B) Cross-linking of Rcf1 was performed in mitochondria isolated from the wild type and from the Δaac2 strain or the Δaac2 strain harboring _HisAac2 protein in a [rho⁺] or [rho⁰] background (top) or from the Δaac2 Δaac1 strain harboring Aac1_His or Aac3_His (bottom). Samples were further analyzed as described for panel A. Only the area of the gel encompassing the 45-kDa Rcf1-containing adduct (***) is shown in both cases. (C) MBS cross-linking of Rcf1_His and Rcf1ΔC_His was performed and further analyzed as described for panel A. The resulting Western blot was immunodecorated with antiserum which was directed against the His epitope but which also recognizes a nonspecific (Non Sp.) band of approximately 15 kDa. *** and **, Rcf1-containing adducts.