Evidence that the assembly of the yeast cytochrome bc1 complex involves the formation of a large core structure in the inner mitochondrial membrane

Abstract

The assembly status of the cytochrome bc(1) complex has been analyzed in distinct yeast deletion strains in which genes for one or more of the bc(1) subunits were deleted. In all the yeast strains tested, a bc(1) sub-complex of approximately 500 kDa was found when the mitochondrial membranes were analyzed by blue native electrophoresis. The subsequent molecular characterization of this sub-complex, carried out in the second dimension by SDS/PAGE and immunodecoration, revealed the presence of the two catalytic subunits, cytochrome b and cytochrome c(1), associated with the noncatalytic subunits core protein 1, core protein 2, Qcr7p and Qcr8p. Together, these bc(1) subunits build up the core structure of the cytochrome bc(1) complex, which is then able to sequentially bind the remaining subunits, such as Qcr6p, Qcr9p, the Rieske iron-sulfur protein and Qcr10p. This bc(1) core structure may represent a true assembly intermediate during the maturation of the bc(1) complex; first, because of its wide distribution in distinct yeast deletion strains and, second, for its characteristics of stability, which resemble those of the intact homodimeric bc(1) complex. By contrast, the bc(1) core structure is unable to interact with the cytochrome c oxidase complex to form respiratory supercomplexes. The characterization of this novel core structure of the bc(1) complex provides a number of new elements clarifying the molecular events leading to the maturation of the yeast cytochrome bc(1) complex in the inner mitochondrial membrane.

Figure 1

Characterization of 500 kDa bc₁ sub-complexes in the yeast deletion strains lacking Qcr9p, ISP or Bcs1p. In panel A, mitochondrial membranes from wild type (WT), ΔQCR9, ΔISP and ΔBCS1 strains were solubilized with 1% digitonin and analyzed by BN-PAGE, as described in Experimental Procedures. The protein complexes were detected by immunoblotting with antisera specific for core protein 1 and core protein 2. The calibration markers are indicated on the right side of the gel blot. In panel B, the mitochondrial membranes from the three yeast deletion strains were analyzed by SDS-PAGE after BN-PAGE in the first dimension. The gel was blotted and probed with antibodies to the proteins indicated on the left side of the gel blot. Cyt c₁, cytochrome c₁; cyt b, cytochrome b; ISP, Rieske iron-sulfur protein; core 1, core protein 1; core 2, core protein 2; Qcr6p, Qcr7p, Qcr8p, Qcr9p and Qcr10p, subunits 6, 7, 8, 9 and 10 of the yeast bc₁ complex, respectively. Cox1p and Cox6bp, subunits 1 and 6b of the yeast cytochrome c oxidase complex, respectively.

Figure 1

Characterization of 500 kDa bc₁ sub-complexes in the yeast deletion strains lacking Qcr9p, ISP or Bcs1p. In panel A, mitochondrial membranes from wild type (WT), ΔQCR9, ΔISP and ΔBCS1 strains were solubilized with 1% digitonin and analyzed by BN-PAGE, as described in Experimental Procedures. The protein complexes were detected by immunoblotting with antisera specific for core protein 1 and core protein 2. The calibration markers are indicated on the right side of the gel blot. In panel B, the mitochondrial membranes from the three yeast deletion strains were analyzed by SDS-PAGE after BN-PAGE in the first dimension. The gel was blotted and probed with antibodies to the proteins indicated on the left side of the gel blot. Cyt c₁, cytochrome c₁; cyt b, cytochrome b; ISP, Rieske iron-sulfur protein; core 1, core protein 1; core 2, core protein 2; Qcr6p, Qcr7p, Qcr8p, Qcr9p and Qcr10p, subunits 6, 7, 8, 9 and 10 of the yeast bc₁ complex, respectively. Cox1p and Cox6bp, subunits 1 and 6b of the yeast cytochrome c oxidase complex, respectively.

Figure 2

Resolution of mitochondrial membranes from WT and ΔQCR10 yeast strains by BN-PAGE and SDS-PAGE. In Panel A, mitochondrial membranes were analyzed by BN-PAGE, as described in Fig. 1A. Panel B shows the SDS-PAGE of the subunit 10 deletion strain membranes after BN-PAGE in the first dimension. The gel was blotted and probed with antibodies to the proteins indicated on the left side of the gel blot. Panel C shows the SDS-PAGE analysis of the mitochondrial membranes from WT and ΔQCR10 yeast strains followed by Western blotting with antibodies to the subunits of the bc₁ complex indicated on the left side of the blots.

Figure 3

Resolution of mitochondrial membranes from WT and ΔISP/ΔQCR9 yeast strains by BN-PAGE and SDS-PAGE. In Panel A, mitochondrial membranes were analyzed by BN-PAGE, as described in Fig. 1A. Panel B shows the SDS-PAGE of the ΔISP/ΔQCR9 deletion strain membranes after BN-PAGE in the first dimension. The gel was blotted and probed with antibodies to the proteins indicated on the left side of the gel blot.

Figure 4

Resolution of mitochondrial membranes from WT, ΔISP/ΔQCR10 andΔQCR9/ΔQCR10 yeast strains by BN-PAGE and SDS-PAGE. In Panel A, mitochondrial membranes were analyzed by BN-PAGE, as described in Fig. 1A. Panels B show the SDS-PAGE of the ΔISP/ΔQCR10 (left) and the ΔQCR9/ΔQCR10 (right) deletion strain membranes after BN-PAGE in the first dimension. The gel was blotted and probed with antibodies to the proteins indicated on the left side of the gel blots.

Figure 5

Resolution of mitochondrial membranes from WT and ΔQCR6/ΔQCR9 yeast strains by BN-PAGE and SDS-PAGE. In Panel A, mitochondrial membranes were analyzed by BN-PAGE, as described in Fig. 1A. Panel B shows the SDS-PAGE of the ΔQCR6/ΔQCR9 deletion strain membranes after BN-PAGE in the first dimension. The gel was blotted and probed with antibodies to the proteins indicated on the left side of the gel blot.

Figure 6

Resolution of mitochondrial membranes from WT and ΔISP/ΔQCR6 yeast strains by BN-PAGE and SDS-PAGE. In Panel A, mitochondrial membranes were analyzed by BN-PAGE, as described in Fig. 1A. Panel B shows the SDS-PAGE of the ΔISP/ΔQCR6 deletion strain membranes after BN-PAGE in the first dimension. The gel was blotted and probed with antibodies to the proteins indicated on the left side of the gel blot.

Figure 7

Stability of the 500 kDa bc₁ sub-complex in different conditions of solubilization. In Panel A, mitochondrial membranes from WT (lanes 1 and 2) and ΔQCR9 (lanes 3 and 4) yeast strains were solubilized with 1% digitonin (lanes 1 and 3) or 1% Triton X-100 (lanes 2 and 4) and protein complexes were analyzed by BN-PAGE, as described in Fig. 1A. In Panel B, mitochondrial membranes from the subunit 9 deletion strain were solubilized with 1% digitonin or 1% Triton X-100 and incubated for 10 min at different temperatures ranging from 0°C to 25°C. After this treatment, mitochondrial lysates were analyzed by BN-PAGE, as described in Fig. 1A. The immunodecorated bc₁ sub-complex of approximately 500 kDa was quantified as described under Experimental Procedures and shown in Panel B; the amount of the 500 kDa bc₁ sub-complex solubilized with 1% digitonin at 0°C was set to 100% (control).

Figure 8

Schematic model depicting the putative pathway of assembly of the yeast cytochrome bc₁ complex. De novo assembly occurs via the association of bc₁ sub-complexes (cytochrome b/Qcr7p/Qcr8p and cytochrome c₁/core protein 1/core protein 2) in a large core structure which also includes the chaperone protein Bcs1p. This core structure is then able to sequentially bind the remaining bc₁ subunits in a process which eventually leads to the formation of the homodimeric bc₁ complex in the inner mitochondrial membrane. As Qcr10p is not essential for the dimerization of the bc₁ complex, it is represented with dashed outlines. Indeed, the bc₁ complex apparently can dimerize without the addition of Qcr10p, since the enzyme from the subunit 10 deletion strain and from the wild type strain were purified by the same chromatography procedure from the mitochondrial membranes of the respective strains [54].