Cox16 protein is physically associated with Cox1p assembly intermediates and with cytochrome oxidase

Abstract

Mitochondrial cytochrome oxidase (COX) catalyzes the last step in the respiratory pathway. In the yeast Saccharomyces cerevisiae, this inner membrane complex is composed of 11 protein subunits. Expression of COX is assisted by some two dozen ancillary proteins that intercede at different stages of the assembly pathway. One such protein, Cox16p, encoded by COX16, was shown to be essential for the activity and assembly of COX. The function of Cox16p, however, has not been determined. We present evidence that Cox16p is present in Cox1p assembly intermediates and in COX. This is based on the finding that Cox16p, tagged with a dual polyhistidine and protein C tag, co-immunopurified with Cox1p assembly intermediates. The pulldown assays also indicated the presence of Cox16p in mature COX and in supercomplexes consisting of COX and the bc₁ complex. From the Western signal strengths, Cox16p appears to be substoichiometric with Cox1p and Cox4p, which could indicate that Cox16p is only present in a fraction of COX. In conclusion, our results indicate that Cox16p is a constituent of several Cox1p assembly intermediates and of COX.

Keywords: cytochrome c oxidase (complex IV); electron transfer complex; membrane biogenesis; mitochondria; yeast.

Figure 1.

Properties of strains expressing COX16-CH. A, the indicated strains were serially diluted and spotted on YPD (rich glucose) and YEPG (rich ethanol plus glycerol) plates. The photograph was taken after 2 days of growth at 30 °C. B, mitochondria (50 μg of protein) prepared from W303/COX16-CH and the parental strain W303-1B were separated by SDS-PAGE on a 12% polyacrylamide gel. The blot was first reacted with a polyclonal antibody to the protein C tag followed by a second anti-rabbit antibody coupled to peroxidase. Proteins were detected with SuperSignal chemiluminescent substrate kit (Pierce). C, mitochondria (500 μg of protein) from the indicated strains were extracted with digitonin, purified on protein C antibody beads, and separated by BN-PAGE on a 4–13% polyacrylamide gel. The gel was stained for COX activity as described previously (13).

Figure 2.

Cox16p is a constituent of the Cox1p module. A, mitochondria were prepared from the wild-type W303-1B and aW303/COX16-CH expressing COX16-CH. The latter strain was grown with and without a terminal 2-h growth in the presence of chloramphenicol (CAP). Mitochondria (250 μg of protein) were labeled with [³⁵S]methionine plus [³⁵S]cysteine for 20 min, extracted with digitonin, and purified on protein C beads as described under “Experimental procedures.” The eluate (10%) was separated by SDS-PAGE on a 12% polyacrylamide gel. The radiolabeled mitochondrial gene products were transferred to a PVDF membrane and exposed to X-ray film. B, BN-PAGE of 30% of the eluates obtained in A were separated on a 4–13% polyacrylamide gel. The positions of the COX intermediates with Cox1p (D2, D3, and D4) are marked in the margin. C, the remaining 60% of the eluate in A was separated in the first dimension by BN-PAGE on a 4–13% polyacrylamide gel, followed by SDS-PAGE on a 12% polyacrylamide gel in the second dimension. The increase in incorporation of radiolabel by mitochondria as a result of the chloramphenicol treatment is seen in increased translation of cytochrome b and Cox1 intermediates D2–D4. The identity of the radioactive band labeled with an asterisk was not studied.

Figure 3.

Cox16p is associated with COX. A, W303/COX16-CH mitochondria were labeled and Cox16p-CH purified on protein C antibody beads as in Fig. 2A. The eluate from the protein C antibody beads was separated by BN-PAGE on a 4–13% polyacrylamide gel and exposed to X-ray film. The positions of the supercomplexes, COX, and D2–D4 intermediates wit Cox1p are indicated in the margin. B, a duplicate of the blue native gel shown in A was transferred to nitrocellulose and reacted with antibody to protein C and further processed as in Fig. 1A. The gel on the right was exposed longer to detect the radiolabel associated with COX. The concentrations of the D2–D4 intermediates were too low to detect on the Western. C, mitochondria of W303/COX16-CH and W303ΔCBP3/COX16-CH carrying a mutation in CBP3 were first grown in rich galactose followed by 2 h in galactose medium containing 2 mg/ml chloramphenicol. Mitochondria were labeled, and the fraction was purified on protein C antibody beads separated by BN-PAGE as in A.

Figure 4.

Purification of COX with Cox16. A, mitochondria were prepared from MRSI^o/COX1-HAC and W303/COX16-CH expressing Cox1p-HAC and Cox16-CH, respectively. The mitochondria (2 mg) were extracted with either 2% lauryl maltoside (L) or 2% digitonin (D). The extracts were purified on protein C antibody beads, and $\raisebox{1ex}{$1$}\!\left/ \!\raisebox{-1ex}{$20$}\right.$ of the eluates were separated by SDS-PAGE on a 15% gel (22). The silver-stained proteins are identified in the margin. Some of the extra bands of the bc₁ complex seen in the digitonin extract are absent in Cox16p-CH purified from lauryl maltoside extracted mitochondria because this detergent dissociates the supercomplexes, and only the Cox16p-CH associated with COX is recovered from the protein C beads. B, to confirm identity of the bands migrating in the region between Cox6p and smaller subunits in A, COX was purified on protein C antibody beads from strains expressing Cox1p-HAC, Cox4p-HAC, and Cox16p-CH. Proteins were separated and silver-stained as in A. A duplicate gel was transferred to a PVDF membrane and probed with an antibody to the protein C epitope as in Fig. 1A. C, Western analysis of COX affinity-purified from strains expressing Cox1p-HAC with and without a cox16 null mutation and from strains expressing either Cox4p-HAC Cox5p-HAC, Cox6p-HAC, and Cox16p-CH.

Figure 5.

Homology and Kyte–Doolittle hydrophobicity plots of yeast, mushroom and human Cox16p. A, proteins were aligned by the Clustal Omega and formatted with BlockShade. B, the Kyte–Doolittle parameters with window size of 9 was used to plot the hydrophobicity of yeast Cox16p and of the homologous mushroom and human proteins.