Mutational analysis of the Saccharomyces cerevisiae cytochrome c oxidase assembly protein Cox11p

Abstract

Cox11p is an integral protein of the inner mitochondrial membrane that is essential for cytochrome c oxidase assembly. The bulk of the protein is located in the intermembrane space and displays high levels of evolutionary conservation. We have analyzed a collection of site-directed and random cox11 mutants in an effort to further define essential portions of the molecule. Of the alleles studied, more than half had no apparent effect on Cox11p function. Among the respiration deficiency-encoding alleles, we identified three distinct phenotypes, which included a set of mutants with a misassembled or partially assembled cytochrome oxidase, as indicated by a blue-shifted cytochrome aa(3) peak. In addition to the shifted spectral signal, these mutants also display a specific reduction in the levels of subunit 1 (Cox1p). Two of these mutations are likely to occlude a surface pocket behind the copper-binding domain in Cox11p, based on analogy with the Sinorhizobium meliloti Cox11 solution structure, thereby suggesting that this pocket is crucial for Cox11p function. Sequential deletions of the matrix portion of Cox11p suggest that this domain is not functional beyond the residues involved in mitochondrial targeting and membrane insertion. In addition, our studies indicate that Deltacox11, like Deltasco1, displays a specific hypersensitivity to hydrogen peroxide. Our studies provide the first evidence at the level of the cytochrome oxidase holoenzyme that Cox1p is the in vivo target for Cox11p and suggest that Cox11p may also have a role in the response to hydrogen peroxide exposure.

FIG. 1.

Alignment of multiple Cox11p sequences from 10 disparate species with a summary of Cox11p mutations generated in yeast. ClustalX was used to align Cox11p sequences from 10 species. Residues identical across all 10 species are highlighted in red, while residues conserved across all 10 species are highlighted in green. The putative TM domain of the yeast sequence is shown overlined in black. The 10 β-strands assigned by Banci et al. (1) are overlined and labeled. The predicted MPP cleavage site for yeast Cox11p is denoted by the arrowhead. Point mutations generated in yeast Cox11p are shown below the alignment and are color coded as follows on the basis of the phenotypes they encode: brown, respiration competent; blue, respiration deficient (petite). Deletions generated in yeast Cox11p are shown above the alignment by dotted lines and are color coded as are the single-residue mutations. Symbols: *, mutations that synthetically interact to produce complete respiratory deficiency; #, mutations that produce a synthetic partial respiratory deficiency. The S. meliloti sequence represents only the soluble domain of Cox11.

FIG. 2.

Comparative stereo views of two cox11 alleles generated based on molecular modeling of the S. meliloti Cox11 three-dimensional structure. (A) Model of S. meliloti Cox11 (Protein Data Bank code, 1SO9). The side chains of residues F84 (Y192 in yeast) and V118 (V226 in yeast) are shown in green. These residues border a pocket in the protein behind the CFCF copper-binding loop, which is labeled. The boxed region was used to generate stereo views. (B to D) Stereo views of the wild-type (B) and theoretical F84R (C) and V118W (D) cox11 mutant alleles from S. meliloti. In panels C and D, only the mutated residue is green. Mutations were generated in 1SO9 with the Swiss Protein Data Bank viewer, followed by energy minimization. Stereo views were subsequently generated with Pymol.

FIG.3.

Mitochondrial cytochrome spectra of cox11 mutants fall into three categories. Mitochondria were solubilized in 1% deoxycholate, and the difference spectra of mitochondrial extracts reduced with sodium dithionite and oxidized with potassium ferricyanide are shown. Parent strain W303 displays a typical cytochrome aa₃ peak at 603 nm, which is denoted by the vertical line. The Δcox11 mutant and most of the respiration-deficient cox11 mutants lack a peak at 603 nm. Several of the cox11 allele mutants with partial respiratory deficiency display aa₃ peaks that are reduced and shifted 1 to 3 nm to the blue range. The Y192R and V226W alleles are the only complete respiration deficiency- encoding alleles for which an aa₃ peak is observed. Mutants are labeled according to the mutations they bear.

FIG. 4.

Western blot analysis of Cox11p, Por1p, and COX catalytic core proteins from cox11 alleles. Mitochondria were purified from strains expressing each cox11 allele on a low-copy-number plasmid. Mitochondrial proteins (20 μg) were separated on 12% polyacrylamide gels and transferred to nitrocellulose membrane prior to probing with antibodies specific to Cox1p, Cox2p, Cox3p, Cox11p, and Por1p. (A) Steady-state levels of COX subunits 1, 2, and 3. Mitochondria from all mutants, except those with the Δ84-88 and E212K/M240K mutations, were electrophoresed and blotted together. The mutants with Δ84-88 and E212K/M240K were analyzed separately but with the same wild-type (W303) and null mutant (ΔCOX11) mitochondrial samples to allow comparison. (B) Steady-state levels of Cox11p were determined in the same experiment for all mutants except those with the Δ84-88 and E212K/M240K mutations, which were analyzed separately as described for panel A. Cox11p instability is observed in several of the complete respiratory deficiency-encoding alleles (C208A, F209A, C210A, P225R, L106P/S150P, and Δ84-88). (C) All samples were blotted for mitochondrial porin (Por1p) to ensure loading of uniform amounts of protein. (D) The two cox11 mutants that result from occlusion of the pocket behind the copper-binding domain were analyzed for steady-state levels of Cox1p, Cox2p, Cox3p, Cox11p, and Por1p. The misassembled mutants (Y192R, V226W) have proportionally less Cox1p than Cox2p or Cox3p, with uniform levels of Por1p.

FIG. 5.

The Δcox11 mutant is hypersensitive to hydrogen peroxide. Exponential-phase liquid cultures were exposed to 6 mM H₂O₂ (+) for 2 h, serially diluted, and spotted onto 1% yeast extract-2% peptone-2% dextrose plates along with untreated controls (−) and allowed to grow for 36 to 48 h to determine colony-forming potential. The W303 parent strain and the COX mutants aW303ΔCOX4, aW303ΔCOX6, aW303ΔCOX9, and aW303ΔCOX17, in addition to aW303 ρ⁰, do not display sensitivity to H₂O₂, suggesting that loss of COX itself does not impart a peroxide-sensitive phenotype. aW303ΔCOX11 and aW303ΔSCO1 are hypersensitive to H₂O₂, and this phenotype can be rescued by expression of COX11 and SCO1, respectively, from a low-copy-number plasmid.