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. 2012 Oct 5;287(41):34484-93.
doi: 10.1074/jbc.M112.382630. Epub 2012 Aug 17.

Oxa1-ribosome complexes coordinate the assembly of cytochrome C oxidase in mitochondria

Melanie Keil  1 , Bettina Bareth, Michael W Woellhaf, Valentina Peleh, Martin Prestele, Peter Rehling, Johannes M Herrmann
Affiliations

Oxa1-ribosome complexes coordinate the assembly of cytochrome C oxidase in mitochondria

Melanie Keil et al. J Biol Chem. .

Abstract

The terminal enzyme of the respiratory chain, cytochrome c oxidase, consists of a hydrophobic reaction center formed by three mitochondrially encoded subunits with which 9-10 nuclear encoded subunits are associated. The three core subunits are synthesized on mitochondrial ribosomes and inserted into the inner membrane in a co-translational reaction facilitated by the Oxa1 insertase. Oxa1 consists of an N-terminal insertase domain and a C-terminal ribosome-binding region. Mutants lacking the C-terminal region show specific defects in co-translational insertion, suggesting that the close contact of the ribosome with the insertase promotes co-translational insertion of nascent chains. In this study, we inserted flexible linkers of 100 or 200 amino acid residues between the insertase domain and ribosome-binding region of Oxa1 of Saccharomyces cerevisiae. In the absence of the ribosome receptor Mba1, these linkers caused a length-dependent decrease in mitochondrial respiratory activity caused by diminished levels of cytochrome c oxidase. Interestingly, considerable amounts of mitochondrial translation products were still integrated into the inner membrane in these linker mutants. However, they showed severe defects in later stages of the biogenesis process, presumably during assembly into functional complexes. Our observations suggest that the close proximity of Oxa1 to ribosomes is not only used to improve membrane insertion but is also critical for the productive assembly of the subunits of the cytochrome c oxidase. This points to a role for Oxa1 in the spatial coordination of the ribosome with assembly factors that are critical for enzyme biogenesis.

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Figures

FIGURE 1.
FIGURE 1.
In the Oxa1 linker mutants, the ribosome-binding and insertase domains of Oxa1 are separated. A, structure of the Oxa1 linker mutants. The mitochondrial targeting sequence (MTS) and the five transmembrane domains are shown as boxes. Flexible regions derived from residues 2–101 or 2–201 of an Nsp1FS mutant (23) were inserted between the membrane-embedded insertase domain and the ribosome-binding domain of Oxa1. B, the linker mutants of Oxa1 are expressed to similar levels as wild-type Oxa1. Oxa1 and the mitochondrial protein MrpL36 were detected by Western blotting in mitochondria isolated from the wild-type and mutant strains. C and D, cells of the mutants indicated were grown overnight to log phase. Serial dilutions (10-fold) were spotted on 1% yeast extract and 2% peptone plates containing glucose or galactose as the carbon source and incubated at 30 °C for 2 and 3 days, respectively.
FIGURE 2.
FIGURE 2.
Insertion of linkers into Oxa1 leads to reduced levels of respiratory chain complexes. A, mitochondria (10 μg) from the indicated strains were incubated in the presence of 7 mm NADH. The oxygen consumption per time was measured in three independent measurements. B, the activity of cytochrome c oxidase was analyzed in 500 μg of mitochondria after lysis with Triton X-100. C, steady-state levels of mitochondrial proteins were assessed by Western blotting. The positions of molecular mass markers are indicated. D, quantification of Cox1 and Cox2 levels in Δoxa1 Δmba1 strains expressing the indicated proteins from plasmids.
FIGURE 3.
FIGURE 3.
Insertion of linkers into Oxa1 does not impair processing of Cox2. A, mitochondrial translation products were radiolabeled with [35S]methionine in yeast cells of the indicated strains after inhibition of cytosolic protein synthesis with cycloheximide. The inset shows the topology of Cox2. The arrowhead depicts the low levels of Cox2 precursor that accumulate in Δoxa1 mutants. B, translation products were radiolabeled as described for A in Mba1-deficient strains. Note that the levels of mature Cox2 are similar between the linkerless and linker-containing Oxa1 variants. C, mitochondria were isolated from the indicated strains and incubated in the presence of different concentrations of valinomycin and [35S]methionine. After incubation for 15 min at 30 °C, radiolabeling was stopped by the addition of unlabeled methionine, and the samples were analyzed by SDS-PAGE and autoradiography. Cyt b, cytochrome b.
FIGURE 4.
FIGURE 4.
Insertion of linkers into Oxa1 does not prevent protein insertion into the inner membrane. A, structure of the maleimide derivatives mPEG-12 and mPEG-24. B, wild-type mitochondria were incubated in the presence of 1 mm mPEG-12 or mPEG-24 or mock-treated. After quenching the alkylating reagents by the addition of 2 mm DTT, mitochondria were reisolated, washed, and analyzed by Western blotting. The total numbers of cysteine residues in the proteins are indicated. The mPEG-24-treated sample was loaded twice to see the migration differences more clearly. C, mitochondrial translation products were radiolabeled in isolated mitochondria before mitochondria were incubated with mPEG-12 and mPEG-24 as described for B. D, following translation and treatment with mPEG-24, mitochondria were lysed and either directly applied to the gel (10% total, T) or used for immunoprecipitation (IP) with Cox2-specific antibodies. As shown in the inset, completely inserted Cox2 exposes four cysteine residues to the IMS. Fully or partially modified Cox2 species are indicated by one or two asterisks, respectively. Cyt b, cytochrome b.
FIGURE 5.
FIGURE 5.
Insertion of linkers into Oxa1 does not prevent binding of Cox2 to Cox20. Translation products were radiolabeled in isolated mitochondria. Mitochondria were lysed with Triton X-100 and used for co-immunoprecipitation with serum against Cox20 (αCox20) or with preimmune (pi) serum as a control. The total lanes (T) show 10% of the material used for the co-immunoprecipitations. Arrows depict the Cox2 and Cox3 proteins that were co-immunoprecipitated with anti-Cox20 antibodies. Cyt b, cytochrome b.
FIGURE 6.
FIGURE 6.
Insertion of linkers into Oxa1 impairs the assembly of the cytochrome c oxidase complex. 35S-Labeled precursor forms of Cox5a (A) and Cox13 (B) were imported into isolated mitochondria for the indicated times. Non-imported material was removed by treatment with proteinase K. Mitochondria were lysed with digitonin before protein complexes were separated by BN-PAGE (upper panels) or SDS-PAGE (lower panels). pre, precursor; m, mature.
FIGURE 7.
FIGURE 7.
Oxa1 linker mutants contain reduced levels of cytochrome c oxidase-containing supercomplexes. A, digitonin extracts of mitochondria were analyzed by BN-PAGE and Western blotting with antibodies against subunits of the cytochrome c oxidase complex (Cox1, Cox2, and Cox4), the cytochrome bc1 complex (Rip1), or the ATPase (Atp5). B, model of mitochondrial ribosomes serving as a binding platform for assembly factors. The model proposes that the close contact of mitochondrial ribosomes with the inner membrane allows the binding of assembly factors to the sites at which newly synthesized proteins are inserted into the membrane. Accordingly, spatial separation of the membrane-embedded insertase region and the ribosome-binding region of Oxa1 interferes with the coordination of assembly factors and therefore reduces the efficiency of cytochrome c oxidase assembly.
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