Abstract
We have identified Cox20p, a 23.8-kDa protein of the mitochondrial inner membrane that is involved in the biogenesis of the yeast cytochrome oxidase complex. Cytochrome oxidase subunit 2 (Cox2p) accumulates as a precursor in cox20 mutants, suggesting a defect in biogenesis of this mitochondrially encoded protein. The inability of cox20 mutants to process the subunit 2 precursor (pCox2p) is not due to impaired export of the protein across the inner membrane or to an inactive Imp1p/Imp2p peptidase. Rather, Cox20p specifically binds the newly synthesized pCox2p, a step required to present the exported pCox2p as a substrate to the Imp1p peptidase. All of the endogenous pCox2p accumulated in an Deltaimp1 mutant, and a small fraction of Cox2p in wild type yeast, is detected in a complex with Cox20p. Following maturation Cox2p remained associated with Cox20p, prior to assembling into the cytochrome oxidase complex. We propose that Cox20p acts as a membrane-bound chaperone necessary for cleavage of pCox2p and for interaction of the mature protein with other subunits of cytochrome oxidase in a later step of the assembly process.
Highlights
An important event in the biogenesis of cytochrome oxidase is the sorting and processing of the subunit 2 precursor1 after its synthesis by mitochondrial ribosomes (1–5)
We have identified Cox20p, a 23.8-kDa protein of the mitochondrial inner membrane that is involved in the biogenesis of the yeast cytochrome oxidase complex
Interaction of newly synthesized Cox2p with Cox20p was observed in ⌬imp1 mitochondria (Fig. 7B). In this case the cross-linked adduct was slightly larger (53 kDa) than that observed in wild type mitochondria, indicating the non-processed precursor form of Cox2p was associated with Cox20p
Summary
The OXA1 disruption/deletion strain (aW303⌬OXA1) (⌬oxa1) was constructed by replacing the nucleotides 220 –1038 of the open reading frame encoding Oxa1p, with the HIS3 gene, as described (32). Cloning of COX20 and Construction of a cox Null Allele—C176/L6 (MAT␣ leu 112, cox20-1) was transformed by the method of Beggs (33) with a yeast genomic library constructed in the shuttle plasmid.
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