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.
Highlights
Cytochrome oxidase (COX)2 is a hetero-oligomeric complex of the mitochondrial inner membrane that catalyzes the last step of the respiratory pathway by oxidizing ferrocytochrome c and reducing molecular oxygen to water
The results presented here indicate that Cox16p is a constituent of several Cox1p assembly intermediates and of cytochrome oxidase (COX)
The presence of Cox16p in the Cox1p module was assessed in a strain of yeast (W303/COX16-CH) expressing Cox16p with a C-terminal double tag consisting of the protein C epitope followed by polyhistidine (CH)
Summary
Was reported to be associated with yeast COX in the supercomplexes. This subunit encoded by COX26 does not affect biogenesis of COX. Cox16p is a low-molecular-weight protein present in all fungi, insect, and mammalian mitochondria where it is a component of the inner membrane. It is essential for COX assembly and activity. Mutations in the yeast COX16 gene elicit a phenotype characteristic of other COX mutants, highlighted by the absence of enzyme activity and cytochromes a and a3 and extensive proteolysis of the catalytic core subunits [16]. The results presented here indicate that Cox16p is a constituent of several Cox1p assembly intermediates and of COX
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