Analysis Of The Assembly Of Saccharomyces Cerevisiae Cytochrome C Oxidase Subunit Cox2 By The Oxa1 And Cox18 Translocases
Oxa1 and Cox18 are members of a conserved protein family of integral inner membrane translocases responsible for insertion of other proteins into the membranes of chloroplasts, bacteria and mitochondria. In Saccharomyces cerevisiae both Oxa1 and Cox18 are required for the translocation of the hydrophilic domains of cytochrome c oxidase subunit Cox2 into the inner membrane space. Deletions in either oxa1 or cox18 result in yeast strains unable to respire. Over-expression of OXA1 does not allow for respiration in strains with deletions in cox18, but does allow for export of the C-terminus of Cox2. This suggests that Cox18 also has a role in the assembly of Cox2 into functional cytochrome c oxidase following the translocation of the C-terminus. I identified a physical interaction between Cox18 and the noncanonical chaperone Cox20, which is dependent on the presence of the Cox2 peptide. I isolated spontaneous respiring pseudorevertants from cox18 deletion strains carrying OXA1 on a high copy plasmid. Several of these pseudorevertants have mutations in residue 291 of the plasmid borne Oxa1. Substitution of charged residues, both positive and negative, at position 291 results in OXA1 alleles that can compensate for a deletion in cox18 when present in high copy number or when expressed from the chromosomal locus. Neutral substitutions at residue 291 of Oxa1 result in alleles that fail to compliment cox18 when present in high copy. These results show that the presence of a charged residue at position 291 allows Oxa1 to assemble Cox2 independent of Cox18 and its partner Mss2. The mutant alleles all retain Oxa1 function to various degrees. In addition, the alleles with charged residues compensate for a mss2 deletion. Cox20 is not required for assembly by these mutant proteins, although Cox20's chaperoning of Cox2 processing is required. Therefore, these mutant alleles are not simply replacing Cox18 in its normal complexes. Rather, they may interact with the Cox2 peptide in a novel way that makes it competent for assembly.
cytochrome c oxidase; mitochondria; Cox20
Fox, Thomas D.
Hanson, Maureen R; Pleiss, Jeffrey A.
Ph.D. of Genetics
Doctor of Philosophy
dissertation or thesis