Mutations in the membrane anchor of yeast cytochrome c1 compensate for the absence of Oxa1p and generate carbonate-extractable forms of cytochrome c1.

Oxa1p is a mitochondrial inner membrane protein that is mainly required for the insertion/assembly of complex IV and ATP synthase and is functionally conserved in yeasts, humans, and plants. We have isolated several independent suppressors that compensate for the absence of Oxa1p. Molecular cloning and sequencing reveal that the suppressor mutations (CYT1-1 to -6) correspond to amino acid substitutions that are all located in the membrane anchor of cytochrome c1 and decrease the hydrophobicity of this anchor. Cytochrome c1 is a catalytic subunit of complex III, but the CYT1-1 mutation does not seem to affect the electron transfer activity. The double-mutant cyt1-1,164, which has a drastically reduced electron transfer activity, still retains the suppressor activity. Altogether, these results suggest that the suppressor function of cytochrome c1 is independent of its electron transfer activity. In addition to the membrane-bound cytochrome c1, carbonate-extractable forms accumulate in all the suppressor strains. We propose that these carbonate-extractable forms of cytochrome c1 are responsible for the suppressor function by preventing the degradation of the respiratory complex subunits that occur in the absence of Oxa1p

Non-native intragenic reversions selected from Saccharomyces cerevisiae cytochrome b-deficient mutants. Structural and functional features of the catalytic center N domain.

A total of 110 revertants have been isolated from two well-characterized cytochrome b deficient (mit-) mutants. The mit- mutations are located in an extramembranous loop linking the transmembrane alpha-helices IV and V of cytochrome b which has been postulated to be part of the catalytic center QN and therefore is assumed to be essential for the functioning of the bc1 complex. The molecular bases of the reversions were identified by sequencing the cytochrome b mRNAs. This allowed us to identify seven new structures of cytochrome b which are more or less compatible with its catalytic activity. The secondary mutations occurred either at the level of the original site mutation or at adjacent positions (region 204-208 of the polypeptide chain), or even at a distance of more than 150 amino acids (position 30) suggesting topological interaction between these two areas. All the revertants recovered cytochrome contents and phosphorylation efficiencies similar to the wild-type ones, albeit differences appeared in their specific growth rates and NADH respirations. The failure in bc1 complex functioning induced by the mutation S206L and its restoration by non native reversions are tentatively explained