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The nuclear gene OXA1encodes a protein located within the mitochondrial inner membrane that is required for the biogenesis of both cytochrome coxidase (Cox) and ATPase. In the absence of Oxa1p, the translocation of the mitochondrially encoded subunit Cox2p to the intermembrane space (also referred to as export) is prevented, and it has been proposed that Oxa1p could be a component of a general mitochondrial export machinery. We have examined the role of Oxa1p in light of its relationships with two mitochondrial proteases, the matrix protease Afg3p-Rca1p and the intermembrane space protease Yme1p, by analyzing the assembly and activity of the Cox and ATPase complexes in Δoxa1,Δoxa1Δafg3, and Δoxa1Δyme1mutants. We show that membrane subunits of both complexes are specifically degraded in the absence of Oxa1p. Neither Afg3p nor Yme1p is responsible for the degradation of Cox subunits. However, the F0subunits Atp4p, Atp6p, and Atp17p are stabilized in the Δoxa1Δyme1double mutant, and oligomycin-sensitive ATPase activity is restored, showing that the increased stability of the ATPase subunits allows significant translocation and assembly to occur even in the absence of Oxa1p. These results suggest that Oxa1p is not essential for the export of ATPase subunits. In addition, although respiratory function is dispensable in Saccharomyces cerevisiae, we show that the simultaneous inactivation of AFG3and YME1is lethal and that the essential function does not reside in their protease activity.

We have analyzed the heme-associated peroxidase activity in thylakoid membranes from the green algae Chlamydomonas reinhardtiiafter electrophoresis in the presence of sodium dodecyl sulfate. Besides cytochrome fand cytochrome b6, we observed peroxidase activity in two other bands, of 34 and 11 kDa, of unknown origin. Characterization of the b6/fcomplex subunits was undertaken by means of a comparison of the polypeptide deficiencies in several b6/fmutants with the polypeptide content of preparations enriched in b6/fcomplexes. We conclude that the b6/fcomplex consists of five subunits. Using site-specific translation inhibitors, we show that cytochrome f, cytochrome b6and subunit IV are of chloroplast origin, whereas the Rieske protein and probably subunit V are translated on cytoplasmic ribosomes. A model of assembly of the complex is proposed: a cytochrome moiety, comprising the subunits of chloroplast origin, is assembled in the thylakoid membranes prior to the insertion and assembly of the subunits encoded in the nuclear genome.

We have analysed the precise location of a large number (170) of mutations affecting the structural gene for subunit 1 of the cytochrome c oxidase complex. This gene, COXI, is 12.9 kb long and the major part of the sequence (i.e. 11.3 kb) is composed of introns. Several conclusions can be drawn from this study: (1) A significant proportion (84/170) of the mutations cannot be assigned to a single position within the gene by deletion mapping, in spite of clearly being located in it. These mutations are probably large deletions or multiple mutations. (2) Four mutants carry distant double mutations, which have been individually localized. (3) Eighty-two mutants have lesions that are restricted to very short regions of the gene and we therefore conclude that they are most probably due to single hits; amongst these single mutations, 41 are unambiguously located in exons and 28 in introns. This result implies that, at least in this particular split gene, the probability of selection of a mutant phenotype in an exon is, on the average, 13.3 times greater than in an intron, in spite of the existence, within most of these introns, of open reading frames specifying intronic proteins. The evolutionary significance and biological implications of these results are discussed.