Cytochrome c Oxidase Subunits in Nuclear and Extranuclear Cytochrome‐aa3‐Deficient Mutants of Neurospora crassa

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Abstract
The mitochondria of cytochrome‐aa3‐deficient Neurospora crassa mutants were screened for the seven polypeptide constituents of cytochrome c oxidase. The polypeptides of the holoenzyme and the unassembled or partially assembled subunits were detected by sodium dodecyl sulfate/acrylamide gel electrophoresis of immunoprecipitates obtained with antiserum to the holoenzyme as well as to several individual subunits. With respect to the mitochondrially synthesized polypeptides of the oxidase, subunits 1 to 3, the results obtained from the analysis of immunoprecipitates were confirmed through the direct electrophoretic analysis of mitochondrial translation products. The results were as follows. The mitochondria of the cya‐2‐8 and cya‐3‐16 nuclear mutants and the [exn‐5] cytoplasmic mutant contained a protein complex immunoprecipitated by anti‐holoenzyme antibody and composed of the complete set of the seven cytochrome oxidase polypeptides. Only the oxidase sub‐units 5 and 6 were immunoprecipitated by anti‐holoenzyme antibody from the mitochondria of the cyt‐2‐1 and 299‐1 nuclear mutants, even though at least some of the mitochondrially synthesized polypeptides were detected in both mutants by subunit specific immunoprecipitation. A ‘subunit 1’ polypeptide larger than the authentic subunit‐1 polypeptide of wild‐type cytochrome oxidase was found in the mitochondria from two nuclear mutants, cyt‐2‐1, and 299‐1 and the [mi‐3] cytoplasmic mutant. This larger polypeptide may be an unprocessed precursor of the ‘mature’ subunit 1 protein of the holoenzyme. No changes in the apparent molecular weights were found for the polypeptide subunits of cytochrome oxidase in mitochondria of the [exn‐5] cytoplasmic mutant and the cya‐2‐8 and cya‐4‐23 nuclear mutants. A nuclear mutant, 299‐1, lacks the mitochondrially synthesized subunit‐2 polypeptide of cytochrome oxidase. When cells were labelled in the presence of cycloheximide, the subunit 2 content of mitochondria from mutants [exn‐5], cya‐2‐8, cya‐3‐16 and cya‐4‐23 was lower than in mitochondria from wild‐type. This deficiency, however, does not appear to be sufficiently severe to fully account for the lack of cytochrome aa3 in these mutants. The cya‐4‐23 nuclear mutant either is severely deficient in or lacks cytochrome oxidase subunits 5 and 6.On the basis of these and previously reported observations, it is proposed that the cytochrome oxidase deficiencies of as many as seven of the eight N. crassa cytochrome‐aa3‐deficient mutants could be caused by genetically imposed alterations in regulatory systems controlling the production of different components of the enzyme.