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

Abstract

The mitochondria of cytochrome-aa3-deficient N. crassa mutants were screened for the 7 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 1-3, the results obtained from the analysis of immunoprecipitates were confirmed through the direct electrophoretic analysis of mitochondrial translation products. 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 7 cytochrome oxidase polypeptides. Only the oxidase subunits 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 2 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 MW 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 labeled 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, apparently is not 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. The cytochrome oxidase deficiencies of as many as 7 of the 8 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.