Characterization of a 45-kDa polypeptide as the precursor of subunit 1 of cytochrome c oxidase in Neurospora crassa.
In a previous paper (Van 't Sant, P., Mak, J.F.C. and Kroon, A.M. (1981) Eur. J. Biochem. 121, 21-26) we showed the existence of three elongated precursor proteins (45, 36 and 25 kDa) of mitochondrial translation products in Neurospora crassa. We presented some indications that the largest precursor could be related to subunit 1 of cytochrome c oxidase. Here we present conclusive evidence that the 45-kDa polypeptide is indeed this precursor by demonstrating that an immunodetectable 45-kDa polypeptide displays the same behaviour as the labeled 45-kDa precursor; both accumulate after long incubation with cycloheximide or by decreasing the temperature and both are not tightly membrane bound. Moreover the antibody against subunit 1 of cytochrome c oxidase also recognizes, in immunoadsorption experiments, besides subunit 1, the 45-kDa polypeptide accumulated by cycloheximide incubation. Furthermore, we developed a small scale purification of antibodies against subunit 1 of cytochrome c oxidase. By means of these purified antibodies it is demonstrated that the 45-kDa polypeptide and subunit 1 have corresponding antigenic determinants. Under the various conditions tested, all three precursors are less firmly membrane-bound than the mature subunits. Finally, it is observed that in short incubations in vivo, chloramphenicol inhibits the processing of the mitochondrially synthesized precursors, under conditions where mitochondrial translation is only partially inhibited.
D003576:Electron Transport Complex IV / Q000235:genetics* / Q000302:isolation & purification; D004792:Enzyme Precursors / Q000235:genetics* / Q000302:isolation & purification; D046911:Macromolecular Substances; D008928:Mitochondria / Q000201:enzymology; D008970:Molecular Weight; D009491:Neurospora / Q000201:enzymology*; D009492:Neurospora crassa / Q000201:enzymology*; D014176:Protein Biosynthesis
D004792:Enzyme Precursors; D046911:Macromolecular Substances; D003576:Electron Transport Complex IV