Bertrand H, Werner S
Eur J Biochem. 1979 Jul;98(1):9-18. doi: 10.1111/j.1432-1033.1979.tb13154.x.
The mitochondria of cytochrome-aa3-deficient Neurospora crassa mutants were screened for the seven polypeptide constiuents 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. 1. 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 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. 2. 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. 3. 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.
对细胞色素aa3缺陷型粗糙脉孢菌突变体的线粒体进行筛选，以寻找细胞色素c氧化酶的七种多肽成分。通过用全酶抗血清以及几种单个亚基的抗血清获得的免疫沉淀物进行十二烷基硫酸钠/丙烯酰胺凝胶电泳，检测全酶以及未组装或部分组装亚基的多肽。关于氧化酶的线粒体合成多肽亚基1至3，通过对线粒体翻译产物的直接电泳分析，证实了从免疫沉淀物分析中获得的结果。结果如下：1. cya - 2 - 8和cya - 3 - 16核突变体以及[exn - 5]细胞质突变体的线粒体含有一种被抗全酶抗体免疫沉淀的蛋白质复合物，该复合物由细胞色素氧化酶的全套七种多肽组成。即使通过亚基特异性免疫沉淀在cyt - 2 - 1和299 - 1核突变体的线粒体中检测到至少一些线粒体合成的多肽，但只有氧化酶亚基5和6被抗全酶抗体从这两个突变体的线粒体中免疫沉淀。2. 在cyt - 2 - 1、299 - 1这两个核突变体以及[mi - 3]细胞质突变体的线粒体中发现了一种比野生型细胞色素氧化酶的真实亚基1多肽更大的“亚基1”多肽。这种更大的多肽可能是全酶“成熟”亚基1蛋白的未加工前体。在[exn - 5]细胞质突变体以及cya - 2 - 8和cya - 4 - 23核突变体的线粒体中，细胞色素氧化酶的多肽亚基的表观分子量没有变化。3. 核突变体299 - 1缺乏细胞色素氧化酶的线粒体合成亚基2多肽。当细胞在放线菌酮存在下进行标记时，[exn - 5]、cya - 2 - 8、cya - 3 - 16和cya - 4 - 23突变体线粒体中的亚基2含量低于野生型线粒体。然而，这种缺陷似乎不够严重，不足以完全解释这些突变体中细胞色素aa3的缺乏。cya - 4 - 23核突变体要么严重缺乏细胞色素氧化酶亚基5和6，要么完全没有。基于这些以及先前报道的观察结果，有人提出，在粗糙脉孢菌的八个细胞色素aa3缺陷型突变体中，多达七个突变体的细胞色素氧化酶缺陷可能是由控制该酶不同成分产生的调节系统的基因改变引起的。
