Schoppink P J, Berden J A, Grivell L A
Department of Molecular Cell Biology, University of Amsterdam, The Netherlands.
Eur J Biochem. 1989 May 1;181(2):475-83. doi: 10.1111/j.1432-1033.1989.tb14749.x.
The single nuclear gene encoding the 14-kDa subunit VII of yeast ubiquinol:cytochrome c oxidoreductase has been inactivated by one-step gene disruption, as verified by Southern blot analysis and immunoblotting. The resulting mutant has no ubiquinol:cytochrome c oxidoreductase activity and is respiratory-deficient. Immunoblotting shows that cells lacking the 14-kDa protein, also have lowered steady-state levels of other subunits of complex III, the nuclear-encoded 11-kDa subunit VIII, the Rieske Fe-S protein and the mitochondrially encoded cytochrome b. No cytochrome b can be detected spectrally. The steady-state levels of the transcripts from genes encoding these proteins are not reduced, implying that the mutation exerts its pleiotropic effects at a post-transcriptional level. The residual amounts of subunits of complex III are recovered in the mutant mitochondria, suggesting that import is unaffected. The results strongly suggest that the 14-kDa protein plays an essential role in the biosynthesis of the complex, most probably at the level of assembly. Field-inversion gel electrophoresis was used to separate chromosomes of HR2 wild type and the (14-kDa-protein) degrees mutant, after which the gene encoding the 14-kDa protein was located on chromosome IV by Southern blot analysis.
细胞色素c氧化还原酶14 kDa亚基VII的单核基因已通过一步基因破坏失活，这通过Southern印迹分析和免疫印迹得到验证。产生的突变体没有泛醇：细胞色素c氧化还原酶活性，并且呼吸缺陷。免疫印迹显示，缺乏14 kDa蛋白的细胞中，复合物III的其他亚基、核编码的11 kDa亚基VIII、 Rieske铁硫蛋白和线粒体编码的细胞色素b的稳态水平也降低。光谱法无法检测到细胞色素b。编码这些蛋白质的基因转录本的稳态水平没有降低，这意味着该突变在转录后水平发挥其多效性作用。复合物III亚基的残留量在突变体线粒体中得以恢复，表明导入不受影响。结果强烈表明，14 kDa蛋白在复合物的生物合成中起关键作用，很可能在组装水平上。使用场反转凝胶电泳分离HR2野生型和（14 kDa蛋白）缺失突变体的染色体，然后通过Southern印迹分析将编码14 kDa蛋白的基因定位在第四条染色体上。
