Nijtmans L G, Spelbrink J N, Van Galen M J, Zwaan M, Klement P, Van den Bogert C
Department of Neurology, University of Amsterdam, Academic Medical Center, The Netherlands.
Biochim Biophys Acta. 1995 Mar 16;1265(2-3):117-26. doi: 10.1016/0167-4889(94)00203-q.
Synthesis, import, assembly and turnover of the nuclearly encoded subunits of cytochrome-c oxidase were investigated in cultured human cells depleted of mitochondrial gene products by continuous inhibition of mitochondrial protein synthesis (OP- cells). Immunoprecipitation after pulse labeling demonstrated that the synthesis of the nuclear subunits was not preferentially inhibited, implying that there is no tight regulation in the synthesis of mitochondrial and nuclear subunits of mitochondrial enzyme complexes. Quantitative analysis of the mitochondrial membrane potential in OP- cells indicated that its magnitude was about 30% of that in control cells. This explains the normal import of the nuclearly encoded subunits of cytochrome-c oxidase and other nuclearly encoded mitochondrial proteins into the mitochondria that was found in OP- cells. The turnover rate of nuclear subunits of cytochrome-c oxidase, determined in pulse-chase experiments, showed a specific increase in OP- cells. Moreover, immunoblotting demonstrated that the steady-state levels of nuclear subunits of cytochrome-c oxidase were severely reduced in these cells, in contrast to those of the F1 part of complex V. Native electrophoresis of mitochondrial enzyme complexes showed that assembly of the nuclear subunits of cytochrome-c oxidase did not occur in OP- cells, whereas the (nuclear) subunits of F1 were assembled. The increased turnover of the nuclear subunits of cytochrome-c oxidase in OP- cells is, therefore, most likely due to an increased susceptibility of unassembled subunits to intra-mitochondrial degradation.
通过持续抑制线粒体蛋白质合成（OP-细胞），在耗尽线粒体基因产物的培养人细胞中研究了细胞色素c氧化酶核编码亚基的合成、导入、组装和周转。脉冲标记后的免疫沉淀表明，核亚基的合成没有受到优先抑制，这意味着线粒体酶复合物的线粒体亚基和核亚基的合成没有严格调控。对OP-细胞中线粒体膜电位的定量分析表明，其幅度约为对照细胞的30%。这解释了在OP-细胞中发现的细胞色素c氧化酶的核编码亚基和其他核编码线粒体蛋白正常导入线粒体的现象。在脉冲追踪实验中测定的细胞色素c氧化酶核亚基的周转速率显示，OP-细胞中有特异性增加。此外，免疫印迹表明，与复合物V的F1部分相比，这些细胞中细胞色素c氧化酶核亚基的稳态水平严重降低。线粒体酶复合物的天然电泳显示，OP-细胞中细胞色素c氧化酶的核亚基没有组装，而F1的（核）亚基则组装完成。因此，OP-细胞中细胞色素c氧化酶核亚基周转增加最可能是由于未组装亚基对线粒体内降解的敏感性增加。
