In vivo regulation of oxidative phosphorylation in cells harboring a stop-codon mutation in mitochondrial DNA-encoded cytochrome c oxidase subunit I
In vivo regulation of oxidative phosphorylation in cells harboring a stop-codon mutation in mitochondrial DNA-encoded cytochrome c oxidase subunit I
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DOI：
10.1074/jbc.m106429200
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发表时间：
2001-12-14
影响因子：
4.8
通讯作者：
Manfredi, G
中科院分区：
文献类型：
--
作者：
D'Aurelio, M;Pallotti, F;Manfredi, G
调控哺乳动物细胞氧化磷酸化的机制在很大程度上是未知的。为了解决这个问题，通过将缺乏线粒体DNA(MtDNA)的骨肉瘤细胞与来自细胞色素C氧化酶亚基I(COX I)终止密码子突变的患者的血小板融合而产生了胞质杂交。对线粒体DNA突变水平不同的线虫的分子和生化特性进行了研究。我们发现突变的COX I DNA和突变的COX I mRNA之间存在直接的相关性，而COX I总的mRNA水平没有变化。COX I多肽合成和稳态水平与突变水平成反比。细胞色素c氧化酶亚基Il与COX I成比例减少，表明复杂组装中的损伤。COX酶活性与mtDNA突变水平成反比。然而，在突变基因组比例较低的细胞中，细胞呼吸和ATP合成都得到了保护，阈值接近40%，并随着突变mtDNA的增加而线性下降。这些结果表明，突变细胞中的COX水平不受转录、翻译和翻译后水平的调节。由于COX能力有少量过剩，COX亚基的表达水平对氧化磷酸化起到了相对严格的控制作用。
The mechanisms that regulate oxidative phosphorylation in mammalian cells are largely unknown. To address this issue, cybrids were generated by fusing osteosarcoma cells devoid of mitochondrial DNA (mtDNA) with platelets from a patient with a stop-codon mutation in cytochrome c oxidase subunit I (COX I). The molecular and biochemical characteristics of cybrids harboring varying levels of mutated mitochondrial DNA were studied. We found a direct correlation between the levels of mutated COX I DNA and mutated COX I mRNA, whereas the levels of COX I total mRNA were unchanged. COX I polypeptide synthesis and steady-state levels were inversely proportional to mutation levels. Cytochrome c oxidase subunit Il was reduced proportionally to COX I, indicating impairment in complex assembly. COX enzymatic activity was inversely proportional to the levels of mutated mtDNA. However, both cell respiration and ATP synthesis were preserved in cells with lower proportions of mutated genomes, with a threshold at similar to 40%, and decreased linearly with increasing mutated mtDNA. These results indicate that COX levels in mutated cells were not regulated at the transcriptional, translational, and post-translational levels. Because of a small excess of COX capacity, the levels of expression of COX subunits exerted a relatively tight control on oxidative phosphorylation.