Novel insights into the assembly and function of human nuclear-encoded cytochrome c oxidase subunits 4, 5a, 6a, 7a and 7b
Novel insights into the assembly and function of human nuclear-encoded cytochrome c oxidase subunits 4, 5a, 6a, 7a and 7b
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DOI：
10.1042/bj20091714
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发表时间：
2010-06-15
影响因子：
4.1
通讯作者：
Zeman, Jiri
中科院分区：
文献类型：
--
作者：
Fornuskova, Daniela;Stiburek, Lukas;Zeman, Jiri
哺乳动物细胞色素c氧化酶(CcO)是由线粒体和核基因组编码的13个结构亚基组成的异寡聚蛋白质复合体。为了研究核编码的CcO亚基在人类复合体的组装和功能中的作用，我们使用了稳定的COX4、COX5a和COX6A1的RNA干扰以及表位标记的Cox6a、Cox7a和Cox7b在HEK-293细胞中的表达。Cox4、Cox5a和Cox6a基因敲除后，CcO活性降低，残存酶对氧的亲和力降低，全CcO和CcO二聚体水平降低，CcO亚复合体积累增加，呼吸超复合体模式发生改变。通过对敲除细胞和过表达细胞中CcO亚复合体模式的分析，确定了一种新的CcO组装中间体，确定了三个晚期组装亚基的入口点，并直接证明了Cox4和Cox5a组装的基本特征和相互依赖。心脏/肌肉特异性COX6亚基(COX6A2)的异位表达导致了COX6A1基因敲除细胞中CcO全酶和活性的恢复。这与这些细胞中COX6A2 mRNA的水平没有变化形成了鲜明的对比，表明存在一个固定的表达程序。我们所有的CcO缺陷敲除细胞系中呼吸复合体I的正常数量和功能表明，与非人类CcO缺陷模型不同，即使是相对少量的CcO也可以在培养的人类细胞中维持这种呼吸复合体的正常生物发生。
Mammalian CcO (cytochrome c oxidase) is a hetero-oligomeric protein complex composed of 13 structural subunits encoded by both the mitochondrial and nuclear genomes. To study the role of nuclear-encoded CcO subunits in the assembly and function of the human complex, we used stable RNA interference of COX4, COX5A and COX6A1, as well as expression of epitope-tagged Cox6a, Cox7a and Cox7b, in HEK (human embryonic kidney)-293 cells. Knockdown of Cox4, Cox5a and Cox6a resulted in reduced CcO activity, diminished affinity of the residual enzyme for oxygen, decreased holoCcO and CcO dimer levels, increased accumulation of CcO subcomplexes and gave rise to an altered pattern of respiratory supercomplexes. An analysis of the patterns of CcO subcomplexes found in both knockdown and overexpressing cells identified a novel CcO assembly intermediate, identified the entry points of three late-assembled subunits and demonstrated directly the essential character as well as the interdependence of the assembly of Cox4 and Cox5a. The ectopic expression of the heart/muscle-specific isoform of the Cox6 subunit (COX6A2) resulted in restoration of both CcO holoenzyme and activity in COX6A1-knockdown cells. This was in sharp contrast with the unaltered levels of COX6A2 mRNA in these cells, suggesting the existence of a fixed expression programme. The normal amount and function of respiratory complex I in all of our CcO-deficient knockdown cell lines suggest that, unlike non-human CcO-deficient models, even relatively small amounts of CcO can maintain the normal biogenesis of this respiratory complex in cultured human cells.