Identification and characterization of a common set of complex I assembly intermediates in mitochondria from patients with complex I deficiency
Identification and characterization of a common set of complex I assembly intermediates in mitochondria from patients with complex I deficiency
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DOI：
10.1074/jbc.m304998200
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发表时间：
2003-10-31
影响因子：
4.8
通讯作者：
Shoubridge, EA
中科院分区：
文献类型：
--
作者：
Antonicka, H;Ogilvie, I;Shoubridge, EA
复合物I （NADH：泛醌氧化还原酶）活性的缺乏是人类线粒体疾病的重要原因。复合体I由至少46个结构亚基组成，这些亚基编码于核DNA和线粒体DNA中。酶缺乏症可由催化效率受损或不能组装全酶复合物引起；然而，组装过程仍然知之甚少。我们使用二维Blue-Native/SDS凝胶电泳和一组针对酶结构亚基的11种抗体来研究4例由线粒体或核基因缺陷引起的复合物I缺乏患者肌肉线粒体中的复合物I组装。第二次元变性凝胶的免疫印迹分析在研究的患者中鉴定出7种不同的复合物I亚复合物，其中5种也可以在第一次元非变性凝胶中检测到。虽然这些中间产物的丰度在不同的患者中有所不同，但在所有病例中都观察到一个共同的亚复合物星座。类似的亚复合物存在于人/小鼠杂交成纤维细胞系中，由于几乎完全缺乏全酶复合物的组装，严重缺乏复合物I。复合物I缺乏的不同原因产生相似的复合物I亚复合物模式的发现表明，这些是全酶复合物组装的中间产物。我们提出了一种可能的复合物组装途径，它与目前复合物I组装模型Neurospora的组装途径有很大不同。
Deficiencies in the activity of complex I (NADH: ubiquinone oxidoreductase) are an important cause of human mitochondrial disease. Complex I is composed of at least 46 structural subunits that are encoded in both nuclear and mitochondrial DNA. Enzyme deficiency can result from either impaired catalytic efficiency or an inability to assemble the holoenzyme complex; however, the assembly process remains poorly understood. We have used two-dimensional Blue-Native/SDS gel electrophoresis and a panel of 11 antibodies directed against structural subunits of the enzyme to investigate complex I assembly in the muscle mitochondria from four patients with complex I deficiency caused by either mitochondrial or nuclear gene defects. Immunoblot analyses of second dimension denaturing gels identified seven distinct complex I subcomplexes in the patients studied, five of which could also be detected in non-denaturing gels in the first dimension. Although the abundance of these intermediates varied among the different patients, a common constellation of subcomplexes was observed in all cases. A similar profile of subcomplexes was present in a human/mouse hybrid fibroblast cell line with a severe complex I deficiency due to an almost complete lack of assembly of the holoenzyme complex. The finding that diverse causes of complex I deficiency produce a similar pattern of complex I subcomplexes suggests that these are intermediates in the assembly of the holoenzyme complex. We propose a possible assembly pathway for the complex, which differs significantly from that proposed for Neurospora, the current model for complex I assembly.