Marques Isabel, Dencher Norbert A, Videira Arnaldo, Krause Frank
Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal.
Eukaryot Cell. 2007 Dec;6(12):2391-405. doi: 10.1128/EC.00149-07. Epub 2007 Sep 14.
The existence of specific respiratory supercomplexes in mitochondria of most organisms has gained much momentum. However, its functional significance is still poorly understood. The availability of many deletion mutants in complex I (NADH:ubiquinone oxidoreductase) of Neurospora crassa, distinctly affected in the assembly process, offers unique opportunities to analyze the biogenesis of respiratory supercomplexes. Herein, we describe the role of complex I in assembly of respiratory complexes and supercomplexes as suggested by blue and colorless native polyacrylamide gel electrophoresis and mass spectrometry analyses of mildly solubilized mitochondria from the wild type and eight deletion mutants. As an important refinement of the fungal respirasome model, we found that the standard respiratory chain of N. crassa comprises putative complex I dimers in addition to I-III-IV and III-IV supercomplexes. Three Neurospora mutants able to assemble a complete complex I, lacking only the disrupted subunit, have respiratory supercomplexes, in particular I-III-IV supercomplexes and complex I dimers, like the wild-type strain. Furthermore, we were able to detect the I-III-IV supercomplexes in the nuo51 mutant with no overall enzymatic activity, representing the first example of inactive respirasomes. In addition, III-IV supercomplexes were also present in strains lacking an assembled complex I, namely, in four membrane arm subunit mutants as well as in the peripheral arm nuo30.4 mutant. In membrane arm mutants, high-molecular-mass species of the 30.4-kDa peripheral arm subunit comigrating with III-IV supercomplexes and/or the prohibitin complex were detected. The data presented herein suggest that the biogenesis of complex I is linked with its assembly into supercomplexes.
大多数生物体线粒体中特定呼吸超级复合体的存在已备受关注。然而，其功能意义仍知之甚少。粗糙脉孢菌复合体I（NADH：泛醌氧化还原酶）中有许多缺失突变体，其组装过程明显受到影响，这为分析呼吸超级复合体的生物发生提供了独特的机会。在此，我们通过对野生型和八个缺失突变体轻度溶解的线粒体进行蓝色和无色原聚丙烯酰胺凝胶电泳及质谱分析，描述了复合体I在呼吸复合体和超级复合体组装中的作用。作为真菌呼吸体模型的一项重要改进，我们发现粗糙脉孢菌的标准呼吸链除了包含I-III-IV和III-IV超级复合体外，还包括假定的复合体I二聚体。三个能够组装完整复合体I且仅缺少被破坏亚基的脉孢菌突变体，具有呼吸超级复合体，特别是I-III-IV超级复合体和复合体I二聚体，与野生型菌株类似。此外，我们能够在没有整体酶活性的nuo51突变体中检测到I-III-IV超级复合体，这代表了无活性呼吸体的首个实例。此外，III-IV超级复合体也存在于缺乏组装好的复合体I的菌株中，即在四个膜臂亚基突变体以及外周臂nuo30.4突变体中。在膜臂突变体中，检测到与III-IV超级复合体和/或禁止素复合体共迁移的30.4 kDa外周臂亚基的高分子量物种。本文提供的数据表明，复合体I的生物发生与其组装成超级复合体有关。
