Department of Molecular Genetics and Department of Molecular Cellular Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA.
Genetics. 2011 Jun;188(2):349-58. doi: 10.1534/genetics.111.128827. Epub 2011 Apr 5.
Mitochondrial complex I is the largest multimeric enzyme of the respiratory chain. The lack of a model system with facile genetics has limited the molecular dissection of complex I assembly. Using Chlamydomonas reinhardtii as an experimental system to screen for complex I defects, we isolated, via forward genetics, amc1-7 nuclear mutants (for assembly of mitochondrial complex I) displaying reduced or no complex I activity. Blue native (BN)-PAGE and immunoblot analyses revealed that amc3 and amc4 accumulate reduced levels of the complex I holoenzyme (950 kDa) while all other amc mutants fail to accumulate a mature complex. In amc1, -2, -5-7, the detection of a 700 kDa subcomplex retaining NADH dehydrogenase activity indicates an arrest in the assembly process. Genetic analyses established that amc5 and amc7 are alleles of the same locus while amc1-4 and amc6 define distinct complementation groups. The locus defined by the amc5 and amc7 alleles corresponds to the NUOB10 gene, encoding PDSW, a subunit of the membrane arm of complex I. This is the first report of a forward genetic screen yielding the isolation of complex I mutants. This work illustrates the potential of using Chlamydomonas as a genetically tractable organism to decipher complex I manufacture.
线粒体复合物 I 是呼吸链中最大的多聚酶。由于缺乏具有简便遗传学的模型系统，限制了对复合物 I 组装的分子剖析。我们使用莱茵衣藻作为实验系统，通过正向遗传学筛选复合物 I 缺陷，分离出了 amc1-7 核突变体（用于线粒体复合物 I 的组装），其表现出复合物 I 活性降低或缺失。蓝色非变性（BN）-PAGE 和免疫印迹分析表明，amc3 和 amc4 积累的复合物 I 全酶（950 kDa）水平降低，而所有其他 amc 突变体都无法积累成熟的复合物。在 amc1、-2、-5-7 中，检测到保留 NADH 脱氢酶活性的 700 kDa 亚基表明组装过程停滞。遗传分析表明，amc5 和 amc7 是同一基因座的等位基因，而 amc1-4 和 amc6 则定义了不同的互补群。由 amc5 和 amc7 等位基因定义的基因座与编码复合物 I 膜臂亚基 PDSW 的 NUOB10 基因相对应。这是首次通过正向遗传筛选分离出复合物 I 突变体的报道。这项工作说明了利用衣藻作为遗传上易于处理的生物体来阐明复合物 I 制造的潜力。
