A Forward Genetic Screen Identifies Mutants Deficient for Mitochondrial Complex I Assembly in Chlamydomonas reinhardtii
A Forward Genetic Screen Identifies Mutants Deficient for Mitochondrial Complex I Assembly in Chlamydomonas reinhardtii
复制标题
DOI：
10.1534/genetics.111.128827
复制
发表时间：
2011-06-01
期刊：
影响因子：
3.3
通讯作者：
Hamel, Patrice P.
中科院分区：
文献类型：
--
作者：
Barbieri, M. Rosario;Larosa, Veronique;Hamel, Patrice P.
线粒体复合物 I 是呼吸链中最大的多聚酶。缺乏具有简单遗传学的模型系统限制了复杂 I 组装的分子解剖。使用莱茵衣藻作为筛选复合物 I 缺陷的实验系统，我们通过正向遗传学分离出 amc1-7 核突变体（用于组装线粒体复合物 I），显示复合物 I 活性降低或无复合物 I 活性。蓝色天然 (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 突变体均无法积累成熟复合物。在 amc1、-2、-5-7 中，检测到保留 NADH 脱氢酶活性的 700 kDa 亚复合物表明组装过程发生停滞。遗传分析确定 amc5 和 amc7 是同一基因座的等位基因，而 amc1-4 和 amc6 定义不同的互补组。 amc5 和 amc7 等位基因定义的基因座对应于 NUOB10 基因，编码 PDSW（复合物 I 膜臂的一个亚基）。这是分离出复合物 I 突变体的正向遗传筛选的第一份报告。这项工作说明了使用衣藻作为遗传上易处理的生物体来破译复合物 I 制造的潜力。
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.