Montreal Neurological Institute and Department of Human Genetics, McGill University, Montreal, QC, Canada H3A 2B4.
Hum Mol Genet. 2012 Sep 1;21(17):3815-24. doi: 10.1093/hmg/dds209. Epub 2012 May 31.
Complex I (CI, NADH ubiquinone oxidoreductase), the largest complex of the respiratory chain, is composed of 45 structural subunits, 7 of which are encoded in mtDNA. At least 10 factors necessary for holoenzyme assembly have been identified; however, the specific roles of most of them are not well understood. We investigated the role of NDUFAF3, NDUFAF4, C8orf38 and C20orf7, four early assembly factors, in the translation of the mtDNA-encoded CI structural subunits. Transient, or stable, siRNA-mediated knock-down of any of these factors abrogated the assembly of CI, and resulted in a specific decrease in the labeling of the ND1 subunit in a pulse translation experiment, whereas knock-down of NDUFAF2, a late assembly factor, did not affect ND1 translation. Pulse-chase experiments in cells knocked down for NDUFAF3 showed that the half-life of ND1 in the chase was reduced 4-fold, fully accounting for the decrease in pulse labeling. Transient, short-term knock-down of the m-AAA protease AGF3L2 in cells that had been depleted of any of the early CI assembly factors completely rescued the ND1 labeling phenotype, confirming that it is not a synthesis defect, but rather results from rapid proteolysis of newly synthesized ND1. NDUFAF3 co-immunoprecipitated with NDUFAF4, and three matrix arm structural subunits (NDUFS2, NDUFA9, NDUFS3) that are found in a 400 kDa assembly intermediate containing ND1. These data suggest that the four early CI assembly factors have non-redundant functions in the assembly of a module that docks and stabilizes newly synthesized ND1, nucleating assembly of the holoenzyme.
复合体 I（CI，NADH 泛醌氧化还原酶）是呼吸链中最大的复合物，由 45 个结构亚基组成，其中 7 个由 mtDNA 编码。已经鉴定出至少 10 种组装全酶所必需的因子；然而，它们大多数的具体作用还不是很清楚。我们研究了四个早期组装因子 NDUFAF3、NDUFAF4、C8orf38 和 C20orf7 在 mtDNA 编码的 CI 结构亚基翻译中的作用。瞬时或稳定的 siRNA 介导的这些因子之一的敲低会破坏 CI 的组装，并导致脉冲翻译实验中 ND1 亚基的标记特异性减少，而敲低晚期组装因子 NDUFAF2 则不会影响 ND1 的翻译。在敲低 NDUFAF3 的细胞中进行的脉冲追踪实验表明，ND1 在追踪过程中的半衰期减少了 4 倍，完全解释了脉冲标记的减少。在耗尽任何早期 CI 组装因子的细胞中瞬时、短期敲低 m-AAA 蛋白酶 AGF3L2 完全挽救了 ND1 标记表型，证实这不是合成缺陷，而是由于新合成的 ND1 的快速蛋白水解。NDUFAF3 与 NDUFAF4 共免疫沉淀，并与三个基质臂结构亚基（NDUFS2、NDUFA9、NDUFS3）共沉淀，这些亚基存在于包含 ND1 的 400 kDa 组装中间物中。这些数据表明，这四个早期的 CI 组装因子在一个模块的组装中具有非冗余的功能，该模块对接并稳定新合成的 ND1，为全酶的组装提供起始点。
