Mammalian mitochondria assemble four complexes of the respiratory chain (RCI, RCIII, RCIV, and RCV) by combining 13 polypeptides synthesized within mitochondria on mitochondrial ribosomes (mitoribosomes) with over 70 polypeptides encoded in nuclear DNA, translated on cytoplasmic ribosomes, and imported into mitochondria. We have previously observed that mitoribosome assembly is inefficient because some mitoribosomal proteins are produced in excess, but whether this is the case for other mitochondrial assemblies such as the RCs is unclear. We report here that pulse-chase stable isotope labeling with amino acids in cell culture (SILAC) is a valuable technique to study RC assembly because it can reveal considerable differences in the assembly rates and efficiencies of the different complexes. The SILAC analyses of HeLa cells indicated that assembly of RCV, comprising F1/Fo-ATPase, is rapid with little excess subunit synthesis, but that assembly of RCI (i.e. NADH dehydrogenase) is far less efficient, with dramatic oversynthesis of numerous proteins, particularly in the matrix-exposed N and Q domains. Unassembled subunits were generally degraded within 3 h. We also observed differential assembly kinetics for individual complexes that were immunoprecipitated with complex-specific antibodies. Immunoprecipitation with an antibody that recognizes the ND1 subunit of RCI co-precipitated a number of proteins implicated in FeS cluster assembly and newly synthesized ubiquinol-cytochrome c reductase Rieske iron-sulfur polypeptide 1 (UQCRFS1), the Rieske FeS protein in RCIII, reflecting some coordination between RCI and RCIII assemblies. We propose that pulse-chase SILAC labeling is a useful tool for studying rates of protein complex assembly and degradation.

中文翻译：

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基于脉冲追踪SILAC的分析揭示了呼吸复合物的选择性过度合成和线粒体蛋白组分的快速更新。

哺乳动物的线粒体通过将线粒体内在线粒体核糖体（线粒体）上合成的13种多肽与在核DNA中编码的70多种多肽结合在一起，在呼吸道链中合成四种呼吸链（RCI，RCIII，RCIV和RCV），并翻译成胞质核糖体，然后导入线粒体。以前我们已经观察到线粒体装配效率低下，因为某些线粒体蛋白的产量过高，但是对于其他线粒体装配（如RC）是否如此尚不清楚。我们在此报告，在细胞培养物中用氨基酸进行脉冲追逐稳定同位素标记（SILAC）是研究RC组装的有价值的技术，因为它可以揭示不同复合物的组装速率和效率的显着差异。SILAC对HeLa细胞的分析表明，包含F1 / Fo-ATPase的RCV的组装快速，几乎没有多余的亚基合成，但是RCI（即NADH脱氢酶）的组装效率低得多，大量蛋白质特别是过度合成。在矩阵暴露的N和Q域中。未组装的亚基通常在3小时内降解。我们还观察到了用复合物特异性抗体免疫沉淀的单个复合物的组装动力学差异。用识别RCI的ND1亚基的抗体进行免疫沉淀，共同沉淀了许多与FeS簇组装有关的蛋白质和新合成的泛醇-细胞色素c还原酶Rieske铁-硫多肽1（UQCRFS1），这是RCIII中的Rieske FeS蛋白，反映了一些RCI和RCIII大会之间的协调。