Sodium carbonate extraction mass spectrometry reveals novel steps in mitochondrial respiratory chain complex III assembly

Mitochondria are complex organelles containing 13 proteins encoded by mitochondrial DNA and over 1000 proteins encoded on nuclear DNA. Many mitochondrial proteins are associated with the inner or outer mitochondrial membranes, either peripherally or as integral membrane proteins, while others reside in either of the two soluble mitochondrial compartments, the mitochondrial matrix and the intermembrane space. The biogenesis of the five complexes of the oxidative phosphorylation system are exemplars of this complexity. These large multi-subunit complexes are built through the tightly controlled coalescence of more than 80 proteins with both membrane integral and peripheral associations, with progression between different assembly steps dependent on soluble, membrane integral and peripherally associated assembly factor proteins. Understanding the membrane association of subunits and assembly factors during each assembly step is critical to understanding OXPHOS complex biogenesis. Here we couple sodium carbonate extraction with quantitative mass spectrometry to track changes in the membrane association of the mitochondrial proteome across multiple human complex III knockout cell lines. In addition to identifying the membrane association status of over 840 human mitochondrial proteins, we identify a previously undetected intermediate step of complex III biogenesis. Sodium carbonate extraction with quantitative mass spectrometry (SCE-MS) is thus an easy to apply tool with general utility in the study of mitochondrial respiratory chain biogenesis.

线粒体是一类复杂的细胞器，其包含13种由线粒体DNA（mitochondrial DNA, mtDNA）编码的蛋白质，以及千余种由核DNA编码的蛋白质。诸多线粒体蛋白质可通过外周结合或整合膜蛋白的形式定位于线粒体内膜或外膜；其余蛋白质则分布于线粒体的两个可溶性区室——线粒体基质（mitochondrial matrix）与膜间隙（intermembrane space）中。氧化磷酸化系统（oxidative phosphorylation system）五种复合物的生物发生过程，便是这种复杂性的典型范例。这些大型多亚基复合物，需通过受严格调控的组装过程形成：该过程涉及超过80种兼具膜整合与外周结合特性的蛋白质，不同组装步骤间的推进则依赖于可溶性、膜整合型及外周结合型组装因子蛋白。解析各组装步骤中亚基与组装因子的膜结合特性，对于理解氧化磷酸化复合物的生物发生过程至关重要。本研究将碳酸钠抽提（sodium carbonate extraction）与定量质谱（quantitative mass spectrometry）技术相结合，以追踪多株人类复合物III（complex III）敲除细胞系中线粒体蛋白质组（mitochondrial proteome）的膜结合特性变化。本研究不仅鉴定了超过840种人类线粒体蛋白质的膜结合状态，还发现了一条此前未被报道的复合物III生物发生中间步骤。综上，碳酸钠抽提联合定量质谱（sodium carbonate extraction with quantitative mass spectrometry, SCE-MS）是一种易于操作、可广泛应用于线粒体呼吸链生物发生研究的工具。