Robinson David R L, Hock Daniella H, Muellner-Wong Linden, Kugapreethan Roopasingam, Reljic Boris, Surgenor Elliot E, Rodrigues Carlos H M, Caruana Nikeisha J, Stroud David A
Department of Biochemistry and Pharmacology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, VIC, Australia.
The Royal Children's Hospital, Murdoch Children's Research Institute, Parkville, VIC, Australia.
Front Cell Dev Biol. 2022 Mar 1;10:786268. doi: 10.3389/fcell.2022.786268. eCollection 2022.
Mitochondria are complex organelles containing 13 proteins encoded by mitochondrial DNA and over 1,000 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 comprised of more than 80 proteins with both membrane integral and peripheral associations and require soluble, membrane integral and peripherally associated assembly factor proteins for their biogenesis. Mutations causing human mitochondrial disease can lead to defective complex assembly due to the loss or altered function of the affected protein and subsequent destabilization of its interactors. Here we couple sodium carbonate extraction with quantitative mass spectrometry (SCE-MS) to track changes in the membrane association of the mitochondrial proteome across multiple human knockout cell lines. In addition to identifying the membrane association status of over 840 human mitochondrial proteins, we show how SCE-MS can be used to understand the impacts of defective complex assembly on protein solubility, giving insights into how specific subunits and sub-complexes become destabilized.
线粒体是复杂的细胞器，包含13种由线粒体DNA编码的蛋白质和1000多种由核DNA编码的蛋白质。许多线粒体蛋白与线粒体内膜或外膜相关联，要么是外周关联，要么作为整合膜蛋白，而其他蛋白则存在于两个可溶性线粒体区室之一，即线粒体基质和膜间隙中。氧化磷酸化系统的五个复合物的生物发生就是这种复杂性的例证。这些大型多亚基复合物由80多种蛋白质组成，既有膜整合关联又有外周关联，其生物发生需要可溶性、膜整合和外周关联的组装因子蛋白。导致人类线粒体疾病的突变可由于受影响蛋白质的功能丧失或改变以及随后其相互作用分子的不稳定而导致复合物组装缺陷。在这里，我们将碳酸钠提取与定量质谱联用（SCE-MS），以追踪多种人类基因敲除细胞系中线粒体蛋白质组膜关联的变化。除了确定840多种人类线粒体蛋白的膜关联状态外，我们还展示了SCE-MS如何用于了解复合物组装缺陷对蛋白质溶解度的影响，从而深入了解特定亚基和亚复合物如何变得不稳定。
