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

ABSTRACT: 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.

INSTRUMENT(S): Orbitrap Eclipse, Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

SUBMITTER: David Stroud  

LAB HEAD: David Stroud

PROVIDER: PXD028935 | Pride | 2022-03-23

REPOSITORIES: Pride