Applying Sodium Carbonate Extraction Mass Spectrometry to Investigate Defects in the Mitochondrial Respiratory Chain.

David R. L. Robinson,Daniella H. Hock,Nikeisha J. Caruana,Linden Muellner-Wong,Elliot E. Surgenor,Boris Reljic,David A. Stroud,Carlos H. M. Rodrigues,Roopasingam Kugapreethan

doi:10.3389/fcell.2022.786268

Abstract

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.

Highlights

Mitochondria are double membrane-bound eukaryotic organelles that perform critical cellular functions including producing the bulk of cellular energy, acting as hubs for synthesis of various biomolecules, and driving the apoptotic response
Complex I N-module/matrix arm subunit NDUFV1 and assembly factor NDUFAF1, both peripherally associated with the membrane (Stroud et al, 2016), along with import translocase components TIMM50 and mt-HSP70 are found in both fractions at pH 11.5
At pH 9.5, a reduction in carbonate extraction is observed for each protein, with only cytochrome c and mt-HSP70 found in equal proportions in both the pellet and supernatant fractions, possibly due to their localization in both soluble and peripherally membrane associated pools (Kang et al, 2018;Timón-Gómez et al, 2018)

Summary

Introduction

Mitochondria are double membrane-bound eukaryotic organelles that perform critical cellular functions including producing the bulk of cellular energy, acting as hubs for synthesis of various biomolecules, and driving the apoptotic response. Mitochondria produce the majority of the cell’s ATP through oxidative phosphorylation (OXPHOS), which involves five multi-protein complexes (complexes I-IV and the F1-F0 ATP Synthase, or complex V) found within the inner mitochondrial. Mitochondria evolved from endosymbiotic prokaryotes, and have their own genome, known as mitochondrial DNA (mtDNA), and their own ribosomes, known as mitoribosomes. Following synthesis in the cytosol, nuclear encoded proteins are imported into the mitochondria through translocases of the inner and outer membranes, TIM and TOM respectively (Jackson et al, 2018; Pfanner et al, 2019). 13 proteins are encoded by mtDNA, all hydrophobic transmembrane subunits of the OXPHOS complexes that are translated on mitoribosomes and co-translationally inserted into the IMM (Ott and Herrmann, 2010; Thompson et al, 2018)

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Conclusion