Endogenous TOM20 Proximity Labeling: A Swiss-Knife for the Study of Mitochondrial Proteins in Human Cells

Biotin-based proximity labeling approaches, such as BioID, have demonstrated their use for the study of mitochondria proteomes in living cells. The use of genetically engineered BioID cell lines enables the detailed characterization of poorly characterized processes such as mitochondrial co-translational import. In this process, translation is coupled to the translocation of the mitochondrial proteins, alleviating the energy cost typically associated with the post-translational import relying on chaperone systems. However, the mechanisms are still unclear with only few actors identified but none that have been described in mammals yet. We thus profiled the TOM20 proxisome using BioID, assuming that some of the identified proteins could be molecular actors of the co-translational import in human cells. The obtained results showed a high enrichment of RNA binding proteins close to the TOM complex. However, for the few selected candidates, we could not demonstrate a role in the mitochondrial co-translational import process. Nonetheless, we were able to demonstrate additional uses of our BioID cell line. Indeed, the experimental approach used in this study is thus proposed for the identification of mitochondrial co-translational import effectors and for the monitoring of protein entry inside mitochondria with a potential application in the prediction of mitochondrial protein half-life.

Automated summary

Biotin-based proximity labeling approaches, specifically BioID, have been used to study mitochondria proteomes in living cells. Genetically engineered cell lines enable detailed characterization of poorly understood processes (e.g., mitochondrial co-translational import). However, the mechanisms are still unclear, and few actors have been identified in mammals. Using BioID, the TOM20 proxosome was profiled, revealing enrichment of RNA binding proteins, although their role in the co-translational import process was not demonstrated for selected candidates.