This thesis was divided in two different parts dedicated to the study of a mitochondrial process and of a mitochondrial protein using a proximity labelling assay called BioID.
The first project was dedicated to the study of a poorly characterized process, the 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 endogenously the TOM20 proxisome using BioID. Despite the enrichment of RNA binding proteins in the TOM20 proxisome, we could not demonstrate a role for a selected candidate, LARP4, in the mitochondrial co-translational import process. Nonetheless, additional uses of this BioID cell line were highlighted such as the monitoring of protein entry inside mitochondria and a potential application in the prediction of mitochondrial protein half-life.
The second project was dedicated to the study of MPV17, a protein of the inner mitochondrial membrane whose gene is associated with mitochondrial DNA depletion syndrome. However, the exact molecular function of the protein is still unclear. The approach used in this project was to identify the interacting partners of MPV17, using BioID, to get additional clues about the protein function. In this project we demonstrated an interaction of MPV17 with the MICOS complex but the KO of MPV17 didn’t impact mitochondria ultrastructure. However, the depletion of MPV17 protein led to increased mitochondria-derived vesicles formation. Therefore, we investigated a potential degradation of the mtDNA as the cause of the mtDNA depletion observed in MPV17 KO cells but, despite a higher mitophagy level in KO cells, the blockade of the lysosomal activity didn’t prevent the depletion. Additional in silico analyses suggested a channel activity of MPV17 further supported by its direct interaction with the Cyclophilin D, a protein of the mitochondrial permeability transition pore. Interestingly, MPV17 KO cells also display higher level of mitochondrial calcium which would be related to the degradation of the mtDNA since the blockade of mitochondrial calcium entry prevents the depletion. We thus propose a role of MPV17 as a potential new member of the mitochondrial permeability transition pore whereas in the absence of the protein, the build-up of calcium inside the mitochondria would lead to the observed mtDNA degradation.
