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

Abstract

The majority of mitochondrial proteins are encoded by the nuclear genome and must be imported into the mitochondria. There are two main paths for mitochondrial protein import: post-translational and co-translational import. Co-translational import couples the translation and the translocation of the mitochondrial proteins, alleviating the energy cost typically associated with the post-translational import relying on chaperone systems. The mitochondrial co-translational import mechanisms are still unclear with few actors identified but none have been described in mammals yet. We thus profiled the TOM20 proxisome using BioID, assuming that some of 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 a new mitochondrial localization for nuclear proteins. Besides, additional analyses revealed a negative correlation between the abundance of mitochondrial proteins and their reported half-life. This experimental approach is thus proposed to potentially characterize mitochondrial co-translational import effectors in human cells and to monitor protein entry inside mitochondria with a potential application in the prediction of mitochondrial protein half-life.

In-depth study of MPV17: a molecular travel unveiling a mitochondrial calcium regulation function

“…KO of MPV17 in HEK293T cells was performed using CRISPR-Cas9 technology as described previously (Meurant et al, 2023). Briefly, crRNAs targeting the exons 2 (5'-TTTCCACGGGTGAGCGGCCAGGG-3') (Integrated DNA Technologies (IDT), Coralville, USA) or 4 (5'-GGTTTGGATCGGTTCATCCCTGG-3') of the MPV17 gene were designed using CRISPOR bioinformatic resource (Concordet & Haeussler, 2018) and used separately or in combination.…”

“…Immunoprecipitation was performed as previously described (Meurant et al, 2023). HEK293T cells were scraped in RIPA buffer and the lysate was incubated for 30 min on a rotating wheel at 4 °C before a centrifugation of 10 min at 16.000× g at 4 °C.…”

“…For the mass spectrometry analyses performed on total protein lysates, 10 µg of proteins were digested by Filter Aided Sample Preparation (FASP) as previously described (Nicolas et al, 2023). All mass spectrometry analyses were performed as previously described (Meurant et al, 2023). Briefly, digested proteins were analyzed using nanoLC-ESI-MS/MS tims TOF Pro (Bruker, Billerica, USA) coupled with an UHPLC nanoElute (Bruker).…”

“…The BioID vectors, MPV17-BirA*, preOTC-BirA* and TWNK-BirA*, were constructed using gateway cloning, as described previously (Liu et al, 2018). Briefly, the Coding DNA Sequence (CDS) of human MPV17 was picked by PCR from HEK293T total cDNA whereas the presequence of the Ornithine Transcarbamylase was picked by PCR from the previously generated preOTC-DHFR plasmid (Meurant et al, 2023). Similarly, the CDS of the human TWNK gene was picked by PCR using the Twinkle-APEX2-V5 plasmid, a generous gift from Dr. Alice Ting (Han et al, 2017) (Addgene plasmid # 129705 ; http://n2t.net/addgene:129705 ; RRID:Addgene_129705).…”

Localization of RNAs to the Mitochondria – Mechanisms and Functions

“…Knockout of SYNJ2B abolished the localization of these mRNAs at the OMM and impaired the cellular stress response by compromising the function of oxidative phosphorylation complexes. Another recent study identified the proteins in proximity to TOM20 (OMM translocase) in human cells and found an overall enrichment of RBPs near TOM20, specifically proteins like AKAP1, LARP4, MED15, and CPSF2, thereby providing a candidate list of RBPs that might be involved in guiding mRNAs to the OMM 25 . Thus, although many RBPs are known to interact with nuclear-encoded mitochondrial transcripts, further studies are required to show that these RBPs play a role in RNA localization.…”