The oxidative phosphorylation (OXPHOS) system is composed of five multimeric complexes that work together to produce the bulk of cellular energy. Mutations in subunits of any of these complexes or proteins required for their biogenesis may lead to primary mitochondrial diseases. While most OXPHOS subunits are nuclear-encoded, 13 proteins are encoded by mitochondrial DNA (mtDNA). These mtDNA-encoded proteins are highly hydrophobic, thus, upon their translation by the mitochondrial ribosome (mitoribosome), the nascent polypeptides must simultaneously be inserted into the inner mitochondrial membrane, a process achieved when the mitoribosome binds the general insertase OXA1L during active translation. Helper proteins called assembly factors are then involved to incorporate these mtDNA-encoded subunits into the different OXPHOS complexes.
Although prior studies have provided insights to the players involved in the biogenesis and assembly of mtDNA-encoded proteins into their respective complexes, those associated with the co-translational insertion of these subunits remain to be fully elucidated. We hypothesise there exists novel proteins involved in this process either as general translation factors or subunit-specific assembly factors. Here, we employed Split-TurboID (sTID), a two-fragment proximity labelling system that requires the interaction of two proteins, to identify the transient interactors of the co-translational insertion machinery. We generated a cell line that co-expresses sTID-tagged OXA1L and one other bait protein, either an assembly factor or a mitoribosome protein (MRP). Interaction between the bait proteins allowed reconstitution of the TurboID enzyme, leading to the biotinylation of proximal proteins upon induction. Proteomic analyses performed on the enriched biotinylated proteins have revealed novel candidate proteins at the translation-membrane insertion site, providing the basis for future investigations into their roles in the co-translational insertion of mtDNA-encoded proteins.