A novel connection between Trypanosoma brucei mitochondrial proteins TbTim17 and TbTRAP1 is discovered using Biotinylation Identification (BioID)

The protein translocase of the mitochondrial inner mem-brane in Trypanosoma brucei, TbTIM17, forms a modular complex in association with several other trypanosome -specific proteins. To identify transiently interacting proximal partner(s) of TbTim17, we used Biotinylation Identification (BioID) by expressing a modified biotin ligase-TbTim17 (BirA*-TbTim17) fusion protein in T. brucei. BirA*-TbTim17 was targeted to mitochondria and assembled in the TbTIM complex. In the presence of biotin, BirA*-TbTim17 bio-tinylated several mitochondrial proteins. Interestingly, TbHsp84/TbTRAP1, a mitochondrial Hsp90 homolog, was identified as the highest enriched biotinylated proteins. We validated that interaction and colocalization of TbTim17 and TbHsp84 in T. brucei mitochondria by coimmunoprecipita-tion analysis and confocal microscopy, respectively. TbTim17 association with TbTRAP1 increased several folds during denaturation/renaturation of mitochondrial proteins in vitro, suggesting TbTRAP1 acts as a chaperone for TbTim17 refolding. We demonstrated that knockdown of TbTRAP1 reduced cell growth and decreased the levels of the TbTIM17, TbTim62, and mitochondrial (m)Hsp70 complexes. However, ATPase, VDAC, and Atom69 complexes were minimally affected. Additionally, the steady state levels of TbTim17, TbTim62, and mHsp70 were reduced significantly, but Atom69, ATPase beta, and RBP16 were mostly unaltered due to TbTRAP1 knockdown. Quantitative proteomics analysis also showed significant reduction of TbTim62 along with a few other mitochondrial proteins due to TbTRAP1 knockdown. Finally, TbTRAP1 depletion did not hamper the import of the ectopically expressed TbTim17-2xMyc into mitochondria but reduced its assembly into the TbTIM17 complex, indicating TbTRAP1 is critical for assembly of TbTim17. This is the first report showing the role of TRAP1 in the TIM complex as-sembly in eukaryotes.

Automated summary

In this study, the interaction and colocalization of TbTIM17 and TbTRAP1 in Trypanosoma brucei mitochondria were identified. TbTRAP1 plays a critical role in the refolding of TbTim17 and the assembly of the TbTIM17 complex. Knockdown of TbTRAP1 leads to reduced cell growth and decreased levels of mitochondrial complexes.