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A novel connection between Trypanosoma brucei mitochondrial proteins TbTim17 and TbTRAP1 is discovered using Biotinylation Identification (BioID)
The protein translocase of the mitochondrial inner membrane 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 exp...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Society for Biochemistry and Molecular Biology
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9694106/ https://www.ncbi.nlm.nih.gov/pubmed/36309084 http://dx.doi.org/10.1016/j.jbc.2022.102647 |
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author | Soto-Gonzalez, Fidel Tripathi, Anuj Cooley, Ayorinde Paromov, Victor Rana, Tanu Chaudhuri, Minu |
author_facet | Soto-Gonzalez, Fidel Tripathi, Anuj Cooley, Ayorinde Paromov, Victor Rana, Tanu Chaudhuri, Minu |
author_sort | Soto-Gonzalez, Fidel |
collection | PubMed |
description | The protein translocase of the mitochondrial inner membrane 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 biotinylated 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 coimmunoprecipitation 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 β, 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 assembly in eukaryotes. |
format | Online Article Text |
id | pubmed-9694106 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | American Society for Biochemistry and Molecular Biology |
record_format | MEDLINE/PubMed |
spelling | pubmed-96941062022-11-28 A novel connection between Trypanosoma brucei mitochondrial proteins TbTim17 and TbTRAP1 is discovered using Biotinylation Identification (BioID) Soto-Gonzalez, Fidel Tripathi, Anuj Cooley, Ayorinde Paromov, Victor Rana, Tanu Chaudhuri, Minu J Biol Chem Research Article The protein translocase of the mitochondrial inner membrane 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 biotinylated 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 coimmunoprecipitation 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 β, 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 assembly in eukaryotes. American Society for Biochemistry and Molecular Biology 2022-10-26 /pmc/articles/PMC9694106/ /pubmed/36309084 http://dx.doi.org/10.1016/j.jbc.2022.102647 Text en © 2022 The Authors https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). |
spellingShingle | Research Article Soto-Gonzalez, Fidel Tripathi, Anuj Cooley, Ayorinde Paromov, Victor Rana, Tanu Chaudhuri, Minu A novel connection between Trypanosoma brucei mitochondrial proteins TbTim17 and TbTRAP1 is discovered using Biotinylation Identification (BioID) |
title | A novel connection between Trypanosoma brucei mitochondrial proteins TbTim17 and TbTRAP1 is discovered using Biotinylation Identification (BioID) |
title_full | A novel connection between Trypanosoma brucei mitochondrial proteins TbTim17 and TbTRAP1 is discovered using Biotinylation Identification (BioID) |
title_fullStr | A novel connection between Trypanosoma brucei mitochondrial proteins TbTim17 and TbTRAP1 is discovered using Biotinylation Identification (BioID) |
title_full_unstemmed | A novel connection between Trypanosoma brucei mitochondrial proteins TbTim17 and TbTRAP1 is discovered using Biotinylation Identification (BioID) |
title_short | A novel connection between Trypanosoma brucei mitochondrial proteins TbTim17 and TbTRAP1 is discovered using Biotinylation Identification (BioID) |
title_sort | novel connection between trypanosoma brucei mitochondrial proteins tbtim17 and tbtrap1 is discovered using biotinylation identification (bioid) |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9694106/ https://www.ncbi.nlm.nih.gov/pubmed/36309084 http://dx.doi.org/10.1016/j.jbc.2022.102647 |
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