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Unique Interactions of the Small Translocases of the Mitochondrial Inner Membrane (Tims) in Trypanosoma brucei.

ABSTRACT: The infectious agent for African trypanosomiasis, Trypanosoma brucei, possesses a unique and essential translocase of the mitochondrial inner membrane, known as the TbTIM17 complex. TbTim17 associates with six small TbTims (TbTim9, TbTim10, TbTim11, TbTim12, TbTim13, and TbTim8/13). However, the interaction patterns of these smaller TbTims with each other and TbTim17 are not clear. Through yeast two-hybrid (Y2H) and co-immunoprecipitation analyses, we demonstrate that all six small TbTims interact with each other. Stronger interactions were found among TbTim8/13, TbTim9, and TbTim10. However, TbTim10 shows weaker associations with TbTim13, which has a stronger connection with TbTim17. Each of the small TbTims also interacts strongly with the C-terminal region of TbTim17. RNAi studies indicated that among all small TbTims, TbTim13 is most crucial for maintaining the steady-state levels of the TbTIM17 complex. Further analysis of the small TbTim complexes by size exclusion chromatography revealed that each small TbTim, except for TbTim13, is present in ~70 kDa complexes, possibly existing in heterohexameric forms. In contrast, TbTim13 is primarily present in the larger complex (>800 kDa) and co-fractionates with TbTim17. Altogether, our results demonstrate that, relative to other eukaryotes, the architecture and function of the small TbTim complexes are specific to T. brucei.

SUBMITTER: Quinones LS

PROVIDER: S-EPMC10855554 | biostudies-literature | 2024 Jan

REPOSITORIES: biostudies-literature

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Unique Interactions of the Small Translocases of the Mitochondrial Inner Membrane (Tims) in <i>Trypanosoma brucei</i>.

Quiñones Linda S LS Gonzalez Fidel Soto FS Darden Chauncey C Khan Muhammad M Tripathi Anuj A Smith Joseph T JT Davis Jamaine J Misra Smita S Chaudhuri Minu M

International journal of molecular sciences 20240124 3

The infectious agent for African trypanosomiasis, <i>Trypanosoma brucei</i>, possesses a unique and essential translocase of the mitochondrial inner membrane, known as the TbTIM17 complex. TbTim17 associates with six small TbTims (TbTim9, TbTim10, TbTim11, TbTim12, TbTim13, and TbTim8/13). However, the interaction patterns of these smaller TbTims with each other and TbTim17 are not clear. Through yeast two-hybrid (Y2H) and co-immunoprecipitation analyses, we demonstrate that all six small TbTims ...[more]

PMID: 38338692

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Project description:Trypanosomatids are unicellular parasitic protozoa. Many of the species of this genera cause severe diseases in human, such as Leishmaniasis, African trypanosomiasis and Chagas disease. These parasites possess a single reticular mitochondrion with a concatenated structure of mitochondrial DNA known as kinetoplast or kDNA. kDNA encodes few essential mitochondrial proteins but no tRNAs. Therefore, trypanosomatid mitochondrion import a full set of nucleus-encoded tRNAs for mitochondrial translation. Recent advances indicated that mitochondrial protein translocases, particularly the subunits of the ATOM complex, are involved in the import of a tRNA in Trypanosoma brucei. However, the global picture and the role of the translocase components of the mitochondrial inner membrane (TbTims) are not well understood. Here we investigated the relative abundance of 16 different tRNAs in the cytosolic and mitochondrial fractions isolated from the six TbTims knockdown cell lines. We found that knockdown of TbTim17, one of the primary components of the TbTIM complex, reduced the abundance of all of these tRNAs into mitochondria and increased their abundance in the cytosol. Depletion of TbTim62, a TbTim17 associated proteins, also reduced the relative abundance of most of these tRNAs into mitochondria except for tRNAleu, tRNAmet, and tRNAglu. Whereas, knockdown of other TbTims, like TbTim50 and two small TbTims, TbTim10 and TbTim8/13, didn't have any effect on tRNA abundance either in the cytosol or mitochondria. Depletion of any of these TbTims showed minimal effect on the levels of total tRNAs in T. brucei. Absolute quantification of tRNA levels revealed that TbTim17 knockdown reduced the levels of different tRNAs in mitochondria from 3-6% to 0.8-1.4%, which is equivalent to ~70% reduction in average. Whereas, TbTim62 depletion showed somewhat selective effect. Overall, our results suggest that TbTim17 and TbTim62 are essential for tRNA import that further makes a connection between the tRNA and protein import into mitochondria in T. brucei.

Dataset Information

Unique Interactions of the Small Translocases of the Mitochondrial Inner Membrane (Tims) in Trypanosoma brucei.

Publications

Unique Interactions of the Small Translocases of the Mitochondrial Inner Membrane (Tims) in <i>Trypanosoma brucei</i>.

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