Divergent Small Tim Homologues Are Associated with TbTim17 and Critical for the Biogenesis of TbTim17 Protein Complexes in <italic toggle="yes">Trypanosoma brucei</italic>

ABSTRACT The small Tim proteins belong to a group of mitochondrial intermembrane space chaperones that aid in the import of mitochondrial inner membrane proteins with internal targeting signals. Trypanosoma brucei, the protozoan parasite that causes African trypanosomiasis, possesses multiple small...

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Autores principales:	Joseph T. Smith, Ujjal K. Singha, Smita Misra, Minu Chaudhuri
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2018
Materias:	TbTim17 Trypanosoma brucei mitochondria protein import small Tims Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/43bbb2ae141f42e1b880b53895b28cce
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spelling	oai:doaj.org-article:43bbb2ae141f42e1b880b53895b28cce2021-11-15T15:24:22ZDivergent Small Tim Homologues Are Associated with TbTim17 and Critical for the Biogenesis of TbTim17 Protein Complexes in <italic toggle="yes">Trypanosoma brucei</italic>10.1128/mSphere.00204-182379-5042https://doaj.org/article/43bbb2ae141f42e1b880b53895b28cce2018-06-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00204-18https://doaj.org/toc/2379-5042ABSTRACT The small Tim proteins belong to a group of mitochondrial intermembrane space chaperones that aid in the import of mitochondrial inner membrane proteins with internal targeting signals. Trypanosoma brucei, the protozoan parasite that causes African trypanosomiasis, possesses multiple small Tim proteins that include homologues of T. brucei Tim9 (TbTim9) and Tim10 (TbTim10) and a unique small Tim that shares homology with both Tim8 and Tim13 (TbTim8/13). Here, we found that these three small TbTims are expressed as soluble mitochondrial intermembrane space proteins. Coimmunoprecipitation and mass spectrometry analysis showed that the small TbTims stably associated with each other and with TbTim17, the major component of the mitochondrial inner membrane translocase in T. brucei. Yeast two-hybrid analysis indicated direct interactions among the small TbTims; however, their interaction patterns appeared to be different from those of their counterparts in yeast and humans. Knockdown of the small TbTims reduced cell growth and decreased the steady-state level of TbTim17 and T. brucei ADP/ATP carrier (TbAAC), two polytopic mitochondrial inner membrane proteins. Knockdown of small TbTims also reduced the matured complexes of TbTim17 in mitochondria. Depletion of any of the small TbTims reduced TbTim17 import moderately but greatly hampered the stability of the TbTim17 complexes in T. brucei. Altogether, our results revealed that TbTim9, TbTim10, and TbTim8/13 interact with each other, associate with TbTim17, and play a crucial role in the integrity and maintenance of the levels of TbTim17 complexes. IMPORTANCE Trypanosoma brucei is the causative agent of African sleeping sickness. The parasite’s mitochondrion represents a useful source for potential chemotherapeutic targets. Similarly to yeast and humans, mitochondrial functions depend on the import of proteins that are encoded in the nucleus and made in the cytosol. Even though the machinery involved in this mitochondrial protein import process is becoming clearer in T. brucei, a comprehensive picture of protein complex composition and function is still lacking. In this study, we characterized three T. brucei small Tim proteins, TbTim9, TbTim10, and TbTim8/13. Although the parasite does not have the classical TIM22 complex that imports mitochondrial inner membrane proteins containing internal targeting signals in yeast or humans, we found that these small TbTims associate with TbTim17, the major subunit of the TbTIM complex in T. brucei, and play an essential role in the stability of the TbTim17 complexes. Therefore, these divergent proteins are critical for mitochondrial protein biogenesis in T. brucei.Joseph T. SmithUjjal K. SinghaSmita MisraMinu ChaudhuriAmerican Society for MicrobiologyarticleTbTim17Trypanosoma bruceimitochondriaprotein importsmall TimsMicrobiologyQR1-502ENmSphere, Vol 3, Iss 3 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	TbTim17 Trypanosoma brucei mitochondria protein import small Tims Microbiology QR1-502
spellingShingle	TbTim17 Trypanosoma brucei mitochondria protein import small Tims Microbiology QR1-502 Joseph T. Smith Ujjal K. Singha Smita Misra Minu Chaudhuri Divergent Small Tim Homologues Are Associated with TbTim17 and Critical for the Biogenesis of TbTim17 Protein Complexes in <italic toggle="yes">Trypanosoma brucei</italic>
description	ABSTRACT The small Tim proteins belong to a group of mitochondrial intermembrane space chaperones that aid in the import of mitochondrial inner membrane proteins with internal targeting signals. Trypanosoma brucei, the protozoan parasite that causes African trypanosomiasis, possesses multiple small Tim proteins that include homologues of T. brucei Tim9 (TbTim9) and Tim10 (TbTim10) and a unique small Tim that shares homology with both Tim8 and Tim13 (TbTim8/13). Here, we found that these three small TbTims are expressed as soluble mitochondrial intermembrane space proteins. Coimmunoprecipitation and mass spectrometry analysis showed that the small TbTims stably associated with each other and with TbTim17, the major component of the mitochondrial inner membrane translocase in T. brucei. Yeast two-hybrid analysis indicated direct interactions among the small TbTims; however, their interaction patterns appeared to be different from those of their counterparts in yeast and humans. Knockdown of the small TbTims reduced cell growth and decreased the steady-state level of TbTim17 and T. brucei ADP/ATP carrier (TbAAC), two polytopic mitochondrial inner membrane proteins. Knockdown of small TbTims also reduced the matured complexes of TbTim17 in mitochondria. Depletion of any of the small TbTims reduced TbTim17 import moderately but greatly hampered the stability of the TbTim17 complexes in T. brucei. Altogether, our results revealed that TbTim9, TbTim10, and TbTim8/13 interact with each other, associate with TbTim17, and play a crucial role in the integrity and maintenance of the levels of TbTim17 complexes. IMPORTANCE Trypanosoma brucei is the causative agent of African sleeping sickness. The parasite’s mitochondrion represents a useful source for potential chemotherapeutic targets. Similarly to yeast and humans, mitochondrial functions depend on the import of proteins that are encoded in the nucleus and made in the cytosol. Even though the machinery involved in this mitochondrial protein import process is becoming clearer in T. brucei, a comprehensive picture of protein complex composition and function is still lacking. In this study, we characterized three T. brucei small Tim proteins, TbTim9, TbTim10, and TbTim8/13. Although the parasite does not have the classical TIM22 complex that imports mitochondrial inner membrane proteins containing internal targeting signals in yeast or humans, we found that these small TbTims associate with TbTim17, the major subunit of the TbTIM complex in T. brucei, and play an essential role in the stability of the TbTim17 complexes. Therefore, these divergent proteins are critical for mitochondrial protein biogenesis in T. brucei.
format	article
author	Joseph T. Smith Ujjal K. Singha Smita Misra Minu Chaudhuri
author_facet	Joseph T. Smith Ujjal K. Singha Smita Misra Minu Chaudhuri
author_sort	Joseph T. Smith
title	Divergent Small Tim Homologues Are Associated with TbTim17 and Critical for the Biogenesis of TbTim17 Protein Complexes in <italic toggle="yes">Trypanosoma brucei</italic>
title_short	Divergent Small Tim Homologues Are Associated with TbTim17 and Critical for the Biogenesis of TbTim17 Protein Complexes in <italic toggle="yes">Trypanosoma brucei</italic>
title_full	Divergent Small Tim Homologues Are Associated with TbTim17 and Critical for the Biogenesis of TbTim17 Protein Complexes in <italic toggle="yes">Trypanosoma brucei</italic>
title_fullStr	Divergent Small Tim Homologues Are Associated with TbTim17 and Critical for the Biogenesis of TbTim17 Protein Complexes in <italic toggle="yes">Trypanosoma brucei</italic>
title_full_unstemmed	Divergent Small Tim Homologues Are Associated with TbTim17 and Critical for the Biogenesis of TbTim17 Protein Complexes in <italic toggle="yes">Trypanosoma brucei</italic>
title_sort	divergent small tim homologues are associated with tbtim17 and critical for the biogenesis of tbtim17 protein complexes in <italic toggle="yes">trypanosoma brucei</italic>
publisher	American Society for Microbiology
publishDate	2018
url	https://doaj.org/article/43bbb2ae141f42e1b880b53895b28cce
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Divergent Small Tim Homologues Are Associated with TbTim17 and Critical for the Biogenesis of TbTim17 Protein Complexes in <italic toggle="yes">Trypanosoma brucei</italic>

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