Journal
MOLECULAR AND BIOCHEMICAL PARASITOLOGY
Volume 218, Issue -, Pages 4-15Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.molbiopara.2017.09.003
Keywords
Mitochondria; Mitochondrial inner membrane; N-terminal mutants; Protein import; Protein assembly; Tim17; Trypanosoma brucei
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Funding
- National Institute of Health [1RO1AI125662, 2SC1GM081146]
- NIH [U54RR026140/U54MD007593]
- [T32HL007737]
- [T32AI007281]
- [2R25GM059994]
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Trypanosoma brucei Tim17 (TbTim17), the single member of the Tim17/23/22 protein family, is an essential component of the translocase of the mitochondrial inner membrane (TIM). In spite of the conserved secondary structure, the primary sequence of TbTim17, particularly the N-terminal hydrophilic region, is significantly divergent. In order to understand the function of this region we expressed two N-terminal deletion mutants (Delta 20 and Delta 30) of TbTim17 in T. brucei. Both of these mutants of TbTim17 were targeted to mitochondria, however, they failed to complement the growth defect of TbTim17 RNAi cells. In addition, the import defect of other nuclear encoded proteins into TbTim17 knockdown mitochondria were not restored by expression of the N terminal deletion mutants but complemented by knock-in of the full-length protein. Further analysis revealed that Delta 20-TbTim17 and Delta 30-TbTim17 mutants were not localized in the mitochondrial inner membrane. Analysis of the protein complexes in the wild type and'mutant mitochondria by two-dimensional Blue-native/SDS-PAGE revealed that none of these mutants are assembled into the TbTim17 protein complex. However, FL-TbTim17 was integrated into the mitochondrial inner membrane and assembled into TbTim17 complex. Co-immunoprecipitation analysis showed that unlike the FL-TbTim17, mutant proteins are not associated with the endogenous TbTim17 as well as its interacting partner TbTim62, a novel trypanosome specific Tim. Together, these results show that the N-terminal domain of TbTim17 plays unique and essential roles for its sorting and assembly into the TbTim17 protein complex.
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