Essential Functions of the N‐terminal Domain of Tim17 in Trypanosoma brucei

Abstract

The majority of proteins essential for mitochondrial functions are encoded in the nuclear DNA and subsequently imported from the cytosol by translocases of the mitochondrial outer and inner membranes, termed TOM and TIM, respectively. These translocases are multimeric protein complexes, which are overall conserved from fungi to human. However, mitochondrial protein import machinery in trypanosomatids, a group of ancient and early branching unicellular eukaryotes that causes a fatal human disease African trypanosomiasis, is significantly different. As a part of this machinery, we previously characterized Tim17 from Trypanosoma brucei. TbTim17 is critical for mitochondrial protein import and thus essential for parasite survival. Here we further investigate the function of the divergent N‐terminal domain of this protein. We found that deletion of the first 20 or 30 amino acid residues (Δ20‐ and Δ30‐) of TbTim17 is detrimental for its function. These mutant proteins neither complement the growth defect of TbTim17 RNAi cells nor the import defect of other nuclear encoded proteins into TbTim17 knockdown mitochondria. However, knock‐in of the full‐length protein (FL‐TbTim17) completely restored these functions in RNAi cells. Further analysis showed that both of these mutants of TbTim17 were properly targeted to mitochondria, however, Δ20‐TbTim17 was not membrane integrated and Δ30‐mutant, though integrated into the mitochondrial membrane was sensitive to protease digestion of the isolated mitochondria, indicating that the N‐terminal region is necessary for proper integration of TbTim17 into the mitochondrial inner membrane. Analysis of the protein complexes in the wild type and mutant mitochondria by Blue‐native gel electrophoresis revealed that none of these mutants were assembled into the TbTim17 protein complex. Co‐immunoprecipitation analysis showed that unlike the wild type TbTim17, the mutant proteins were 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 is critical for its interaction with TbTim62 and required for the formation of a functional TbTim17 protein complex. To elucidate the assembly of the TIM complex in T. brucei, analysis is under progress to identify essential amino acids residues of this region by site‐directed mutagenesis.Support or Funding InformationNIH Grants 2SC1GM081146‐05, T32HL007727, and G12RR003032