The divergent N-terminal domain of Tim17 is critical for its assembly in the TIM complex in Trypanosoma brucei

Abstract

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 (Δ20 and Δ30) 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 Δ20-TbTim17 and Δ30-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 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.

Novel IM‐associated protein Tim54 plays a role in the mitochondrial import of internal signal‐containing proteins in Trypanosoma brucei

“…To investigate if TbTim54 exists in a protein complex similar to TbTim17, we performed Blue‐native (BN)‐PAGE analysis of mitochondrial protein complexes, followed by immunoblotting with TbTim54 and TbTim17 antibodies. The TbTim17 antibody recognised TbTim17 protein complexes with molecular weights ranging from 300 to 1100 kDa in the parental cells, as expected [Weems et al., 2015; Weems et al., 2017]. TbTim17 KD reduced the levels of these complexes by more than 60% (Figures 2C, first panel and 2D).…”

Tim17 Updates: A Comprehensive Review of an Ancient Mitochondrial Protein Translocator

“…A recent study from Chaudhuri-lab on T. brucei revealed that deletion of the N-terminal 30 AAs of TbTim17 did not hinder its targeting to mitochondria but largely hampered its assembly into the TbTIM complex [120]. These results suggest that TbTim17 possesses internal mitochondrial targeting signal(s) but the N-terminal region is possibly critical for interaction with other proteins necessary for its assembly.…”

Divergent Small Tim Homologues Are Associated with TbTim17 and Critical for the Biogenesis of TbTim17 Protein Complexes in Trypanosoma brucei

“…For generation of TbTim9-Myc, TbTim10-Myc, or TbTim8/13-Myc expression constructs, the open reading frames (ORFs) of TbTim9, TbTim10, or TbTim8/13 were subjected to PCR amplification using T. brucei 427 genomic DNA as the template and the corresponding sequence-specific primers (see Table S4 in the supplemental material). TbTim17 (Tb927.11.13290) was previously cloned in the same manner ( 51 , 58 ). The forward and reverse primers were designed to add restriction sites for HindIII and XbaI at the 5′ and 3′ ends of the ORFs, respectively.…”

“…Total transfected cell populations that were resistant to selective antibiotics after several subcultures were used for this study. We then transfected the TbTim17-Myc cells that had been established previously in the laboratory ( 51 , 58 ) with the puromycin-selective small TbTim RNAi constructs for generation of small TbTim RNAi cells that simultaneously expressed the double-stranded RNA for the specific small TbTim and TbTim17-Myc transcript.…”