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

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.

Distinct structural motifs are necessary for targeting and import of Tim17 in Trypanosoma brucei mitochondrion.

Nuclear-encoded mitochondrial proteins are correctly translocated to their proper sub-mitochondrial destination using location-specific mitochondrial targeting signals and via multi-protein import machineries (translocases) in the outer and inner mitochondrial membranes (TOM and TIMs, respectively). However, targeting signals of multi-pass Tims are less defined. Here, we report the characterization of the targeting signals of Trypanosoma brucei Tim17 (TbTim17), an essential component of the most divergent TIM complex. TbTim17 possesses a characteristic secondary structure including four predicted transmembrane (TM) domains in the center with hydrophilic N- and C-termini. After examining mitochondrial localization of various deletion and site-directed mutants of TbTim17 in T. brucei using subcellular fractionation and confocal microscopy, we located at least two internal targeting signals (ITS): (i) within TM1 (31-50 AAs) and (ii) TM4 + loop 3 (120-136 AAs). Both signals are required for proper targeting and integration of TbTim17 in the membrane. Furthermore, a positively charged residue (K122) is critical for mitochondrial localization of TbTim17. This is the first report of characterizing the ITS for a multipass inner membrane protein in a divergent eukaryote, like T. brucei.IMPORTANCEAfrican trypanosomiasis (AT) is a deadly disease in human and domestic animals, caused by the parasitic protozoan Trypanosoma brucei. Therefore, AT is not only a concern for human health but also for economic development in the vast area of sub-Saharan Africa. T. brucei possesses a single mitochondrion per cell that imports hundreds of nuclear-encoded mitochondrial proteins for its functions. T. brucei Tim17 (TbTim17), an essential component of the TbTIM17 complex, is a nuclear-encoded protein; thus, it is necessary to be imported from the cytosol to form the TbTIM17 complex. Here, we demonstrated that the internal targeting signals within the transmembrane 1 (TM1) and TM4 with loop 3, and residue K122 are required collectively for import and integration of TbTim17 in the T. brucei mitochondrion. This information could be utilized to block TbTim17 function and parasite growth.

Unique interactions and functions of the mitochondrial small Tims in Trypanosoma brucei.

Trypanosoma brucei is an early divergent parasitic protozoan that causes a fatal disease, African trypanosomiasis. T. brucei possesses a unique and essential translocase of the mitochondrial inner membrane, the TbTIM17 complex. TbTim17 associates with 6 small TbTims, (TbTim9, TbTim10, TbTim11, TbTim12, TbTim13, and TbTim8/13). However, the interaction pattern of the small TbTims with each other and TbTim17 are not clear. Here, we demonstrated by yeast two-hybrid (Y2H) analysis that all six small TbTims interact with each other, but stronger interactions were found among TbTim8/13, TbTim9, and TbTim10. Each of the small TbTims also interact directly with the C-terminal region of TbTim17. RNAi studies indicated that among all small TbTims, TbTim13 is most crucial to maintain the steady-state levels of the TbTIM17 complex. Co-immunoprecipitation analyses from T. brucei mitochondrial extracts also showed that TbTim10 has a stronger association with TbTim9 and TbTim8/13, but a weaker association with TbTim13, whereas TbTim13 has a stronger connection with TbTim17. Analysis of the small TbTim complexes by size exclusion chromatography revealed that each small TbTim, except TbTim13, is present in ∼70 kDa complexes, which could be heterohexameric forms of the small TbTims. However, TbTim13 is primarily present in the larger complex (>800 kDa) and co-fractionated with TbTim17. Altogether, our results demonstrated that TbTim13 is a part of the TbTIM complex and the smaller complexes of the small TbTims likely interact with the larger complex dynamically. Therefore, relative to other eukaryotes, the architecture and function of the small TbTim complexes are specific in T. brucei .

Distinct structural motifs are necessary for targeting and import of Tim17 in Trypanosoma brucei mitochondrion.

Nuclear-encoded mitochondrial proteins are correctly translocated to their proper sub-mitochondrial destination using location specific mitochondrial targeting signals (MTSs) and via multi-protein import machineries (translocases) in the outer and inner mitochondrial membranes (TOM and TIMs, respectively). However, MTSs of multi-pass Tims are less defined. Here we report the characterization of the MTSs of Trypanosoma brucei Tim17 (TbTim17), an essential component of the most divergent TIM complex. TbTim17 possesses a characteristic secondary structure including four predicted transmembrane (TM) domains in the center with hydrophilic N- and C-termini. After examining mitochondrial localization of various deletion and site-directed mutants of TbTim17 in T. brucei using subcellular fractionation and confocal microscopy we located at least two internal signals, 1) within TM1 (31-50 AAs) and 2) TM4 + Loop 3 (120-136 AAs). Both signals are required for proper targeting and integration of TbTim17 in the membrane. Furthermore, a positively charged residue (K 122 ) is critical for mitochondrial localization of TbTim17. This is the first report of characterizing the internal mitochondrial targeting signals (ITS) for a multipass inner membrane protein in a divergent eukaryote, like T. brucei . Summary: Internal targeting signals within the TM1, TM4 with Loop 3, and residue K122 are required collectively for import and integration of TbTim17 in the T. brucei mitochondrion. This information could be utilized to block parasite growth.

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

The translocases of the mitochondrial outer and inner membranes, the TOM and TIMs, import hundreds of nucleus-encoded proteins into mitochondria. TOM and TIMs are multi-subunit protein complexes that work in cooperation with other complexes to import proteins in different sub-mitochondrial destinations. The overall architecture of these protein complexes is conserved among yeast/fungi, animals, and plants. Recent studies have revealed unique characteristics of this machinery, particularly in the eukaryotic supergroup Excavata. Despite multiple differences, homologues of Tim17, an essential component of one of the TIM complexes and a member of the Tim17/Tim22/Tim23 family, have been found in all eukaryotes. Here, we review the structure and function of Tim17 and Tim17-containing protein complexes in different eukaryotes, and then compare them to the single homologue of this protein found in Trypanosoma brucei, a unicellular parasitic protozoan.