Distinct structural motifs are necessary for targeting and import of Tim17 in 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 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 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 (K) 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 .IMPORTANCEAfrican trypanosomiasis (AT) is a deadly disease in human and domestic animals, caused by the parasitic protozoan . Therefore, AT is not only a concern for human health but also for economic development in the vast area of sub-Saharan Africa. possesses a single mitochondrion per cell that imports hundreds of nuclear-encoded mitochondrial proteins for its functions. 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 mitochondrion. This information could be utilized to block TbTim17 function and parasite growth.