Trypanosoma brucei causes a deadly disease known as trypanosomiasis that affects the rural population in Sub-Saharan Africa. T. brucei contains a single mitochondrion which needs to import thousands of proteins for its function, making mitochondria protein import essential. In eukaryotes, the mitochondria protein import machinery is comprised of three major complexes: the translocase of the outer membrane (TOM) and two translocases of the inner membrane (TIM). A group of small Tim proteins of the intermembrane space form 2-3 heterohexameric complexes and are essential for translocation of the hydrophobic inner membrane proteins through one of the TIM. Mitochondrial protein import machinery in T. brucei is significantly divergent. Although T. brucei possesses 6 homologues of the small Tims (TbTim9, TbTim10, TbTim11, TbTim12, TbTim13, and TbTim8/13), each with a characteristic secondary structure, their functions and interaction patterns are not well understood. In contrast to yeast, all the small TbTims are associated with the single TIM complex in T. brucei and critical for the stability of this complex. Here, we analyze the interactions of small TbTims by yeast two-hybrid (Y2-H) analysis. Results showed that all small TbTims directly interact with each other; however, stronger interactions were found between TbTim8/13 with TbTim9 and TbTim10. To determine the structural domain(s) necessary for their interaction, the small TbTims were split into their N-terminal and C-terminal helices to use for Y2-H analysis in all sorts of combinations. Results show that both helices of TbTim9, TbTim10, and TbTim8/13 are involved in interaction among themselves and with the other small TbTims, like TbTim11, TbTim12, and TbTim13, suggesting that TbTim9, TbTim10, and TbTim8/13 are the core of the small TbTim complex.
