Protein interactions studies of the small TbTims chaperons in the mitochondrion of Trypanosoma brucei

Abstract

Trypanosoma brucei causes a neglected tropical disease known as trypanosomiasis that causes death in population of Sub‐Sahara Africa. T. brucei possesses a single mitochondrion and like other eukaryotes it imports most of its proteins into the mitochondria, however mitochondrial protein import machinery is divergent in this parasite, which makes it a useful target to control this disease. Nucleus‐encoded proteins are imported into T. brucei mitochondrion via two major heteromeric protein complexes in the outer and inner membrane, ATOM and TbTIM, respectively. Besides the membrane integral protein components of these complexes, TbTIM is also peripherally associated with 6 small TbTims (Tim9, 10, 8/13, 11, 12, and 13). At least three of these small TbTims (TbTim8/13, 11, & 12) are unique for T. brucei. Knock‐down of any of these 6 small TbTims destabilized the TbTIM17 complex therefore these proteins are essential in T. brucei. In fungi and human, the small Tims are soluble proteins that form hetero‐hexameric complexes. Each small Tims possesses two pairs of CX3C motifs that are required for proper folding of the monomers by formation of the two intramolecular disulfide bonds. T. brucei small Tims also possess these conserved motifs, except for TbTim12, which has only one pair. However, it is not clear how small TbTims interact with each other and what type of complex structure they form. Thus, our goal for these studies is to find the direct interaction pattern for the small TbTims. To complete this goal, we use yeast two hybrid (Y2H) analysis. Results showed that although all small TbTims can interact with one another, some of these interactions are stronger than others. Overall, we found that TbTim8/13 interacts strongly with TbTim9, TbTim10, and TbTim8/13 while TbTim13 interacts strongly with TbTim13. Based on these results we proposed 3 possible model for small TbTim heterohexameric complexes; 1) random order, and 2) defined order, and 3) modular with different sub‐complexes. We are now testing these models by structure‐modeling and site‐directed mutagenesis studies.Support or Funding InformationThis work is supported by the following NIH grants: 5RO1AI125666, 2SC1GM081146, and R25 GM05994