Project Summary:
The majority of proteins essential for mitochondrial functions are encoded in the nuclear DNA and
subsequently imported from the cytosol by translocases of the mitochondrial outer and inner membranes,
termed TOM and TIM, respectively. These translocases are multimeric protein complexes, which are
extensively studied in fungi and later in humans and plants. However, mitochondrial protein import machinery
has been poorly characterized in trypanosomatids, a group of ancient and early branching unicellular
eukaryotes that possesses a single mitochondrion per cell. Recent studies by our laboratory and others
identified a number of non-canonical protein components for this essential cellular process in Trypanosoma
brucei. As part of this machinery, we previously characterized TbTim17, which is critical for mitochondrial
protein import and thus essential for parasite survival in two major developmental forms, the procyclic and
bloodstream forms, found in the insect vector and the mammalian bloodstream, respectively. TbTim17 is
present in multiple protein complexes within the range of 300-1100 kDa. Using pull down assays we identified
one relatively conserved protein, Tim50, and several other novel proteins. Among these, we found TbTim62, a
non-canonical Tim protein, act as a critical assembly factor of the TbTim17 protein complexes and have a
major effect on TbTim17 stability. In addition, we found that Tim50 interacts with TbTim17 and is involved in
the import of N-terminal signal containing mitochondrial proteins. Besides, TbTim17 also associates with a
group soluble small TbTims. In contrast to the two TIM complexes (TIM23 and TIM22) with different substrate
specificities in fungi to mammals, trypanosomatids most likely possess a single TIM (TbTIM17) that is capable
of importing various types of mitochondrial proteins. Based on these results we hypothesized that TbTim17
forms an evolutionarily divergent and modular type TIM complex by association with novel TbTims and imports
different types of substrate protein into mitochondria. We will test this hypothesis by three aims; 1) to define the
structural components of the TbTim17 protein complex; 2) to determine the unique and interactive functions of
TbTim17, TbTim62, TbTim50, TbTim54, and small TbTims in T. brucei, and 3) to determine the structural
domains of TbTims critical for their functions. We will use many conventional and specialized techniques to
evaluate the properties of these TbTims, including their substrate specificities, ability to assemble into the TIM
complex, role in the stability of this complex, protein-protein interactions, and unique structure-function
relations. Together, these studies will elucidate the subunit composition and function of the divergent TbTIM
complex in T. brucei that can also be extrapolated for other trypanosomatid parasites and thus illuminate the
mechanism of mitochondrial protein import in a group of the earliest eukaryotes.