Trypanosoma brucei, a protozoan parasite from the kinetoplastid family, is the causative agent of African sleeping sickness, a re-emerging disease of epidemic proportions in sub-Saharan Africa. Currently 66 million people are at risk of infection, which is transmitted by infected tsetse flies. Trypanosomes have a complex life cycle involving differentiation from an insect vector life form into a mammalian host, which is accompanied by profound alterations in both ultrastructure and metabolism. They have one of the smallest mitochondrial genomes and none of the transfer RNAs (tRNA) necessary for protein translation are encoded. The goal of this project is to identify the mechanism of tRNA translocation into the mitochondria. Little is currently known about the mechanism of tRNA import. The specific hypothesis of this project is that there are unique receptors for tRNA import on the mitochondrial membranes. We will address this hypothesis with the following specific aims: 1) Validate the tRNA-binding proteins identified using tRNA affinity chromatography and mass spectrometry as (i) specific tRNA binding proteins using recombinant proteins in electrophoretic mobility shift assays (EMSA), (ii) mitochondrially localized using immunofluorescence assays, and (iii) found in a complex using blue native electrophoretic gels and probing for the protein complexes using specific antibodies. 2) Verify the protein complexes form in vivo by tagging the tRNA with two stable RNA hairpins and isolate the associated proteins from the purified mitochondria with high affinity ligands for these RNA binding domains, and 3) Functionally characterize these proteins as playing a role in tRNA import into the mitochondria using a combination of in vivo and in vitro techniques, including using RNAi to downregulate protein expression, (i) The RNAi cells can then be used to purify the mitochondria and perform in vitro import assays, (ii) These cells can also be used to directly monitoring import using a tagged-tRNA regulated by tetracycline-inducible promoter. Since transport of tRNA has been shown to occur in a range of organisms, this study should provide us with data to formulate a general model for tRNA transport. Furthermore, this could have therapeutic potential since mammalian mitochondrial tRNAs, unlike in trypanosomes, are encoded within the mitochondria and does not rely on import. [unreadable] [unreadable] [unreadable]