Protein and transfer RNA are imported into the mitochondria of Trypanosoma brucei by use of a shared mitochondrial membrane protein complex
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Unlike most other eukaryotic organisms, the mitochondrial genome of Trypanosoma brucei does not contain genes coding for tRNAs; nuclear encoded tRNAs must be imported in order to maintain active translation within the mitochondria. Translocation of tRNA into the mitochondria requires ATP hydrolysis, an active membrane potential as well as proteinaceous membrane components. Although much is known about the biophysical requirements for mitochondrial tRNA import, very little is known about the proteins that are involved in this translocation process. Similar to tRNA import, the identity of the mitochondrial protein import machinery of Trypanosoma brucei is unknown as the canonical Translocase Outer Membrane and Translocase Inner Membrane have yet to be identified. Through the work presented in this dissertation, a mitochondrial protein complex that is functional in the translocation of tRNA and protein into the mitochondria has been identified. By use of affinity purification and mass spectrometry, we were able to identify mitochondrial membrane complexes that specifically bind tRNA. Candidate proteins were then characterized for their role in mitochondrial tRNA import by use of an in vivo tRNA import assay in tandem with RNA interference for specific candidates. Dual affinity tagged proteins involved in tRNA import were pulled down and their binding partners were identified by mass spectrometry. From this analysis, we were able to identify shared components between tRNA and protein import machinery (Tim17, mHSP70, mHSP60 and mHSP20). Further analysis of the complex by in vivo protein import assays, showed that the identified protein complex is required for both the mitochondrial import of tRNA and protein. In addition, we are also able to show that mitochondrial tRNA and protein import utilize the same biophysical requirements for import. These results suggest that tRNA and protein are imported into the mitochondria of Trypanosoma brucei by use of the same mitochondrial membrane protein complex and that this pathway may represent a universal import mechanism for all eukaryotic organisms.