Abstract
The mitochondrial genome of Trypanosoma brucei does not contain genes encoding tRNAs; instead this protozoan parasite must import nuclear-encoded tRNAs from the cytosol for mitochondrial translation. Previously, it has been shown that mitochondrial tRNA import requires ATP hydrolysis and a proteinaceous mitochondrial membrane component. However, little is known about the mitochondrial membrane proteins involved in tRNA binding and translocation into the mitochondrion. Here we report the purification of a mitochondrial membrane complex using tRNA affinity purification and have identified several protein components of the putative tRNA translocon by mass spectrometry. Using an in vivo tRNA import assay in combination with RNA interference, we have verified that two of these proteins, Tb11.01.4590 and Tb09.v1.0420, are involved in mitochondrial tRNA import. Using Protein C Epitope -Tobacco Etch Virus-Protein A Epitope (PTP)-tagged Tb11.01.4590, additional associated proteins were identified including Tim17 and other mitochondrial proteins necessary for mitochondrial protein import. Results presented here identify and validate two novel protein components of the putative tRNA translocon and provide additional evidence that mitochondrial tRNA and protein import have shared components in trypanosomes.
Highlights
Mitochondrial tRNA import in T. brucei is essential for mitochondrial biogenesis
The putative tRNA translocon contains the translocase inner membrane 17 (Tim17) protein and other proteins proposed to function in mitochondrial protein import. These findings suggest that mitochondrial tRNA and protein import machinery in trypanosomes may either share protein components or that there is a common translocon for both proteins and tRNAs
Identification and Characterization of tRNA Binding Mitochondrial Membrane Protein Complexes—Previous studies on T. brucei suggested that mitochondrial membrane proteins were necessary for tRNA import [9]
Summary
Mitochondrial tRNA import in T. brucei is essential for mitochondrial biogenesis. Results: Proteins necessary for mitochondrial tRNA import are identified. The mechanism by which these tRNAs are imported into the trypanosome mitochondria is unknown; in vitro studies with isolated organelles have shown that translocation of tRNAs into the mitochondrion requires ATP hydrolysis and a proteinaceous membrane component [9, 16, 17]. Little is known about the mitochondrial tRNA import machinery of African trypanosomes, a cytosolic protein, eukaryotic elongation factor 1a (eEF1a), has recently been shown to play a role in mitochondrial tRNA import in T. brucei. These findings suggest that mitochondrial tRNA and protein import machinery in trypanosomes may either share protein components or that there is a common translocon for both proteins and tRNAs
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