Project

Discipline

trypanosoma; mitochondria; transfer RNA; transport of macromolecules; trypanosomes; tRNA; transport; parasites; organellar translation

Lead
Lay summary
Mitochondria do not form de novo but derive from preexisting organelles by processes collectively referred to as mitochondrial biogenesis. These include import of most mitochondrial proteins and, in most eukaryotes, import of at least some tRNAs. Furthermore, a limited set of proteins that is essential for oxidative phosphorylation need to be synthesized inside mitochondria. My research group is working on mitochondrial biogenesis using the parasitic protozoa Trypanosoma brucei as a model system. T. brucei belongs to a different eukaryotic supergroup than yeast, mammals and most other model organisms in molecular biology. Its mitochondrion therefore shows many pecularities. The most important ones in the context of our studies are a complete lack of mitochondrial tRNA genes that is compensated by import of cytosolic tRNAs and an apparently minimized mitochondrial protein import system. Moreover, T. brucei is the causative agent of human sleeping sickness and therefore of great clinical importance. Why should research of exotic trypanosome-specific features be of interest for basic science in general? The reason is that these superficially trypanosome-specific features are not really unique, but rather represent extreme examples of processes that in a more cryptic way occur in other cells as well. Thus, most eukaryotes import mitochondrial tRNAs, just not the entire set as T. brucei. All eukaryotes import most of their mitochondrial proteins, however, their import machineries are not as minimized as the one in T. brucei. Thus, T. brucei is an attractive experimentally highly tractable system to study basic as yet poorly understood biological processes.

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Last update: 21.02.2013

Active participation

Number	Title	Start	Funding scheme

SummaryMy research group is working on mitochondrial biogenesis using the parasitic protozoa Trypanosoma brucei as a model. T. brucei and its relatives belong to the earliest diverging branches of the eukaryotic evolutionary tree, which have bona fide mitochondria involved in oxidative phosphorylation. Their mitochondrion therefore shows many peculiarities. This proposal focuses on three of them: (i) a complete lack of mitochondrial tRNA genes that is compensated by import of cytosolic tRNAs, (ii) an apparently minimized mitochondrial protein import system, and (iii) a striking expansion - when compared to non-plant eukaryotes - of a protein family, the pentatricopeptide repeat (PPR) proteins, postulated to bind to specific organellar RNAs. Mitochondrial tRNA importWe have shown that cytosolic elongation factor 1a (eEF1a) is necessary to determine the specificity of mitochondrial tRNA import. We would now like to understand the mechanism by which the specificity is determined and whether eEF1a acts in concert with other proteins. Different tRNAs are imported to very different extents. We intend to investigate how these extents are determined. Furthermore, we plan to identify the components of the tRNA import machinery and to investigate the mechanism of the import process. Finally, we want to understand how imported eukaryotic-type tRNAs are integrated into the bacterial-type translation system of the mitochondrion. Here we will focus on the extended substrate specificities of some mitochondrial aminoacyl-tRNA synthetases and on the role of a trypanosomatid-specific insertion in mitochondrial elongation factor Tu. - We expect that studying the questions mentioned above will uncover conserved concepts of mitochondrial tRNA import that are valid in all systems.Mitochondrial protein importA genome analysis revealed two aspects where the protein import pathway of T. brucei appears simpler than what is found in other mitochondria. These are the apparent absence of a conventional outer membrane translocase and the existence of only a single inner membrane translocase. We propose to characterize the translocase that imports proteins across the outer membrane in T. brucei. Furthermore we would like to show that both insertion of membrane proteins and translocation of matrix proteins are mediated by a single inner membrane translocase. - The suggested studies are likely to give us more insight into the enigmatic evolutionary origin of mitochondrial protein import in general. PPR proteinsT. brucei contains almost an order of magnitude more PPR proteins than all other non-plant eukaryotes. We have characterized six trypanosomal PPR proteins that are predicted to bind mitochondrial rRNAs. We intend to test this binding directly and determine the minimal RNA sequences it requires. Furthermore, we intend to characterize interacting partners of PPRs proteins and, finally, would like to initiate structural studies. - We hope that these studies will help to determine whether PPR proteins indeed are sequence specific RNA binding proteins.Key experimental approachesWhile the three subprojects focus on different aspects of mitochondrial biogenesis they are linked by a set of shared experimental approaches. These are: Influence of RNAi-mediated ablation of putative tRNA or protein import factors on import of a newly synthesized tRNA or protein in vivo; In vitro import systems for tRNA and proteins; Blue native gels and tandem affinity purification of tagged proteins to characterize protein complexes and the production of recombinant proteins for various in vitro assays as well as for structural studies.