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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 a mitochondrial protein import system that is very different from other eukaryotes. Moreover, T. brucei is the causative agent of human sleeping sickness and therefore of great clinical importance.  All mitochondria have a double membrane. Thus, tRNAs as well as many proteins needs to be transported across both the outer and inner membrane to reach the matrix, the soluble innermost compartment of mitochondria. In this proposal we focus on the outer membrane of the T. brucei mitochondrion. It is known from other organisms that the outer mitochondrial membrane contains only few dozens of major proteins which include the import machineries for macromolecules. We therefore plan to characterize the outer membrane proteome of T. brucei mitochondria with the ultimate aim to identify the as yet elusive components of the tRNA and protein import machineries.

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Last update: 21.02.2013

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Mitochondrial biogenesis has been investigated since many years. However, with a few exceptions most studies were restricted to yeast and mammals that according to the current eukaryotic phylogeny are closely related. My research group is studying mitochondrial biogenesis in the parasitic protozoa Trypanosoma brucei whose mitochondrion differs in a number ways from the organelles of yeast and mammals. The most important differences in the context of this proposal are: (i) a complete lack of mitochondrial tRNA genes that is compensated for by import of cytosolic tRNAs and (ii) a highly diverged mitochondrial protein import system lacking a Tom40 orthologue.Outer membrane proteome•The focus of this research proposal is to obtain an inventory of the components of macromolecular import systems of the mitochondrial outer membrane. To that end we will purify the mitochondrial outer membrane and determine its proteome by mass spectroscopy. Based on results in other organisms we can expect that it consists of a limited set of a few dozen major proteins that include the components of the tRNA and the protein import systems. Mitochondrial tRNA import•We will use microarrays containing cDNA sequences of all trypanosomal tRNAs to get a global and quantitative view on mitochondrial tRNA import across the life cycle of T. brucei. •We will identify tRNA-binding proteins of the mitochondrial membrane using tRNA affinity chromatography. •We have developed a sensitive in vivo import system that allows to monitor import of a newly synthesized tagged tRNA in any given RNAi cell line. The system is highly flexible since the RNAi and the expression of the tagged tRNA are independently controlled by the tet and the lac repressor, respectively. The system will be used to get an inventory of the proteins that are required for mitochondrial tRNA import in vivo. Candidates to be tested include all major components of the outer membrane proteome and all mitochondrial membrane proteins that bind tRNA. •The current in vitro import system for mitochondrial tRNA import in trypanosomes are devoid of cytosolic factors and do not match the specificity that is seen in vivo. We will attempt to establish a novel in vitro system containing cytosolic extracts. We believe that such a system more closely reflects the physiological situation since the cytosolic tRNA-protein complexes that exist in vivo are maintained. Mitochondrial protein import•We will more precisely define the role of TomX, a component of the outer membrane proteome that according to preliminary experiments is required for mitochondrial protein and tRNA import. •We have developed a sensitive in vivo import system that allows to monitor import of newly synthesized proteins in any given RNAi cell line. The system will be used to get an inventory of the factors that are required for mitochondrial protein import in vivo. Candidates to be tested include all major components of the outer membrane proteome. •Using an already established in vitro protein import system we will produce import arrested precursor proteins that are stuck in the import channel and attempt to characterize the resulting protein complexes.•Candidate protein import channels will be recombinantly expressed and characterized by electrophysiology using planar lipid bilayers.