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Proc Natl Acad Sci U S A, 111, 7624-9.
M. Niemann, S. Wiese, J. Mani, A. Chanfon, C. Jackson, C. Meisinger, B. Warscheid, A. Schneider (2013), Mitochondrial outer membrane proteome of Trypanosoma brucei reveals novel factors required to maintain mitochondrial morphology, in
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M. Cristodero, J. Mani, S. Oeljeklaus, L. Aeberhard, H. Hashimi, D. J. Ramrath, J. Lukeš, B. Warscheid, A. Schneider (2013), Mitochondrial translation factors of Trypanosoma brucei: elongation factor-Tu has a unique subdomain that is essential for its function, in
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M. Pusnik, J. Mani, O. Schmid, M. Niemann, S. Oeljeklaus, F. Schnarwiler, B. Warscheid, T. Lithgow, C. Meisinger, A. Schneider (2012), An essential novel component of the non-canonical mitochondrial outer membrane protein import system of trypanosomatids, in
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A. Harsman, M. Niemann, M. Pusnik, O. Schmidt, B. M. Burmann, S. Hiller, C. Meisinger, A. Schneider, R. Wagner (2012), Bacterial origin of a mitochondrial outer membrane protein translocase: New perspectives from comparative single channel electrophysiology, in
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J. Mani, S. Desy, M. Niemann, A. Chanfon, S. Oeljeklaus, M. Pusnik, O. Schmidt, C. Gerbeth, C. Meisinger, B. Warscheid, A. Schneider, Novel mitochondrial protein import receptors in Kinetoplastids reveal convergent evolution over large phylogenetic distances, in
Nature Commun..
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.