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Mitochondrial biogenesis in T. brucei - import of macromolecules

Applicant Schneider André
Number 138355
Funding scheme Project funding (Div. I-III)
Research institution Departement für Chemie, Biochemie und Pharmazie Universität Bern
Institution of higher education University of Berne - BE
Main discipline Molecular Biology
Start/End 01.01.2012 - 31.12.2014
Approved amount 698'000.00
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All Disciplines (4)

Discipline
Molecular Biology
Biochemistry
Cellular Biology, Cytology
Experimental Microbiology

Lay Summary (English)

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 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.

Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
Parasite reveals mitochondrial inheritance machinery
Schneider A., Schnarwiler F. (2014), Parasite reveals mitochondrial inheritance machinery, in Chimia (Aarau), 68, 749-749.
Trypanosomal TAC40 constitutes a novel subclass of mitochondrial beta-barrel proteins specialized in mitochondrial genome inheritance
Schnarwiler F., Niemann M., Doiron N., Harsman A., Kaser S., Mani J., Chanfon A., Dewar C. E., Oeljeklaus S., Jackson C. B., Pusnik M., Schmidt O., Meisinger C., Hiller S., Warscheid B., Schnaufer A. C., Ochsenreiter T., Schneider A. (2014), Trypanosomal TAC40 constitutes a novel subclass of mitochondrial beta-barrel proteins specialized in mitochondrial genome inheritance, in Proc Natl Acad Sci U S A, 111, 7624-9.
Mitochondrial outer membrane proteome of Trypanosoma brucei reveals novel factors required to maintain mitochondrial morphology
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 Mol. Cell. Proteomics, 12, 515-528.
Mitochondrial translation factors of Trypanosoma brucei: elongation factor-Tu has a unique subdomain that is essential for its function
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 Mol. Microbiol., 90, 744-755.
An essential novel component of the non-canonical mitochondrial outer membrane protein import system of trypanosomatids
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 Mol. Biol. Cell, 23, 3420-3428.
Bacterial origin of a mitochondrial outer membrane protein translocase: New perspectives from comparative single channel electrophysiology
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 J. Biol. Chem., 287, 31437-31445.
Response to Zarsky et al.
M. Pusnik, O. Schmidt, A. J. Perry, S. Oeljeklaus, M. Niemann, B. Warscheid, C. Meisinger, T. Lithgow, A. Schneider (2012), Response to Zarsky et al., in Curr. Biol., 22, 481-482.
Trypanosoma brucei has a canonical mitochondrial processing peptidase
Desy S., Schneider A., Mani J. (2012), Trypanosoma brucei has a canonical mitochondrial processing peptidase, in Mol Biochem Parasitol, 185, 161-4.
Novel mitochondrial protein import receptors in Kinetoplastids reveal convergent evolution over large phylogenetic distances
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..

Collaboration

Group / person Country
Types of collaboration
Prof. Peter Bütikofer/University of Bern Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Prof. Chris Meisinger/University of Freiburg Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
- Exchange of personnel
Dr. Achim Schnaufer, University of Edinburgh Great Britain and Northern Ireland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Prof. Tao Pan/University of Chicago United States of America (North America)
- in-depth/constructive exchanges on approaches, methods or results
Prof. Richard Wagner/University of Osnabrück Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Exchange of personnel
Prof. Sebastian Hiller, Biozentrum Basel Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Prof. Bettina Warscheid/University of Freiburg Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
- Exchange of personnel

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
31st Annual Swiss Trypanosomatid Meeting Talk given at a conference The ATOM translocase: building blocks ans function 23.01.2015 Leysin, Switzerland Mani Jan;
Invited Seminar Individual talk What can a unicellular parasite tell us about mitochondrial biogenesis 04.12.2014 Monash University, Melbourne, Australia Schneider André;
Freudenstadt Meeting Talk given at a conference What can a parasite tell us about mitochondrial biogenesis 06.10.2014 Freudenstadt, Germany Schneider André;
SwissMito Meeting 2014 Talk given at a conference What can a parasite tell us about mitochondrial biogenesis? 02.09.2014 Kandersteg, Switzerland Niemann Moritz; Mani Jan; Schneider André;
Gordon research conference: Mitochondria and Chloroplasts Poster ATOM complex: the gate to the trypanosome mitochondrion 06.07.2014 Il Ciocco, Italy Mani Jan;
Gordon research conference: Mitochondria and Chloroplasts Poster The Tim complex in the T. brucei mitochondrion 06.07.2014 Il Ciocco, Italy Harsman Anke;
Gordon research conference: Host parasite interactions Talk given at a conference What can a parasite tell us about mitochondrial biogenesis? 08.06.2014 Newport, United States of America Schneider André;
Invited Seminar Individual talk What can a parasite tell us about mitochondrial biogenesis? 08.05.2014 Glasgow University, Great Britain and Northern Ireland Schneider André;
31st Annual Swiss Trypanosomatid Meeting Talk given at a conference The ATOM complex: building blocks and function 23.01.2014 Leysn, Switzerland Mani Jan;
31st Annual Swiss Trypanosomatid Meeting Talk given at a conference Towards the characterization of protein import complexes in the inner mitochondrial membrane of T. brucei 23.01.2014 Leysin, Switzerland Harsman Anke;


Self-organised

Title Date Place
31st Annual Swiss Trypanosomatid Meeting 23.01.2014 Leysin, Switzerland

Communication with the public

Communication Title Media Place Year
Media relations: print media, online media Neues Protein für die Weitergabe von Erbmaterial entdeckt Pressemitteilung der Universität Bern Italian-speaking Switzerland German-speaking Switzerland Rhaeto-Romanic Switzerland Western Switzerland 2014

Associated projects

Number Title Start Funding scheme
156940 Mitochondrial biogenesis in the unicellular parasite Trypanosoma brucei 01.01.2015 Project funding (Div. I-III)
121937 Mitochondrial biogenesis in T. brucei: import of macromolecules and organellar gene expression 01.01.2009 Project funding (Div. I-III)

Abstract

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.
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