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      A trypanosomal orthologue of an intermembrane space chaperone has a non-canonical function in biogenesis of the single mitochondrial inner membrane protein translocase

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          Abstract

          Mitochondrial protein import is essential for Trypanosoma brucei across its life cycle and mediated by membrane-embedded heterooligomeric protein complexes, which mainly consist of trypanosomatid-specific subunits. However, trypanosomes contain orthologues of small Tim chaperones that escort hydrophobic proteins across the intermembrane space. Here we have experimentally analyzed three novel trypanosomal small Tim proteins, one of which contains only an incomplete Cx3C motif. RNAi-mediated ablation of TbERV1 shows that their import, as in other organisms, depends on the MIA pathway. Submitochondrial fractionation combined with immunoprecipitation and BN-PAGE reveals two pools of small Tim proteins: a soluble fraction forming 70 kDa complexes, consistent with hexamers and a second fraction that is tightly associated with the single trypanosomal TIM complex. RNAi-mediated ablation of the three proteins leads to a growth arrest and inhibits the formation of the TIM complex. In line with these findings, the changes in the mitochondrial proteome induced by ablation of one small Tim phenocopy the effects observed after ablation of TbTim17. Thus, the trypanosomal small Tims play an unexpected and essential role in the biogenesis of the single TIM complex, which for one of them is not linked to import of TbTim17.

          Author summary

          Trypanosoma brucei and its relatives are prominent pathogens causing human and animal diseases, which mainly affect developing countries. The single mitochondrion of trypanosomes is essential across its entire life cycle. Most organellar proteins are imported by hetero-oligomeric protein complexes in the two mitochondrial membranes. Interestingly, the composition of the two import machineries is remarkably different from their corresponding counterparts in other organisms. In contrast, chaperones termed small Tims that escort hydrophobic proteins across the aqueous intermembrane space are conserved in almost all eukaryotes including trypanosomes. Here we show that a fraction of them interact tightly with the inner membrane translocase. Another fraction is present as soluble 70 kDa complexes, which likely consists of hexamers of small Tims without a defined subunit composition. In other eukaryotes these hexamers are usually composed of two alternating small Tims. Moreover, while some small Tims are involved in import of a core subunit of the inner membrane protein translocase, we found one small Tim that directly mediates the assembly of the translocase complex. Knowing which components of the trypanosomal protein import systems are conserved and which ones are not is essential to evaluate whether mitochondrial protein import might be a suitable drug target.

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          Most cited references 40

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          Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.

          A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h. It is particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.
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            A tightly regulated inducible expression system for conditional gene knock-outs and dominant-negative genetics in Trypanosoma brucei.

            First-generation inducible expression vectors for Trypanosoma brucei utilized a single tetracycline-responsive promoter to drive expression of an experimental gene, in tandem with a drug-resistance marker gene to select for integration (Wirtz E, Clayton CE. Science 1995; 268:1179-1183). Because drug resistance and experimental gene expression both depended upon the activity of the regulated promoter, this approach could not be used for inducible expression of toxic products. We have now developed a dual-promoter approach, for expressing highly toxic products and generating conditional gene knock-outs, using back-to-back constitutive T7 and tetracycline-responsive PARP promoters to drive expression of the selectable marker and test gene, respectively. Transformants are readily obtained with these vectors in the absence of tetracycline, in bloodstream or procyclic T. brucei cell lines co-expressing T7 RNA polymerase and Tet repressor, and consistently show tetracycline-responsive expression through a 10(3)-10(4)-fold range. Uninduced background expression of a luciferase reporter averages no more than one molecule per cell, enabling dominant-negative approaches relying upon inducible expression of toxic products. This tight regulation also permits the production of functional gene knock-outs through regulated expression of an experimental gene in a null-mutant background.
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              The real 'kingdoms' of eukaryotes.

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                Author and article information

                Contributors
                Role: InvestigationRole: MethodologyRole: VisualizationRole: Writing – review & editing
                Role: InvestigationRole: MethodologyRole: VisualizationRole: Writing – review & editing
                Role: Funding acquisitionRole: ResourcesRole: SupervisionRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: Project administrationRole: ResourcesRole: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: SupervisionRole: VisualizationRole: Writing – original draft
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, CA USA )
                1553-7366
                1553-7374
                21 August 2017
                August 2017
                : 13
                : 8
                Affiliations
                [1 ] Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, Bern, Switzerland
                [2 ] Department of Biochemistry and Functional Proteomics, Institute of Biology II, Faculty of Biology, University of Freiburg, Schänzlestr. 1, Freiburg, Germany
                [3 ] BIOSS Centre for Biological Signalling Studies, University of Freiburg, Schänzlestr. 18, Freiburg, Germany
                University of California, Los Angeles, UNITED STATES
                Author notes

                The authors have declared that no competing interests exist.

                Article
                PPATHOGENS-D-17-00527
                10.1371/journal.ppat.1006550
                5584982
                28827831
                © 2017 Wenger et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Page count
                Figures: 6, Tables: 0, Pages: 21
                Product
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100001711, Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung;
                Award ID: 138355
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100001711, Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung;
                Award ID: NCCR "RNA & Disease"
                Award Recipient :
                Funded by: Peter und Traudl Engelhorn Stiftung zur Förderung der Lebenswissenschaften
                Award ID: Postdoc Fellowship
                Award Recipient :
                Funded by: Excellence Initiative of the German Federal & State Governments
                Award ID: EXC 294 BIOSS Centre for Biological Signalling Studies
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100001659, Deutsche Forschungsgemeinschaft;
                Award Recipient :
                AH gratefully acknowledges a fellowship from the Peter und Traudl Engelhorn foundation ( http://ptes.2c4b.de/). Research in the group of BW was funded by the Deutsche Forschungsgemeinschaft and the Excellence Initiative of the German Federal & State Governments (EXC 294 BIOSS Centre for Biological Signalling Studies; http://www.dfg.de/en/research_funding/programmes/list/projectdetails/index.jsp?id=39236281). Research in the lab of AS was supported by grant 138355 ( http://p3.snf.ch/Project-138355) and in part by the NCCR "RNA & Disease" ( http://www.snf.ch/en/researchinFocus/nccr/rna-disease/Pages/default.aspx) both funded by the Swiss National Science Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Biochemistry
                Bioenergetics
                Energy-Producing Organelles
                Mitochondria
                Biology and Life Sciences
                Cell Biology
                Cellular Structures and Organelles
                Energy-Producing Organelles
                Mitochondria
                Biology and life sciences
                Genetics
                Epigenetics
                RNA interference
                Biology and life sciences
                Genetics
                Gene expression
                RNA interference
                Biology and life sciences
                Genetics
                Genetic interference
                RNA interference
                Biology and life sciences
                Biochemistry
                Nucleic acids
                RNA
                RNA interference
                Biology and Life Sciences
                Cell Biology
                Cellular Structures and Organelles
                Cell Membranes
                Membrane Proteins
                Integral Membrane Proteins
                Biology and Life Sciences
                Biochemistry
                Proteins
                Chaperone Proteins
                Research and Analysis Methods
                Electrophoretic Techniques
                Gel Electrophoresis
                Polyacrylamide Gel Electrophoresis
                Blue Native Polyacrylamide Gel Electrophoresis
                Research and Analysis Methods
                Precipitation Techniques
                Immunoprecipitation
                Co-Immunoprecipitation
                Biology and Life Sciences
                Organisms
                Fungi
                Yeast
                Biology and Life Sciences
                Cell Biology
                Cellular Structures and Organelles
                Cell Membranes
                Membrane Proteins
                Custom metadata
                vor-update-to-uncorrected-proof
                2017-09-05
                All relevant data are within the paper and its Supporting Information files.

                Infectious disease & Microbiology

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