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Studying the Mechanisms of RNA Translocation into Mitochondria

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  1. Studying the Mechanisms of RNA Translocation into Mitochondria • T. Schirtz (1), M. Vyssokikh (1,2), O. Kolesnikova (1,2), N. Entelis (1), I. Tarassov (1) • UMR 7156 CNRS – UdS, Strasbourg, France • (2) Moscow State University, Moscow, Russia

  2. Mitochondria : the “powerplant” of the cell Fluorescent staining of mitochondrial networks Localization and features • Organelles present in cytoplasm of every eukaryotic • cell • Arranged in highly complex networks • Endosymbiotic origin • Possess own genetic information and own genetic • code Inner membrane space Electron microscopy Electron microscopy Schematic representation

  3. Adenosine tri-phosphate (ATP) INNER MEMBRANE ATP respiratory chain Functions • Respiration and energy production (ATP) • Apoptosis (programmed cell death) • Metabolism (Krebs cycle, urea cycle…) • Oxidation and synthesis of fatty acids • Synthesis of essential amino acids • Regulation of intracellular calcium pool • … ΔΨ + ΔpH = p.m.f (proton-motive force)

  4. Mitochondrial RNA import is widespread among species • GenusImported RNA'sNumber of imported RNA's • Protozoans : tRNA majority - totality • (>40 species) • Plants : tRNA 2 - 15 • (>20 species) • Mammals: tRNA 1 - 2 • (4 species) 5S rRNA (MRP RNA, RnaseP RNA) • Fungi: tRNA 1 - 20 • (3 species) • Saccharomyces cerevisiae tRNA1-3

  5. Why study import of RNA into mitochondria ? Optical nerve atrophy / Retinitis Pigmentosa Respiratory defects Cerebrovascular diseases Mental retardation Cardiomyopathy Liver defects Deafness Human mitochondrial genome Peripherical myopathies Nanisme defects of bone marrow Diabetes Thyroid gland diseases Myopathies


  6. Saccharomyces cerevisiae mitochondrial (anticodon UUU) cytoplasmic (anticodon UUU) cytoplasmic (anticodon CUU) 3 isoforms of tRNALys tRK1 (partially imported) tRK2 (non-imported) tRK3 Cloverleaf structures of cytoplasmic and mitochondrial tRNALys of S.cerevisiae Import of tRNALys into mitochondria of Saccharomyces cerevisiae

  7. Enolase-2 : forms 1st complex with aminoacylated tRK1 interaction favors complex formation with pre-MSK Pre-MSK : forms 2nd complex with aminoacylated tRK1 interaction necessary for tRK1 importation Cytoplasmic targeting factors of tRK1 to the mitochondrial surface Aim : - identification of proteins of outer and inner membranes of mitochondria implicated in the translocation mechanisms of tRNALys (tRK1) - study the requirements for electrochemical membrane potential (ΔΨ) and ATP level (Entelis et al. 2006)

  8. RNase treatment Isolation mitochondrial RNA Incubation 32°C EDTA washing • Import Mix • ATP 5mM • NADH 3mM • Succinate 10mM • MgCl2 2,5mM • Sorbitol 0,44M • HEPES-NaOH pH6,8 10mM In vitro import assay Fixation and drying of the gel Exposition photosensitive plate 10%PAAG/8M Urea gel electrophoresis Autoradiography Input 5% + RNase (-) mitocondria Δ TOM 70 Δ TOM 20 Wild type Δ TIM 44 Wild type Δ POR 2 Input 5% Δ TOM 5 Δ POR 1 tRK1 In vitro import of tRK1 into mitochondria of strains carrying deletions for porins and for a non essential protein of the pre-protein import machinery tRK1 In vitro import of tRK1 into mitochondria of strains carrying deletions for essential proteins of the pre-protein import machinery (Tarassov et al. 1995) ΔTOM 5 ΔPOR 1 ΔPOR 2 WT The pre-protein import machinery, porins and import of tRNALys metabolites POR2 POR1 Scheme of the pre-protein import machinery (Bolender et al. 2008)

  9. Identification of proteins interacting with tRK1 by North-Western and mass-spectrometry analysis Preparation of mitochondrial outer and inner membranes SDS-Page and transfer to nitrocellulose membrane Renaturation of blotted proteins Probing of the filter with radiolabeled aminoacylated tRK1 Autoradiography Localization of the signals on an identical SDS-Page Identification by nano-LC MS/MS

  10. Mitoribosomal proteins: Matrix enzymes: MRP-L1 (mitochondrial ribosomal protein of the large subunit) KGD 1 (component of the mitochondrial alpha-ketoglutarate dehydrogenase complex) MRP-L3 (mitochondrial ribosomal protein of the large subunit) MRP-L7 (mitochondrial ribosomal protein of the large subunit) KGD 2 (dihydrolipoyl transsuccinylase) MRP-L35 (mitochondrial ribosomal protein of the large subunit) Mitochondrial chaperones: SHM 1 (mitochondrial serine hydroxymethyltransferase) HSP 60 (mitochondrial chaperonin required for ATP-dependent folding of precursor polypeptides and complex assembly) Protein involved in genome maintainance: MIS 1 (mitochondrial C1-tetrahydrofolate synthase) RIM 1 (single-stranded DNA-binding protein essential for mitochondrial genome maintenance) MSS 116 (DEAD-box protein required for efficient splicing of mitochondrial Group I and II introns) ILV 5 (Acetohydroxyacid reductoisomerase) ILV 5 (Acetohydroxyacid reductoisomerase) PDX 1 (lipoamide dehydrogenase) ACO 1 (aconitase) Identification of proteins interacting with tRK1 by crosslinking and SDS-PAGE/Mass-spectrometry analysis

  11. x x x x x x x x x x x x x x x x x x x x x x x x x x 8 1 6 (+) 2 3 4 5 7 9 (+) (-) x x x x ATP x x x x x x x x x x succinate oligomycine FCCP DIDS tRK1 PDE ATP ATP ATP is more important then electrochemical membrane potential ΔΨ POR1 seems to be implicated in the import mechanism Dependence of tRK1 import on the level of ATP and electrochemical membrane potential ΔΨ

  12. - - - + + + + - - - internal ATP - - - + + - - - + + external ATP + - - + - - + + - + Δ Ψ Input tRK1 External or internal ATP ? Internal ATP pool is more important than external pool

  13. Conclusions and perspectives • ATP is indispensable for import compared to electrochemical membrane potential ΔΨ • Internal ATP pool is more important than external ATP pool • - Proteins of the pre-protein import machinery (Tom20, Tom5, Tim44) and porin 1 are • implicated in the translocation mechanism • Improve crosslinking approach • Study import in mutants for proteins identified by North-Western and the crosslinkink method • - Reconstitution of a minimal import machinery in artificial liposomes

  14. Participants and sponsors • Team « Mito » • O. Kolesnikova • O. Karicheva • A.-M. Heckel • N. Entelis • A. Smirnov • C. Comte • T. Schirtz • M. Vyssokikh • Y. Tonin • R. Martin • Y. Kharchenkov • I. Tarassov • Collaborations: • A. Lombès • (Salpétrière, Paris) • A. Dietrich • (IBMP, Strasbourg)