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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)

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Presentation on theme: "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)"— Presentation transcript:

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) (1)UMR 7156 CNRS – UdS, Strasbourg, France (2) Moscow State University, Moscow, Russia

2 Mitochondria : the “powerplant” of the cell 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 Fluorescent staining of mitochondrial networks Electron microscopy Schematic representation Inner membrane space

3 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 - … respiratory chain ATP INNER MEMBRANE Adenosine tri-phosphate (ATP) ΔΨ + ΔpH = p.m.f (proton-motive force)

4 Mitochondrial RNA import is widespread among species Genus Imported RNA's Number of imported RNA's Protozoans : tRNA majority - totality (>40 species) Plants : tRNA (>20 species) Mammals: tRNA (4 species) 5S rRNA (MRP RNA, RnaseP RNA) Fungi: tRNA (3 species) Saccharomyces cerevisiae tRNA 1-3

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

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

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

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

9 Wild type ΔPOR1 Outer membrane: TOM 5 (component of the translocase of outer membrane complex) +- TOM 20 (component of the translocase of outer membrane complex) -+ TOM 22 (component of the translocase of outer membrane complex) -+ TOM 40 (component of the translocase of outer membrane complex) -+ POR 1 (Porin 1) +- Inner membrane : AAC (ADP/ATP carrier) ++ MIR 1 (Phosphate carrier) ++ QCR 2 (subunit 2 of the ubiquinol cytochrome-c reductase complex) ++ PHB 1 (subunit 1 of prohibitin complex) -+ TIM 50 (component of the translocase of inner membrane complex) -+ 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 Identification of proteins interacting with tRK1 by North-Western and mass-spectrometry analysis

10 Mitoribosomal proteins: MRP-L1 (mitochondrial ribosomal protein of the large subunit) MRP-L3 (mitochondrial ribosomal protein of the large subunit) MRP-L7 (mitochondrial ribosomal protein of the large subunit) MRP-L35 (mitochondrial ribosomal protein of the large subunit) Mitochondrial chaperones: HSP 60 (mitochondrial chaperonin required for ATP-dependent folding of precursor polypeptides and complex assembly) Protein involved in genome maintainance : 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) Matrix enzymes : KGD 1 (component of the mitochondrial alpha-ketoglutarate dehydrogenase complex) KGD 2 (dihydrolipoyl transsuccinylase) SHM 1 (mitochondrial serine hydroxymethyltransferase) MIS 1 (mitochondrial C1-tetrahydrofolate synthase) 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 ATP is more important then electrochemical membrane potential ΔΨ POR1 seems to be implicated in the import mechanism (-) (+) xxxxxxxxxx xxxx xxxxx xxxxxxxx xxxxxxxx xxxxx ATP succinate FCCP DIDS PDE oligomycine ATP tRK1 Dependence of tRK1 import on the level of ATP and electrochemical membrane potential ΔΨ

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

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 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) Participants and sponsors

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