Studying the Mechanisms of RNA Translocation into Mitochondria
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Transcript of Studying the Mechanisms of RNA Translocation into Mitochondria
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
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 microscopyElectron microscopy
Schematic representation
Inner membrane space
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
INNERMEMBRANE
Adenosine tri-phosphate (ATP)
ΔΨ + ΔpH = p.m.f (proton-motive force)
Mitochondrial RNA import is widespread among species
Genus Imported RNA's Number 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 tRNA 1-3
Why study import of RNA into mitochondria ?
Liver defects
Nanismedefects of bone
marrow
Cardiomyopathy
Diabetes Thyroid gland diseases
Myopathies
Peripherical myopathies
Deafness
Optical nerve atrophy / Retinitis PigmentosaRespiratory defects
Cerebrovascular diseases
Mental retardation
Human mitochondrialgenome
Import of tRNALys 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 tRNALys
Cloverleaf structures of cytoplasmic and mitochondrial tRNALys of S.cerevisiae
(Entelis et al. 2006)
Cytoplasmic targeting factors of tRK1 to the mitochondrial surface
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
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
The pre-protein import machinery, porins and import of tRNALys
Wild
type
Δ T
OM
70
Δ T
OM
20
Δ T
IM 4
4
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)
Inpu
t 5%
Inpu
t 5%
+ R
Nas
e
Wild
type
Δ T
OM
5
Δ PO
R 1
Δ PO
R 2
(-) m
itoco
ndri
a
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 5 ΔPOR 1 ΔPOR 2
Import Mix
- ATP 5mM- NADH 3mM- Succinate 10mM- MgCl2 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
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
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
ATP is more important then electrochemical membrane potential ΔΨ
POR1 seems to be implicated in the import mechanism
(-) 1 2 3 4 5 6 7 8 9 (+) (+)
xxxxxxxxxx
xxxx
xxxxx
xxxxxxxx
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xxxxxATPsuccinate
FCCPDIDS
PDE
oligomycine
ATP ATP
tRK1
Dependence of tRK1 import on the level of ATP and electrochemical membrane potential ΔΨ
External or internal ATP ?
Internal ATP pool is more important than external pool
+-++-- +--+
++---++---
---++++---
Inpu
t
internal ATP
external ATP
Δ Ψ
tRK1
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
Team « Mito »
O. KolesnikovaO. KarichevaA.-M. HeckelN. EntelisA. SmirnovC. ComteT. SchirtzM. VyssokikhY. ToninR. MartinY. KharchenkovI. Tarassov
Collaborations:
A. Lombès(Salpétrière, Paris)
A. Dietrich(IBMP, Strasbourg)
Participants and sponsors