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
xxxxxxxx
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