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
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
Functions
Adenosine tri-phosphate (ATP)
INNER
MEMBRANE
- Respiration and energy production (ATP)
ATP
- Apoptosis (programmed cell death)
- Metabolism (Krebs cycle, urea cycle…)
respiratory chain
ΔΨ + ΔpH = p.m.f (proton-motive force)
- Oxidation and synthesis of fatty acids
- Synthesis of essential amino acids
- Regulation of intracellular calcium pool
-…
Mitochondrial RNA import is widespread among species
Genus
Imported RNA's
Number of imported RNA's
•
Protozoans :
(>40 species)
tRNA
•
Plants :
(>20 species)
tRNA
2 - 15
•
Mammals:
tRNA
1-2
(4 species)
5S rRNA (MRP RNA, RnaseP RNA)
Fungi:
(3 species)
tRNA
1 - 20
Saccharomyces cerevisiae
tRNA
1-3
•

majority - totality
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
Import of tRNALys into mitochondria of Saccharomyces cerevisiae
Saccharomyces cerevisiae
cytoplasmic
(anticodon CUU)
cytoplasmic
(anticodon UUU)
mitochondrial
(anticodon UUU)
3 isoforms of tRNALys
tRK1
(partially imported)
tRK2
(non-imported)
tRK3
Cloverleaf structures of cytoplasmic and mitochondrial tRNALys of S.cerevisiae
Cytoplasmic targeting factors of tRK1 to the mitochondrial surface
Aim :
- identification
inner
of proteins
membranes
implicated
in
of
outer and
of
mitochondria
the
translocation
mechanisms of tRNALys (tRK1)
-
study
the
electrochemical
(ΔΨ) and ATP level
(Entelis et al. 2006)
Enolase-2 : forms 1st complex with aminoacylated tRK1
Pre-MSK : forms 2nd complex with aminoacylated tRK1
interaction favors complex formation with pre-MSK
interaction necessary for tRK1 importation
requirements
membrane
for
potential
The pre-protein import machinery, porins and import of tRNALys
metabolites
RNase treatment
Incubation 32°C
Isolation mitochondrial RNA
EDTA washing
POR2
Import Mix
In vitro import assay
- ATP 5mM
- NADH 3mM
- Succinate 10mM
- MgCl2 2,5mM
- Sorbitol 0,44M
- HEPES-NaOH pH6,8 10mM
Exposition photosensitive plate
Δ POR 1
Δ TOM 5
Wild type
(-) mitocondria
10%PAAG/8M Urea gel electrophoresis
Autoradiography
Input 5% + RNase
Input 5%
Δ TIM 44
Δ TOM 20
Δ TOM 70
Wild type
Scheme of the pre-protein import machinery (Bolender et al. 2008)
Fixation and drying of the gel
Δ POR 2
POR1
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
% relative import
200
180
160
140
% import
In vitro import of tRK1 into
mitochondria of strains carrying
deletions for essential proteins of the
pre-protein import machinery
(Tarassov et al. 1995)
120
100
80
60
40
20
0
WT
ΔTOM 5
ΔPOR 1
ΔPOR 2
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
Wild
type
ΔPOR1
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)
+
-
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)
-
+
Outer membrane:
Inner membrane :
Identification by nano-LC MS/MS
Identification of proteins interacting with tRK1 by crosslinking and
SDS-PAGE/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)
Matrix enzymes:
KGD 1
(component of the mitochondrial alpha-ketoglutarate
dehydrogenase complex)
Mitochondrial chaperones:
HSP 60 (mitochondrial chaperonin required for ATP-dependent folding of
precursor polypeptides and complex assembly)
KGD 2
(dihydrolipoyl transsuccinylase)
SHM 1
(mitochondrial serine
MIS 1
(mitochondrial C1-tetrahydrofolate synthase)
ILV 5
(Acetohydroxyacid reductoisomerase)
PDX 1
(lipoamide dehydrogenase)
ACO 1
(aconitase)
hydroxymethyltransferase)
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)
Dependence of tRK1 import on the level of ATP and
electrochemical membrane potential ΔΨ
1
7
8
9
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
4
5
x
x
x
x
succinate
x
x
x
oligomycine
x
x
ATP
x
2
6
3
(-)
FCCP
x
DIDS
PDE
x
x
x
x
x
x
x
x
x
x
(+)
(+)
x
x
x
x
tRK1
ATP
ATP
ATP is more important then electrochemical membrane potential ΔΨ
POR1 seems to be implicated in the import mechanism
External or internal ATP ?
- ATPinternal
-
+
+
+
+
-
-
-
external ATP
-
+
+
-
-
-
+
+
+
-
+
-
-
+
+
-
+
ΔΨ
-
tRK1
Internal ATP pool is more important than external pool
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
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)