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

Nanisme

defects 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

typ

e

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

typ

e

Δ T

OM

5

Δ P

OR

1

Δ P

OR

2

(-)

mit

ocon

dria

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

% relative import

0

20

40

60

80

100

120

140

160

180

200

% i

mp

ort

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

xxxxxATP

succinate

FCCP

DIDS

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