COMPARTIMENTS DE LA CELLULE ET TRI DES PROT

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COMPARTIMENTS DE LA CELLULE ET TRI DES PROTÉINES
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COMPARTIMENTS DE LA CELLULE ET TRI DES PROTÉINES

• III - Transport des protéines dans les
  mitochondries et les chloroplastes

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A - Rappels sur la mitochondrie

• Structure
• Les compartiments
• ADN mitochondrial (traité ailleurs)

4
Frey,TG2000 (fig1)

• Schéma classique

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Les compartiments(Capaldi,RA (2002) BBA1555p192)

• Trois compartiments membranaires
• Membrane externe
• Membrane interne
• Crêtes
• Trois espaces
• Espace intermembranaire
• Espaces "intracristal"
• Matrice

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Morphologie

• Forme et distribution variables
• Variable en fonction du stade du cycle cellulaire
• petit organite ovoïde en phase S et M
• réticulaire en G0
• Modèle de Palade (1952) 2 membranes, modèle
  retenu dans les livres
• Modèle de Sjöstrand (1956) 3 membranes

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"Computer Axial Tomography (CAR) scan" 60 à
120 images à 1 ou 2 de -60 à 60

• Crêtes reliées à la membrane interne par des
  tubules de 28 6 nm

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Frey,TG2002p196(fig1)
Une des coupes de a ? aspect tubulaires des
jonctions crête membrane interne
ME 0,5? à 400 kV ? Jonctions tubulaires des
crêtes
La même mitochondrie

• Modèle 3D
• Membrane externe
• Membrane interne
• Crêtes

4 crêtes de 4 couleurs différentes jonctions
tubulaires
http//www.sci.sdsu.edu/TFrey/MitoMovie.htm
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Frey,TG2002p196(fig2)

• a - Même mitochondrie crêtes avec les jonctions
  en rouge
• b - Modèle idéalisé de jonction crête - membrane

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Frey,TG2000 (fig3)

• Computer models generated from segmented 3D
  tomograms of a mitochondrion in chick cerebellum.
  (a) The entire model showing all cristae in
  yellow, the inner boundary membrane in light
  blue, and the outer membrane in dark blue. (b)
  Outer membrane, inner boundary membrane and four
  representative cristae in different colors.
  Quicktime videos of these models can be found at
  http//www.sci.sdsu.edu/tfrey/mitomovie.htm

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Frey,TG2000 (fig5)

• Amibe crêtes paracristallines

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Frey,TG2000 (fig2)

• Foie de rat (normal)

Contacts OM/IM
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Griparic,L2001Traffic
Myopathie mitochondriale

• Reconstruction 3-D d une mitochondrie par
  tomographie électronique
• Suspension de mitochondries congelée,
  cryomicroscopie électronique, tomographie
  assistée par ordinateur

Rouge membrane interne
Autres couleurs crêtes
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Frey,TG2002p196(fig3)

• Mitochondrie de Xenope
• Mitochondrie ayant perdu son cytochrome c
• Mitochondrie n'ayant pas perdu son cytochrome c
• Mitochondrie avec Ca augmenté

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En vidéo microscopie

• Dynamique sauts sur de petits mouvements plus
  continus
• Intervention des microtubules et microfilaments
• http//www.sci.sdsu.edu/TFrey/MitoMovie.htm

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Griparic,L2001Traffic

• Distribution typique des mitochondries dans une
  cellule de mammifère en culture (myoblaste de
  souris observé in vivo par le colorant vital
  MitoTracker)

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Griparic,L2001Traffic

• Réseau de mitochondries dans une cellule
  musculaire de C. elegans mutée.
• Le blocage de la division de la membrane externe
  favorise la fusion des mitochondries aboutissant
  à un réseau.
• La membrane interne continue à se diviser.
• Vert membrane mitochondriale ext
• Rouge matrice mitochondriale
• Cellules vivantes (GFP)

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Griparic,L2001Traffic

• Crêtes mitochondriales de cellules de cortex
  surrénalien.
• A - Rat traîté par corticotropin-releasing
  hormone, (qui stimule la production de stéroïdes)
  les crêtes sont tubulaires.
• B - Cellule de la zone fasciculée aves des
  crêtes vésiculaires.

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Griparic,L2001Traffic

• Inclusions paracristallines dans les
  mitochondries dun enfant atteint de myopathie.
• Mitochondries géantes avec des inclusions
  paracristallines qui seraient des crêtes de la
  membrane interne.
• Se retrouve dans dautres situations
  pathologiques.

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Frey,TG2000 (fig6)

• Model of a mitochondrion displaying highly
  abnormal internal compartmentation from a patient
  with a mitochondrial myopathy. The mitochondrion
  contains numerous small vesicles (luminous green)
  that do not connect to the inner boundary
  membrane (blue). There is a second large internal
  membrane (green), which has a vesicle-like
  protrusion, suggesting either that this unusual
  membrane is formed by fusion of the vesicles or,
  conversely, that the vesicles are formed by
  budding from it. Reproduced, with permission,
  from M. Huizing.

Myopathie mitochondriale
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Rappel de quelques fonctions

• Production d'énergie
• Régulation du calcium intracellulaire
• Oxydation des acides gras
• Synthèse des stéroïdes
• Apoptose

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Localisation des protéines

• La plupart dans un seul compartiment
• Exceptions (crêtes membrane interne)
• Enzymes de la chaîne respiratoire
• ATP synthétase

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Constitution biochimique

• Environ 1000 protéines différentes

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Biogenèse

• Croissance dorganites préexistant
• Suivie de fission ?
• Nécessité dimportation de nouvelles protéines

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Capaldi,RA (2002) BBA1555p192

• Genèse par fragmentation du reticulum
  mitochondrial
• Marquage avec GFP-Pyruvate deshydrogénase
• Mitotraker marque le réseau mitochondrial

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Griparic,L2001Traffic
Cellule musculaire cardiaque de souris normale
Adipocyte de papillon(Calpodes ethlius)

• Clivage de la membrane mitochondriale interne
  sans division de la mb externe

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B - TRANSPORT DES PROTÉINES DANS LES
MITOCHONDRIES ET LES CHOLOROPLASTES
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Schéma du routage
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Schéma du routage partiel
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Mitochondrie (rappel)

• Double membrane
• Synthèse d'ATP
• par transport d'électrons et phosphorylation
  oxydative (mitochondrie)
• par photosynthèse phosphorylante (chloroplaste)
• Contiennent leur ADN et leur machine à synthèse
  protéique
• La plupart de leur protéines est codée par le
  noyau

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Les compartiments de la mitochondrie

• Jeu de protéines différent (1000 protéines
  différentes dans la mitochondrie)
• ? translocation sélective de 1 ou 2 membranes
• Les protéines codées par la mitochondrie sont
  dans la membrane interne
• Coordination des 2 types de protéines ?
• Mitochondriales (13 protéines connues de la
  chaîne respiratoire)
• nucléaires (les autres)

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Fig 12-22
Les 2 compartiments de la mitochondrie
Les 3 compartiments du chloroplaste
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Import dans la matrice(et membrane
mitochondriale externe)

• Protéines de fusion chez la levure
• Import post-traductionnel (quelques secondes ou
  minutes)
• Séquence Signal
• à lextrémité -N
• rapidement retiré (signal peptidase)
• hélice ? amphiphile
• AA d un côté de lhélice
• AA - de lautre côté de lhélice
• reconnu par des récepteurs spécifiques

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Fig 12-23

• Séquence signal pour l'import d'une protéine
  mitochondriale
• eg Cytochrome oxydase du complex IV
• Matrix Targeting Signal

1
5
9
12
12
16
Hélice ? de 3,6 résidus par tour
?

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Les translocateurs TOM TIM

• Complexes protéiques
• Translocator of the Outer Membrane (TOM40)
• Translocators of the Inner Membrane (TIM)
• TIM23
• TIM22
• 2 fonctions
• récepteur pour les protéines mitochondriales
  précurseur
• canal translocateur

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Au total 3 complexes

1. TOM 40
2. TIM 23
3. TIM 22

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Pfanner,N2002p400(fig1)

• Les deux grandes voies d'importation des
  protéines mitochondriales
• Préprotéines à signal qui sera retiré par MPP
  (Mitochondrial Processing Peptidase)
• Précurseur des protéines hydrophobes

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1 . TOM

• Import de toutes les protéines mitochondriales
  codées par le noyau
• Comprend le "General Import Pore" (GIP)
• Transporte le signal dans lespace
  intermembranaire
• Aide à linsertion des protéines
  transmembranaires dans la membrane externe (on
  nen reparlera plus)

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Rapaport,D2002

• Complexe TOM

Récepteurs TOM 20, 22,70
Pore TOM 5, 6 ,7 , 20, 22 ,40
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Pfanner,N2002p400(fig2)

• Système de translocation de la membrane externe
  de la mitochondrie
• TOM
• TOM 20 avec une séquence liée (hélice ?)
• GIP TOM 40, 22, 7, 6, 5, 20

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TOM 40

• Forme le canal aqueux (de 20 nm) du GIP (General
  Import Pore)
• Utilisé par les deux voies de transport
• L'entrée se fait sous forme d'une boucle
• Associé à TOM 20, TOM 22, TOM 70 et TOM 5, TOM 6,
  TOM 7

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2 . TIM 23

• TIM 17,23,44 mtHsp70 co-chaperone Mge1
• Transport de certaines protéines dans la matrice
• Insertion de protéines transmembranaires dans la
  membrane interne

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Pfanner,N2002p400(fig4)

• The Mitochondrial presequence translocase and the
  Processing Peptidase (MPP). (a) The presequence
  translocase consists of an innermembrane-
  integrated pore complex (the Tim23 complex) and
  the peripherally attached import motor at the
  matrix side. Tim23 and Tim17 form a stable
  complex in the inner membrane (IM). The amino
  terminus of Tim23 in the intermembrane space
  (IMS) is important for recognising the
  presequences as they emerge from the TOM complex,
  whereas the carboxy-terminal membrane-integrated
  domain of Tim23 forms a channel for preproteins.
  This channel is activated by the membrane
  potential (??), which also exerts a pulling force
  on the positively charged presequence. The import
  motor consists of mtHsp70, the membrane-associated
  protein Tim44, and the homodimeric co-chaperone
  mitochondrial GrpE (Mge1, a nucleotide exchange
  factor). The motor is driven by hydrolysis of
  ATP. The heterodimeric MPP cleaves the
  presequences. (b) The MPP binds the presequences
  in an extended conformation in a large central
  cavity between the ? and ? subunits (the
  encircled area of MPP of a is shown modelled
  after the structure of yeast MPP). MPP? binds the
  conserved hydrophobic (hydro) sidechain of the
  first amino acid residue of the mature protein in
  a hydrophobic pocket that includes a
  phenylalanine (Phe77). The conserved, positively
  charged arginine residue in the 2 position is
  bound by negatively charged residues (Glu160 and
  Asp164) on MPP?. This brings the scissile peptide
  bond (arrow) close to the essential active-site
  zinc ion. The preprotein with the presequence is
  shown in red and blue. Amino acid residues in
  green belong to MPP? (the numbering starts with
  residue 1 of the preprotein of Saccharomyces
  cerevisiae MPP?).

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3 . TIM 22

• Insertion de certaines protéines dans la membrane
  interne dont
• transporteur de ATP, ADP, Pi

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Pfanner,N2002p400(fig5)

• Import stages of hydrophobic membrane proteins to
  the mitochondrial inner membrane
• Stage I Cytosolic chaperones bind to the
  precursor of a carrier protein (used here as an
  example of a membrane protein) and keep it in an
  import-competent state
• Stage II The precursor protein is bound by
  several Tom70 receptor molecules. The precursor
  is then released in an ATP-dependent step and
  transferred to the GIP.
• Stage III The precursor protein is translocated
  in a loop formation through the GIP and binds to
  the Tim9Tim10 complex (via hydrophobic regions
  of the precursor)
• Stage IV Membrane integration of the carrier
  precursor by the protein insertion complex (Tim22
  complex) of the inner membrane depends on the
  membrane potential (?ø
• Stage V The carrier protein assembles to the
  mature homodimer in the inner membrane.

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Complexe OXA

• Translocateur dans la membrane interne, de
  protéines synthétisées dans la mitochondrie
• Aide à l'insertion transmembranaire (interne) de
  protéines transportées initialement dans la
  matrice

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Fig 12-24

• les 3 translocateurs dans les membranes
  mitochondriales

?
48
Koehler,CM2000
Import des protéines et voies d'exportation dans
la mitochondrie
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Les protéines précurseurs mitochondriales sont
importées sous forme de polypeptides non repliés

• Analyse à partir de systèmes sans cellule
• Des protéines cytosoliques empêchent les
  protéines mitochondriales de se replier sous leur
  forme native
• sont le plus souvent des protéines chaperones
  (appartenant à la famille des hsp 70)
• sont parfois spécifiques de leur signal
• puis sont retirées avant l'engagement dans le TOM

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Passage dans la matrice deux hypothèses

• Traverser deux membranes
• Traverser les deux membranes en même temps

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Fig 12-25

• Protocole expérimental
• la translocation se fait en deux étapes
• les deux membranes sont traversées en même temps

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Passage dans la matrice conclusion

• Traversée des deux membranes en même temps en
  deux étapes
• entrée du signal dans l'espace intermembranaire
  par TOM
• liaison du signal par TIM
• soit entrée dans la matrice
• soit entrée dans la membrane interne
• Présence de sites de contacts entre les deux
  membranes
• TOM et TIM peuvent fonctionner indépendamment les
  uns des autres expérimentalement

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Fig 12-26

• Import des protéines dans la matrice

4 étapes
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Deux conditions pour diriger l'import des
protéines dans la mitochondrie

• Hydrolyse de l ATP
• en dehors de la mitochondrie
• et dans la matrice
• Gradient électrochimique dions H de part et
  dautre de la membrane interne
• pompage dions H à travers la membrane interne
  ce gradient ne sert pas quà la synthèse dATP
• la membrane externe est perméable aux ions en
  raison de la présence de porine (comme les
  bacilles gram -) ? pas de gradient

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Aspect énergétique de la translocation

• Libération des hsp 70 cytosoliques hors de la
  protéine ? ATP
• Translocation à travers un TIM ? gradient H
• Libération des hsp 70 mitochondriales hors de la
  protéine ? ATP

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Fig 12-27
Nécessité -du gradient électrochimique à
travers la membrane interne et de -l'hydrolyse
de l'ATP pour limportation dans la matrice
mitochondriale
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Les hsp 70

• Cytosoliques
• Mitochondriales
• Se lient aux protéines non repliées
• Nécessitent de lATP pour se détacher
• Fournit finalement lénergie pour la
  translocation par TIM 23
• Sont intimement associés à TIM 23

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Les deux modèles énergétiques dimportation

•  Roue à cliquet  thermique aller-retour
  thermique dans le canal de TIM23
•  Roue à cliquet  ponté changement de
  conformation de hsp 70 qui tire sur la chaîne
• Dans les deux cas
•  Roue à cliquet  qui empêche le retour de la
  protéine
• liaison de hsp 70 à TIM 23

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Fig 12-28 (A)

•  Roue à cliquet  thermique

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Fig 12-28 (B)

•  Roue à cliquet  ponté

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Changement de hsp

• Les hsp 70 mitochondriales sont remplacées par
  des hsp 60 mitochondriales dans la matrice

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Transport dans la membrane mitochondriale interne
et espace intermembranaire

• Même mécanisme au départ que pour la matrice
• Deux voies
• a - passage par la matrice
• b - pas de passage par la matrice

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Fig 12-26

• Même mécanisme au départ que pour matrice

4 étapes
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a - Passage par la matrice

• Le premier signal peptide est retiré (par une
  signal peptidase matricielle spécifique)
• 2ème signal peptide hydrophobe
• Transloque la protéine de la matrice
• dans la membrane mitochondriale interne ou
• à travers la membrane mitochondriale interne
• Utilise le complexe OXA comme translocateur

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Fig 12-29 (A)

• a - passage par la matrice

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b - pas de passage par la matrice

• Le 2ème signal se comporte comme un signal stop
  de transfert (pour TOM)
• ce qui arrête la translocation à travers la
  membrane interne
• Clivage du 1er signal peptide
• Glissement de TOM et poursuite de la
  translocation à travers TOM

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Fig 12-29 (B)

• b - pas de passage par la matrice

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Transport dans lespace intermembranaire

• Même mécanisme au départ que pour matrice
• Deux voies
• passage par la matrice
• pas de passage par la matrice
• ? insertion dans la membrane interne (id. supra)
• Action dune peptidase spécifique
• Libération de la protéine soluble

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Fig 12-29 (C)

• Transport dans lespace intermembranaire

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Import des transporteurs métaboliques (ADP, ATP,
Pi, ) dans la membrane interne

• 35 protéines différentes chez la levure
• protéines transmembranaires à plusieurs passages
• sans séquence terminale clivable mais des
  séquences internes
• traversée par TOM
• puis insertion dans la membrane interne par TIM
  22
• nécessite le gradient mais ni ATP ni hsp

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Fig 12-29 (D)

• Insertion des protéines multipass dans la
  membrane interne

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Import dans la membrane externe

• Membrane externe de la mitochondrie
• ? membrane externe des bactéries
• Riche en porine
• protéine qui forme des pores
• perméable aux ions inorganiques, aux métabolites,
  aux petites protéines
• empêche le retour des protéines dans le cytosol
• Contient TOM40
• Insertion des protéines du cytosol
• ATP dépendant
• pas de clivage
• pas besoin d'un potentiel de membrane
• signal de translocation séquence d'ancrage ?

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Ciblage des protéines de la mitochondrie

• Post-traductionnelle
• Co-traductionnelle

74
Résumé
75
Neuperts,W Nature Reviews Molecular Cell Biology
3, 555 -565 (2002)

• La machine dimportation des protéines
  mitochondriales

Neuperts,W Nature Reviews Molecular Cell Biology
3, 555 -565 (2002)
76
Neuperts,W Nature Reviews Molecular Cell Biology
3, 555 -565 (2002)

• Deux modèles de dépliement et de translocation
  des préprotéines à travers les membranes
  mitochondriales

Neuperts,W Nature Reviews Molecular Cell Biology
3, 555 -565 (2002)
77
Neuperts,W Nature Reviews Molecular Cell Biology
3, 555 -565 (2002)

• Dépliement sans traction

Matrix Targeting Signal
Neuperts,W Nature Reviews Molecular Cell Biology
3, 555 -565 (2002)
78
Neuperts,W Nature Reviews Molecular Cell Biology
3, 555 -565 (2002)

• Déploiement local et global d'une protéine repliée

Neuperts,W Nature Reviews Molecular Cell Biology
3, 555 -565 (2002)
79
Neuperts,W Nature Reviews Molecular Cell Biology
3, 555 -565 (2002)

• Une roue à cliquet non ciblée ne peut pas initier
  le dépliement efficacement

Neuperts,W Nature Reviews Molecular Cell Biology
3, 555 -565 (2002)
80
Biogenèse de TOM 40
81
Rapaport,D2002

• Assemblage de TOM 40

82
Pfanner,N2002p400

• Assemblage du complexe de la membrane externe
• Recyclage continu entre le complexe TOM mature et
  l'intermédiaire d'assemblage tardif

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Ciblage des protéines de la mitochondrie

• Translocases in the Outer Mitochondrial Membrane
  (TOM)
• Translocase in the Inner Mitochondrial Membrane
  (TIM)
• Cf schéma général

84
Koehler,CM2000
85
Bauer,MF2000

• Import dans la matrice et la membrane interne

Mge1p
86
Bauer,MF2000

• Transfert de ADP/ATP carrier (AAC) à travers
  lespace inter-membranaire

87
Voos,W1999 BBA
88
Voos,W1999 BBA

• The mitochondrial protein import machinery of S.
  cerevisiae

89
Voos,W1999 BBA
90
Rehling P, Brandner K, Pfanner N. Mitochondrial
import and the twin-pore translocase.Nat Rev Mol
Cell Biol 2004 5(7)519-30

• Figure 1  Protein-import pathways for
  mitochondrial proteins.   Precursor proteins
  (brown) with positively charged amino-terminal
  presequences, -barrel outer-membrane proteins
  (dark green), and multispanning inner-membrane
  proteins (blue) with internal targeting signals
  are recognized by specific receptors of the
  translocase of the outer mitochondrial membrane
  (TOM) that is, by Tom20, Tom22 and/or Tom70. Up
  to three dimers of Tom70 are recruited per
  precursor (each Tom70 structure shown here
  represents a dimer). The precursor proteins are
  then translocated through the Tom40 pore (the
  small Tom proteins of the TOM complex Tom5,
  Tom6 and Tom7 are not shown). The TOM complex
  contains two or three pores. The -barrel
  proteins then require the small Tim proteins
  (Tim9Tim10) to guide them through the
  intermembrane space, and the sorting and assembly
  machinery (SAM complex) for insertion and
  assembly into the outer membrane. Outer-membrane
  proteins with single transmembrane spans can be
  directly inserted into the outer membrane by the
  TOM complex. Presequence-containing preproteins
  use the presequence translocase of the inner
  mitochondrial membrane (the TIM23 complex) for
  transport across the inner membrane. Tim23 forms
  a pore in the inner membrane. Presequence-containi
  ng inner membrane proteins can either be directly
  inserted into the inner membrane by the
  presequence translocase or be translocated to the
  matrix side and exported into the inner
  membrane107. It has been reported that the
  extreme amino terminus of Tim23 spans the outer
  membrane36 (not shown). The membrane potential (
  ) and the function of the presequence-translocase-
  associated import-motor (PAM) complex are
  essential for the translocation of
  presequence-containing proteins into the matrix.
  Mitochondrial heat-shock protein-70 (mtHsp70) is
  the central motor component. It cooperates with
  Tim44, Pam16 and Pam18 at the inner membrane and
  requires the matrix protein Mge1 (mitochondrial
  GrpE-related protein-1) for nucleotide exchange.
  In the matrix, the mitochondrial processing
  peptidase (MPP) cleaves off the presequence.
  Multispanning inner-membrane proteins with
  internal signals require the Tim9Tim10 complex
  for transport across the outer membrane and the
  intermembrane space. The insertion of these
  proteins into the inner membrane is catalysed by
  the twin-pore carrier translocase of the inner
  mitochondrial membrane (the TIM22 complex), which
  uses the membrane potential as an external
  driving force. This translocase contains two
  pores.

(presequence-translocase-associated import-motor)
91
Rehling P, Brandner K, Pfanner N. Mitochondrial
import and the twin-pore translocase. Nat Rev
Mol Cell Biol 2004 5(7)519-30. Fig 2

• Figure 2  A hypothetical model for membrane
  insertion by the twin-pore translocase.  
  Multispanning, inner-membrane proteins that have
  internal targeting signals are inserted into the
  inner membrane by the translocase of the inner
  mitochondrial membrane (TIM)22 complex. The steps
  of transport can be experimentally dissected in
  vitro by modulating the membrane potential ( ).
  a In the absence of a membrane potential, the
  TIM22 complex is inactivated (red traffic light).
  However, initial binding of the precursor to the
  translocase from the intermembrane-space side
  through the small Tim proteins Tim9Tim10 and
  Tim12 can occur. This is the tethering step, and
  it resembles a subset of the stage-III-arrested
  carrier precursors (Box 4). b At a low membrane
  potential (less than 60 mV), the pores of the
  translocation machinery are maintained in a
  partially-activated state (orange traffic light),
  and the low membrane potential is sufficient to
  promote the insertion of one precursor loop into
  the translocase (the membrane potential exerts an
  electrophoretic force on positive charges that
  are found in the matrix-exposed loops of the
  precursor). This is the docking step, and it
  resembles stage IV of carrier import. c A high
  membrane potential ( for example, 120 mV)
  across the membrane, together with the
  recognition of an internal targeting signal in
  the precursor, fully activates the translocase
  (green traffic light). One pore closes tightly
  around the initially inserted terminal hairpin
  loop of the precursor, while the second pore
  mediates the insertion of the next set of
  transmembrane domains. The process leads to
  membrane insertion, which occurs by the lateral
  opening of the translocase (insertion step).

Rehling P, Brandner K, Pfanner N. Mitochondrial
import and the twin-pore translocase. Nat Rev
Mol Cell Biol 2004 5(7)519-30
92
Rehling P, Brandner K, Pfanner N. Mitochondrial
import and the twin-pore translocase. Nat Rev
Mol Cell Biol 2004 5(7)519-30. Fig 3

• Figure 3  The transport of multispanning,
  inner-membrane proteins that have a presequence.
    Presequence-containing, multispanning,
  inner-membrane proteins are transported across
  the outer membrane through the translocase of the
  outer mitochondrial membrane (TOM) complex and
  require the presequence translocase (the
  translocase of the inner mitochondrial membrane
  (TIM)23 complex) for translocation across the
  intermembrane space and inner membrane. Both
  translocases have to cooperate tightly to promote
  precursor translocation. a The absence of a
  membrane potential ( ) at the inner membrane,
  together with the inactivation of the ATP-driven,
  inner-membrane PAM complex (presequence
  translocase-associated motor complex), arrests
  the precursor in the TOM complex (red traffic
  light). The intermembrane-space domain of the
  Tom22 receptor stabilizes this transport
  intermediate of the precursor in the TOM complex.
  b A full membrane potential activates the
  membrane integral module of the TIM23 complex and
  allows initial precursor insertion into the Tim23
  pore. However, this is insufficient to promote
  inner-membrane translocation of the precursor
  (orange traffic light). If the PAM complex is
  inactive, the precursor is maintained in
  association with the TOM complex. c Transport
  of the precursor across the outer membrane and
  the intermembrane space requires both driving
  forces of the inner membrane that is, membrane
  potential and the force provided by the
  ATP-driven PAM complex. The multispanning
  proteins can then enter the matrix space and be
  exported into the inner membrane107. Mge1,
  mitochondrial GrpE-related protein-1 MPP,
  mitochondrial processing peptidase mtHsp70,
  mitochondrial heat-shock protein-70.

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import and the twin-pore translocase. Nat Rev
Mol Cell Biol 2004 5(7)519-30
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Rehling P, Brandner K, Pfanner N. Mitochondrial
import and the twin-pore translocase. Nat Rev
Mol Cell Biol 2004 5(7)519-30. Box 2
Le complexe SAM de la membrane mitochondriale
externe

• Box 2 The SAM complex of the mitochondrial
  outer membrane 
• Although a number of components that mediate
  matrix-protein transport and inner-membrane
  protein sorting have been identified, little is
  known about how proteins are sorted into the
  outer mitochondrial membrane. However, recent
  work has narrowed this gap in our knowledge.
  Studies of the transport of outer-membrane
  proteins that have several -strands, such as
  translocase of the outer mitochondrial membrane
  (Tom)40 or porin, have led to the identification
  of a novel translocation machinery in the outer
  membrane. This SAM complex (sorting and assembly
  machinery) is specifically required for the
  insertion and assembly of -barrel proteins into
  the outer-membrane (see figure). Mas37, a
  peripheral outer-membrane protein, was identified
  as the first component of the SAM complex. Two
  further constituents have been found since
  Sam50 (also known as Tob55/Omp85) and Sam35.
  Sam50 and Sam35 are both essential for cell
  viability, which emphasizes a crucial role for
  the SAM complex in mitochondrial biogenesis.
  Sam50 is conserved from bacteria to eukaryotes,
  which indicates that the mechanism of protein
  insertion has been maintained throughout
  evolution81-83, 85. Analyses of the biogenesis
  pathway of mitochondrial outer-membrane proteins
  indicates that they are first translocated across
  the outer membrane by the TOM complex and are
  then inserted from the intermembrane-space side
  with the aid of SAM. This resembles the insertion
  pathway for bacterial outer-membrane proteins
  (outer-membrane proteins of bacteria are inserted
  from the periplasmic side of the membrane).
  Interestingly, the 'small Tim' (translocase of
  the inner mitochondrial membrane) proteins have
  been found to participate in this process by
  assisting precursor transport through the
  intermembrane space towards the SAM complex.

(Sorting and Assembly Machinery)
Rehling P, Brandner K, Pfanner N. Mitochondrial
import and the twin-pore translocase. Nat Rev
Mol Cell Biol 2004 5(7)519-30
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Rehling P, Brandner K, Pfanner N. Mitochondrial
import and the twin-pore translocase. Nat Rev
Mol Cell Biol 2004 5(7)519-30. Box 3

• Box 3 The presequence-translocase-associated
  import motor PAM 
• The transport of proteins across the inner
  membrane into the matrix requires two energy
  sources the membrane potential ( ) of the
  inner membrane (please refer to the main text for
  further details) and the activity of the
  presequence-translocase-associated import-motor
  (PAM) complex. The mitochondrial heat-shock
  protein-70 (mtHsp70) is the central component of
  the PAM complex and it binds directly to an
  incoming, unfolded polypeptide chain87, 88.
  Translocase of the inner mitochondrial membrane
  (Tim)44, an essential peripheral inner-membrane
  protein, functions as a membrane anchor for
  mtHsp70 (Refs 8993 see figure). The function of
  mtHsp70 has to be tightly regulated to allow it
  to cycle between an ADP-bound state and an
  ATP-bound state, which correspond to states of
  high and low substrate affinity, respectively.
  This regulation requires the cooperation of
  mtHsp70 with cofactors. DNAJ-LIKE PROTEINS
  stimulate ATP hydrolysis by Hsp70 proteins,
  whereas a further exchange factor is needed to
  induce ADP release. Mitochondrial Mge1
  (mitochondrial GrpE-related protein-1)9-11
  functions as the exchange factor for mtHsp70,
  whereas Tim44 was thought to fulfil a function
  that is similar to that of DnaJ-like proteins.
  Recently, though, Tim44 was shown to be unable to
  stimulate the ATPase activity of mtHsp70 (Refs
  94,95). How, then, is the regulated cycling of
  mtHsp70 accomplished?
• The answer to this problem lies in the discovery
  of Pam18 (also known as Tim14)94-96, a new
  component of the PAM complex. Pam18, an integral
  inner-membrane protein, is associated with the
  presequence translocase and exposes a DnaJ-like
  domain to the matrix side of the inner membrane
  that stimulates the ATPase activity of mtHsp70
  (Refs 94,95 see figure). The discovery of Pam18
  as a further component of the PAM complex
  indicates that previous models of how the motor
  translocates polypeptides were premature, because
  they were based on only three motor components
  (Tim44, mtHsp70 and Mge1). This is further
  underlined by the discovery of Pam16 (also known
  as Tim16), another essential component of the PAM
  complex, which cooperates with Pam18 in mtHsp70
  regulation and seems to recruit Pam18 to the
  translocation machinery97, 98

The Presequence-translocase-Associated import
Motor
Rehling P, Brandner K, Pfanner N. Mitochondrial
import and the twin-pore translocase. Nat Rev
Mol Cell Biol 2004 5(7)519-30
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Rehling P, Brandner K, Pfanner N. Mitochondrial
import and the twin-pore translocase. Nat Rev
Mol Cell Biol 2004 5(7)519-30. Box 4
Les étapes de limportation dans la mitochondrie

• Box 4 The stages of carrier import into
  mitochondria 
• Initial analyses, which started in the 1980s,
  showed that the hydrophobic carrier precursors
  are post-translationally imported into
  mitochondria99 and that their transport into the
  inner membrane requires the membrane potential (
  ) as an external driving force62, 99. Our
  subsequent understanding of the transport pathway
  came from detailed in vitro import studies, which
  allowed the transport pathway to be dissected
  into five distinct, successive steps (stages IV
  see figure). Each step represents a clearly
  discernable transport intermediate along the
  import pathway42.
• The first step in transport (stage I) represents
  the soluble, cytosolic, chaperone-bound form of
  the precursor42, 99. Subsequently, at stage II,
  the precursor is recognized by receptors on the
  cytosolic face of mitochondria100. The carrier
  precursor can be arrested at stage II in the
  absence of ATP42, 43. For the subsequent
  transport of the precursor across the outer
  membrane, carrier precursors, as well as
  presequence-containing proteins, use the
  general-import pore (GIP) complex. The TOM
  complex (translocase of the outer mitochondrial
  membrane) contains receptor proteins that bind
  the targeting signals and direct the precursor
  proteins towards this pore-forming core unit or
  GIP complex. However, carrier proteins can be
  arrested at this transport step if the membrane
  potential at the inner membrane is dissipated.
  This stage, stage III, was interpreted to
  represent the carrier when it is deeply inserted
  into the outer-membrane translocase (it is
  inaccessible to externally added protease)42 and
  is already exposed to the intermembrane space101.
  Later it was realized that the stage-III
  intermediate is actually a mixture of two
  populations one set of precursors interacts
  with the 'small Tims' (translocases of the inner
  mitochondrial membrane) in association with the
  outer membrane, whereas a second set of
  precursors is already tethered to the TIM22
  complex.
• It became clear that the insertion of the
  precursor into the inner membrane occurs in a
  membrane-potential-dependent manner, but a
  pore-forming TIM complex was not considered at
  this time. At the inner-membrane insertion step
  (stage IV), no stable transport intermediate
  could be generated until recently68, so this
  stage was initially only inferred from kinetic
  analyses42, 43. However, using ionophores to
  gradually reduce the membrane potential across
  the inner membrane in isolated mitochondria, it
  became possible to arrest a carrier precursor in
  the TIM22 complex that is, to obtain a stage-IV
  intermediate. The insertion process ends with
  stage V, which represents the fully
  membrane-inserted carrier that has assembled into
  a functional dimer.

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import and the twin-pore translocase. Nat Rev
Mol Cell Biol 2004 5(7)519-30
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Rehling P, Brandner K, Pfanner N. Mitochondrial
import and the twin-pore translocase. Nat Rev
Mol Cell Biol 2004 5(7)519-30. Box 5

• Box 5 Analysing protein insertion into
  mitochondrial membranes 
• Analysing protein insertion into the
  mitochondrial membranes is difficult in vivo, as
  kinetic analyses and biochemical-fractionation
  techniques need to be combined. For mitochondrial
  protein transport, an in vitro import system
  which imports in-vitro-synthezised, radiolabelled
  proteins into isolated mitochondria and uses
  blue-native polyacrylamide gel electrophoresis
  (BN-PAGE) to address the formation of protein
  complexes102-104 has proven helpful (see
  figure). The latter technique is especially
  suited to the analysis of membrane-protein
  complexes that have been solubilized from
  membranes. If a radiolabelled, imported protein
  assembles into a multiprotein complex or into an
  oligomeric form (for example, stage V of carrier
  import see figure and Box 4), it is possible to
  separate and visualize such complexes on the gel
  and therefore to assess the final stage of
  transport46, 104. Moreover, in many cases, this
  approach has allowed the identification of
  intermediates in the transport and assembly
  process, as the precursors cooperate with other
  proteins during transport and can therefore be
  found in different complexes of the pathway at
  different kinetic stages24, 46, 68, 70, 80,
  82-84, 86 (Box 4).
• BN-PAGE has also been found to be a helpful tool
  to visualize unknown intermediates in
  protein-insertion pathways68, 70, 80, 86. As the
  precursors used in the import studies are
  imported in vitro, a further level of analysis
  can be applied, because the formation of
  transport intermediates and of the
  membrane-integrated assembled complexes can be
  analysed in a time-dependent manner and under
  different salt, temperature or energetic
  conditions. For example, membrane potential ( )
  can be modulated full membrane potential leads
  to the membrane insertion and assembly of the
  carrier to form a dimer (stage V) dissipation of
  the membrane potential arrests the precursor at
  stage III of carrier import (but this precursor
  is released from its associated components during
  BN-PAGE) and a low membrane potential allows the
  precursor to be arrested at stage IV, in which it
  is found bound to the translocase of the inner
  mitochondrial membrane (TIM)22 complex (see
  figure)46, 68. One important technical
  development that can be used to address the
  composition of complexes following BN-PAGE is the
  so-called antibody-shift assay. The binding of
  antibodies to a component of the protein complex
  increases the size of the complex, which can
  easily be seen on the gel70, 80, 105, 106.

Rehling P, Brandner K, Pfanner N. Mitochondrial
import and the twin-pore translocase. Nat Rev
Mol Cell Biol 2004 5(7)519-30