COMPARTIMENTS DE LA CELLULE ET TRI DES PROTÉINES

COMPARTIMENTS DE LA CELLULE ET TRI DES PROTÉINES IV - Peroxysomes

Lundi 2 octobre 2006 PEROXYSOMES http://www.peroxisome.org/index.html

Peroxysomes Une seule membrane Lundi 2 octobre 2006 Peroxysomes Une seule membrane Pas d'ADN, pas de génome, pas de ribosomes Importation des protéines à partir du cytosol Toutes les protéines doivent être importées (comme le RE) Présents chez tous les eucaryotes Contiennent des enzymes oxydatifs : catalase, urate oxydase … Définition biochimique (1960) une oxydase qui produit H2O2 une catalase pour décomposer H2O2 Utilisent de l'oxygène (vestige d’un ancien organite qui métabolise l’oxygène) Rendus obsolètes par la mitochondrie Libèrent peu (pas) d'énergie

A – Morphologie

Morphologie des peroxysomes Sphérique 0,1  à 1  de diamètre Contenu granulaire, parfois paracristallin Grand polymorphisme taille, nombre, composition en fonction de l'environnement

Application des techniques cytochimiques pour la localisation des activités enzymatiques des peroxysomes PO=peroxysome a - Catalase b - Catalase cd - Urate oxydase e - Urate oxydase f - D-amino acid oxydase gh- Peroxysomes avec plaques marginales Lundi 2 octobre 2006 Fahimia,HD1999 Figure 1. Application of cytochemical techniques for localization of peroxisomal enzyme activity. (a) Rat liver peroxisomes (PO) stained with alkaline DAB technique (pH 10.5) for detection of peroxidatic activity of catalase (CAT). (b) Peroxisomes (PO) in rat myocardium stained for the localization of catalase (CAT). Note the elongated tubular form of some peroxisomes, with evidence of branching (inset) reminiscent of the peroxisomal reticulum found in regenerating rat liver (Yamamoto and Fahimi 1987 ). (c,d) Peroxisomes in rat liver stained for the localization of urate oxidase (UOX) activity by the cerium technique. Note distinct staining of the crystalline core only in some peroxisomes. Other peroxisomes (*) completely lack the cerium reaction product, clearly demonstrating the heterogeneity of peroxisomes (PO) within the same hepatocyte. (e) Immunocytochemical localization of urate oxidase by the protein A– gold technique (PAG). Note the exclusive localization of gold particles to the core region. (f) Rat hepatic peroxisomes (PO) stained for D- amino acid oxidase activity using D-proline (D-AAOX) as substrate. Note the heterogeneity in the intensity of staining between adjacent peroxisomes (*) within the same cell. (g,h) Peroxisomes with marginal plates (MP). In g the MP in rat kidney stained for D-amino acid oxidase (DAA-OX) appears as negative contrast below the peroxisomal membrane (arrows). In h, the immunolabeling with an antibody to - hydroxy acid oxidase-B reveals the curved lentiform appearance of marginal plates in a feline kidney (arrows) (Courtesy Dr. K. Zaar).

Lundi 2 octobre 2006 Application of immunoelectron microscopy for detection of peroxisomal protein antigens Fahimia,HD1999 Figure 2. Application of immunoelectron microscopy for detection of peroxisomal protein antigens. (a) Peroxisomes (PO) and an empty membranous loop (*) from the liver of a rat treated with a hypocholesterolemic drug BM15766. The membranous structures resemble the so-called "peroxisome ghosts" observed in the livers of patients with biogenetic disorders of peroxisomes (Espeel et al. 1995b ). (b,c) Marked induction of the multifunctional protein (MFP) by a peroxisome proliferator (BM 17) in rat liver is clearly demonstrated in this immunocytochemical preparation processed by the protein A–gold method. The alterations of immunogold labeling can be quantified by image analysis (Beier et al. 1988 ). (d–f) Localization of lactate dehydrogenase (LDH) in rat liver sections (d) and in isolated PO fractions (e). The identity of labeled particles as peroxisomes is confirmed by staining of fractions with the catalase antibody (f). (g–i) Application of preembedding immunoelectron microscopy to isolated cell fractions from rat liver. The antibody to the 70-kD peroxisomal membrane protein (PMP- 70) by the preembedding technique labels exclusively peroxisomes (PO) in the peroxisome fraction (g), in the microsomal (MIC) fraction (h), and in the light mitochondrial fraction (i). Other organelles, such as ER, with mitochondria and lysosomes (LYS), are unlabeled.

Fahimia,HD1999 Sections from the livers of PEX 5-/- and control mice Control mice PEX 5-/- La catalase est en dehors des PO Lundi 2 octobre 2006 Sections from the livers of PEX 5-/- and control mice Fahimia,HD1999 Figure 3. Sections from the livers of PEX 5-/- and control mice processed for localization of catalase by the DAB technique and by immunocytochemistry for catalase. Note the prominent staining of peroxisomes (PO) in control mice (a,c) and their absence in Zellweger mice (b,d). In PEX 5-/- mouse, catalase labeled by gold particles (arrowheads) is found in the cytoplasm and also in the nucleus (N) of hepatocytes (d). Note the heterogeneity in the intensity of catalase staining and immunolabeling in different hepatocytes (Cells A, B, and C in b and Cells A and B in d). NBL, normoblast.

Lundi 2 octobre 2006 (a,b) Confocal micrographs of HepG2 cells labeled with antibodies to catalase (rhodamine), and tubulin (fluorescein) a Spherical form of peroxisomes b Tubular form of peroxisomes (c–h) In situ hybridization of rat liver sections with digoxigenin-labeled cRNA probes c-f Albumine mRNA d-g Catalase e-h Acyl Co-A oxydase Fahimia,HD1999 Figure 4. (a,b) Confocal laser scanning micrographs (CLSM) of HepG2 cells labeled with antibodies to catalase (rhodamine), and tubulin (fluorescein), overlaid with a 2- photon micrograph showing nuclei counterstained with DAPI. Note the spherical form of peroxisomes in a in contrast to their tubular form in b. The contact sites between microtubules and peroxisomes are rather rare (arrows in inset to b). (c–h) In situ hybridization of rat liver sections with digoxigenin-labeled cRNA probes. (c,f) Albumin mRNA distribution with stronger periportal (P) staining. (d,g) The mRNA for catalase is uniformly distributed in the rat liver lobule. (e,h) The mRNA for acyl-CoA oxidase is slightly stronger in periportal hepatocytes (P) than in the pericentral region (C) of the liver lobule. (e,inset) Sense control with no evidence of staining.

Flexibilité et dynamisme des peroxysomes levures sur un milieu riche en sucre  petits peroxysomes levures sur un milieu riche en méthanol  gros peroxysomes (pour oxyder le méthanol) levures sur des acides gras  gros peroxysomes pour -oxyder les acides gras

Trois peroxysomes d’hépatocyte de rat en ME Fig 12-31

Aspects des peroxysomes dans différents organismes Titorenko,VI2001(fig1).gif Aspects des peroxysomes dans différents organismes

Peroxysomes RH2 + 02  R + H2O2 2 H2O R'H2 = phénol, acide formique, formaldéhyde, alcool, … Foie, Rein, … rôle de détoxification catalase R'H2 R'

Alcool CH3-CH2OH CH3-CHO  oxydation des acides gras O2 H2O2 2 H2O Catalase R'H2 R'

Formation des plasmalogènes Classe de phospholipides la plus abondante dans la myéline Rôle des peroxysomes dans les premières étapes de la synthèse Fréquence des atteintes neurologiques dans les maladies des peroxysomes

Chez les plantes Type feuille Type graine Photorespiration : consommation d’oxygène pour fixer CO2 dans les hydrates de carbone Type graine conversion des acides gras contenus dans les graines en sucre acide gras  sucre = cycle du glyoxylate  ces peroxysomes s’appellent glyoxysomes

Cellule de feuille de tabac Cellule de graine de tomate Fig 12-33

C - Biogenèse Importation des protéines de la lumière Importation des protéines de la membrane

Schéma du routage

Schéma du routage vers peroxysomes CYTOSOL Schéma du routage vers peroxysomes PLASTIDE MITOCHONDRIE NOYAU PEROXYSOME

Signal d’import dans le peroxysome 3 acides aminés à l ’extrémité -C de la protéine parfois signal -N terminal processus mal connu au moins 23 protéines distinctes (appelées peroxines) nécessité d’ATP pas de nécessité de déplier la protéine  processus différent de celui de la mitochondrie Pex5 (peroxine 5) suit l’import tout au long de son trajet

Peroxines Récepteurs protéiques solubles cytosoliques Protéines ancrées dans la membrane du peroxysome du côté cytosolique Au moins 23 types de protéines connus

Peroxines Protéines nécessaires à la biogenèse des peroxysomes assemblage de la membrane import des protéines de la lumière prolifération des peroxysomes héritage des peroxysomes Codées par des gènes PEX

Gènes PEX L’importance des peroxysomes est soulignée par l’existence de nombreuses maladies génétiques associées à des déficits peroxysomaux qu’on peut classer en deux catégories : Maladies résultant d’un déficit d’une seule enzyme peroxysomale Maladies résultant d’un déficit dans la biogenèse du peroxysome

1 - Maladies résultant d’un déficit d’une seule enzyme peroxysomale Ces maladies résultent du déficit d’une seule enzyme du peroxysome et n’affectent donc en général qu’une seule voie métabolique peroxysomale L’hyperoxalurie de type I (alanine:glyoxylate aminotransferase) La maladie de Refsum (phytanoyl-CoA hydroxylase) L’adrénoleucodystrophie liée à l’X La chondrodysplasie rhizomélique ponctuée type II et III (dihydroxyacetone phosphate acyltransferase) Les déficits de la β-oxidation (acyl-CoA oxidase, bifunctional protein, and thiolase)

2 - Maladies résultant d’un déficit dans la biogenèse du peroxysome Ce sont les Peroxisome Biogenesis Disorders (PBDs) Ces maladies (léthales) affectent toutes les voies métaboliques du peroxysome et peuvent résulter de n’importe quelle mutation dans un au moins 13 gènes PEX connus. Le produit de ces gènes s’appelle les peroxines et tous participent à la biogenèse du peroxysome. Le syndrome de Zellweger L’adrenoleucodystrophie néonatale La maladie de Refsum infantile La chondrodysplasie rhizomélique ponctuée type I.

Les peroxines http://www.peroxisome.org/Scientist/Biogenesis/peroxins/membranebio.html

Syndrome de Zellweger Défaut héréditaire de l’import de protéines dans le peroxysome Peroxysome vides Anomalies du cerveau, foie, rein Mort précoce Dans certaines formes, mutation dans le gène de peroxine 2 (protéine membranaire intégrale de la membrane du peroxysome)

Syndrome de Zellweger An autosomal recessive disorder due to defects in PEROXISOME biogenesis which involves more than 13 genes encoding peroxin proteins of the peroxisomal membrane and matrix. Zellweger syndrome is typically seen in the neonatal period with features such as dysmorphic skull MUSCLE HYPOTONIA SENSORINEURAL HEARING LOSS visual compromise SEIZURES progressive degeneration of the KIDNEYS and the LIVER. Zellweger-like syndrome refers to phenotypes resembling the neonatal Zellweger syndrome but seen in children or adults with apparently intact peroxisome biogenesis.

Peroxysome Membrane Protein Lundi 2 octobre 2006 http://www.peroxisome.org/Scientist/Biogenesis/peroxins/membranebio.html Peroxysome Membrane Protein The process of peroxisome membrane biogenesis is much less well understood than the import of peroxisome matrix proteins. This process must involve the formation of the lipid bilayer, and the import of membrane proteins (PMPs) into that bilayer. Only three proteins have been identified as involved in the process of the formation of peroxisome membranes de novo (PEX3, PEX16, and PEX19). A fourth, PEX11, has been found to be involved in peroxisome proliferation. http://www.peroxisome.org/Scientist/Biogenesis/peroxins/membranebio.html

Import des protéines peroxysomales Lundi 2 octobre 2006 Le ciblage est toujours post traductionnel Ciblage des protéines membranaires (membrane Peroxysomal Targeting Signal [mPTS]) : directement à partir du cytosol (mPTS1) ou indirectement via le RE (mPTS2) La machine d'importation pour la membrane est différente de celle pour la matrice Les protéines de la matrice ont besoin de mPTS 1 et mPTS 2 L'import dans la membrane et la matrice est médiée par leur récepteur qui agit avec le PTS de leur cargo Titorenko,VI2001 Nat Rev Mol Cell Biol 2,(5), 357

Biogenèse Croissance de peroxysomes préexistants puis fission Comme mitochondries ou RE

Fig 12-34

Holroyd,C2001 (fig1) Réponse aux « ? » à la diapositive suivante… Lundi 2 octobre 2006 Holroyd,C2001 (fig1) Réponse aux « ? » à la diapositive suivante… Fig. 1. Model of peroxisome biogenesis. The topogenesis of peroxisomal matrix and membrane proteins is performed by distinct transport machineries. (1) A subset of PMPs, likely peroxins involved in the early stages of peroxisome biogenesis (`early' peroxins), are supposed to insert into endomembranes, presumably the ER. (2) Vesicles harboring these PMPs bud from the ER and fuse with peroxisomes. (3) Peroxisomal matrix proteins and other PMPs are synthesized on free ribosomes in the cytosol and imported post- translationally into peroxisomes. (4) Peroxisomes grow and undergo fission to form new peroxisomes.

Curr Biol. 2005 Sep 20;15(18):R774-6. Kunau,WH2005pR774 Lundi 2 octobre 2006 Curr Biol. 2005 Sep 20;15(18):R774-6. Kunau,WH2005pR774

Organelle biogenesis: Where did I come from Organelle biogenesis: Where did I come from? Rachel Smallridge Nature Reviews Molecular Cell Biology 6, 672 - 673 (01 Sep 2005) How do peroxisomes form in eukaryotic cells? A definitive answer to this question has been elusive, with some believing that, similar to the Golgi, peroxisomes are derived from the endoplasmic reticulum (ER) and others believing that, similar to mitochondria, they are autonomous entities. However, Tabak and colleagues now resolve this issue in Cell by showing that peroxisomes come from the ER. The authors worked in Saccharomyces cerevisiae and created a system that allowed the real-time imaging of peroxisome biogenesis. They focused on peroxin-3 (Pex3), an integral membrane protein that is essential for the biogenesis of these organelles. They made a yeast strain that could be induced to express endogenous levels of fluorescently labelled Pex3 (Pex3– YFP) by putting the gene encoding this construct under the control of a galactose-inducible promoter and exposing cells to galactose for a limited time. In the absence of galactose, Pex3 and peroxisomes were absent from these cells.

Lundi 2 octobre 2006 Holroyd,C2001 (fig2) Fig. 2. Peroxisomal matrix protein import cascade. Proteins harboring one of the two PTSs, PTS1 or PTS2, are recognized in the cytosol by specific signal sequence receptors (Pex5p and Pex7p). The multiple binding sites for peroxisomal signal sequence receptors at the peroxisomal membrane reflect the existence of an import cascade where the cargo-loaded receptors successively interact with different components of the import machinery. These interactions are likely to trigger conformational changes of the proteins within the import cascade which are required for the consecutive steps of peroxisomal protein import: docking, translocation, cargo release and receptor recycling. How protein translocation proceeds through the peroxisomal membrane has not yet been resolved.

Fujiki,Y2000(Fig1) Biogenèse des peroxysomes chez les mammifères Lundi 2 octobre 2006 Fujiki,Y2000(Fig1) Fig. 1. A schematic view of peroxisome biogenesis in mammals. The intracellular locations and molecular properties of peroxins so far identified are shown. Peroxins are divided into four groups: (i) peroxins that are required for matrix protein import; (ii) those including Pex3p, Pex16p and Pex19p, responsible for peroxisome membrane assembly; (iii) those such as Pex1p and Pex6p of the AAA family presumably involved in membrane fusion step and (iv) those such as Pex11p apparently involved in proliferation. Import of matrix proteins has been better understood: matrix proteins, PTS1 and PTS2 proteins, are recognized by Pex5p and Pex7p, respectively. Two isoforms of Pex5p are identified in mammals. PTS1 proteins are transported by Pex5pS and Pex5pL to peroxisomes, where Pex14p functions as a convergent component of `protein import machinery' (for details, see text). Pex5pL directly interacts with the PTS2 receptor, Pex7p, carrying its cargo PTS2 protein in the cytosol and translocates the Pex7p-PTS2 protein complex to the initial docking site Pex14p. Pex5p carrying the cargos subsequently translocates to other components such as Pex13p, Pex2p, Pex10p and Pex12p. PTS1 and PTS2 proteins are then released at the inner surface and/or inside of peroxisomes. Both Pex5p and Pex7p finally shuttle back to the cytosol. Les 4 groupes de peroxine

Peroxisomes: Another Branch of the Secretory Pathway ? Cell, Vol. 122, 1–7, July 15, 2005 Randy Schekman Peroxisomes: Another Branch of the Secretory Pathway? Cell, Vol. 122, 1–7, July 15, 2005 Figure 1. Where Do New Peroxisomes Come from? Peroxisomes may form by the budding of vesicles (pale orange) from the ER in a pathway that is distinct from that producing secretory transport vesicles (green) from the ER. In the secretory pathway, vesicles carry membrane and cargo proteins to the Golgi apparatus; other vesicles retrieve some of the membrane material from the Golgi and return it to the ER. Pex3, an integral protein required for peroxisome formation, appears to originate in the ER and be packaged into vesicles (pale orange) by a budding or blebbing process that requires the Pex19 protein. Small precursor vesicles may fuse under the direction of two AAA ATPase proteins, Pex1 and Pex6, to form the functional large peroxisome (dark orange).

Titorenko,VI2001 (fig2) Nat Rev Mol Cell Biol 2,(5), 357 Lundi 2 octobre 2006 Titorenko,VI2001 (fig2) Nat Rev Mol Cell Biol 2,(5), 357 Titorenko,VI2001 Nat Rev Mol Cell Biol Import des protéines membranaires

Titorenko,VI2001 (fig3) Nat Rev Mol Cell Biol 2,(5), 357 Lundi 2 octobre 2006 Titorenko,VI2001 (fig3) Nat Rev Mol Cell Biol 2,(5), 357 Titorenko,VI2001 Nat Rev Mol Cell Biol Import des protéines luminales

Titorenko,VI2001(fig4) Nat Rev Mol Cell Biol 2,(5), 357 Deux modèles de l'assemblage dynamique des peroxysomes

Titorenko,VI2001(fig5) Nat Rev Mol Cell Biol 2,(5), 357 Deux modèles pour la formation des précurseurs des peroxysomes

Importation des protéines dans la matrice (Peroxisomal Targeting Sequence) Tabak,HF1999 (fig3) TiCB

Lundi 2 octobre 2006 Essential role of The endoplasmic reticulum in peroxisome biogenesis Titorenko,VI1998(fig1).gif Fig. 1. A model for the secretory pathways serving protein export, cell surface enlargement during yeast and mycelial modes of growth, and peroxisome biogenesis in the yeast Yarrowia lipolytica. In this model, four distinct secretory pathways diverge at the level of the endoplasmic reticulum (ER)[10]. Pex5p is the peroxisomal- targeting-signal-1 receptor. Pex8p and Pex16p are intraperoxisomal peripheral membrane proteins. Pex9p is a peroxisomal integral membrane protein. Sec14p is a phosphatidylinositol/phosphatidylcholine-transfer protein associated with the Golgi apparatus. The blue bar represents a partial block; black bars represents complete block.

Lundi 2 octobre 2006 Essential role of The endoplasmic reticulum in peroxisome biogenesis Titorenko,VI1998(fig2).gif Fig. 2. A model for vesicle-mediated anterograde/retrograde protein transport between the endoplasmic reticulum (ER) and peroxisomes. Pex2p and Pex16p are initially targeted to the ER[10, 11] and then exit the ER via distinct vesicles (PV1 and PV2)[16]. Coated vesicles (PV2) contain at least two components of COPII, Sec13p and Sec23p (Ref. [16]). Catalase (CAT) and thiolase (THI) are imported into vesicles (PV1 and PV2, respectively) after vesicular budding from the ER[10, 11, 16]. The import of Pex2p, Pex16p, CAT and THI into peroxisomes occurs through the fusion of vesicles (PV1 and PV2) with the growing peroxisome. The retrieval of ER-resident proteins occurs via COPI-coated vesicles (PV3) that mediate retrograde protein transport between peroxisomes and the ER[17]. Phospholipids are delivered to the peroxisomal membrane from the ER via ER-derived vesicles (PV1 and PV2) and/or distinct peroxisome-associated ER elements[16, 20, 21]. The export of proteins to the external medium and the delivery of proteins for plasma-membrane and cell-wall synthesis during mycelial growth are via the Golgi apparatus and are mediated by distinct ER-derived vesicles (SV1 and SV2, respectively)[10, 11, 16]. ARF, ADP- ribosylation factor.

Lundi 2 octobre 2006 Figure 1. Generalized models for the flow of membrane-enclosed carriers through the peroxisomal endomembrane system in yeast, mammals, and plants. PPT, preperoxisomal template; PPV, preperoxisomal vesicle; SV, secretory vesicle; TBSV p33, TSBV 33-kD replicase protein. Titorenko VI, Mullen RT. Peroxisome biogenesis: the peroxisomal endomembrane system and the role of the ER. J Cell Biol. 2006 Jul 3;174(1):11-7. Generalized models for the flow of membrane-enclosed carriers through the peroxisomal endomembrane system in yeast, mammals, and plants Figure 1. Generalized models for the flow of membrane-enclosed carriers through the peroxisomal endomembrane system in yeast, mammals, and plants. PPT, preperoxisomal template; PPV, preperoxisomal vesicle; SV, secretory vesicle; TBSV p33, TSBV 33-kD replicase protein. PPT, preperoxisomal template PPV, preperoxisomal vesicle SV, secretory vesicle TBSV p33, TSBV 33-kD replicase protein