Plasma membrane transport proteins play a crucial role in all cells by conferring to the cell surface a selective permeability to a wide range of ions and small molecules. The activity of these transporters is often regulated by controlling their amount at the plasma membrane, via intracellular trafficking. The recent boom in the numbers of crystallized transporters shows that many of them that belong to different functional families with little sequence similarity adopt the same structural fold implying a conserved transport mechanism. These proteins belong to the APC (Amino acid-Polyamine-organoCation) superfamily and their fold is typified by the bacterial leucine transporter LeuT. This LeuT fold is characterized by inverted structural repeats of 5 transmembrane domains that harbor the central substrate-binding site and a pseudo-symmetry axis parallel to the membrane. The yeast Saccharomyces cerevisiae possesses about 16 amino acid permeases (yAAPs) that belong to the APC superfamily and that display various substrate specificity ranges and affinities. Topological, mutational analysis and in silico data indicate that yAAPS adopt the LeuT fold.<p><p>In this work we combined computational modeling and yeast genetics to study substrate binding by yAAPs and the endocytosis of these transporters in response to substrate transport. In the first part of this work, we analyzed the selective recognition of arginine by the yeast specific arginine permease, Can1. We constructed three-dimensional models of Can1 using as a template the recently resolved structure of AdiC, the bacterial arginine:agmatine antiporter, which is also a member of the APC superfamily. By comparison of the binding pockets of Can1 and Lyp1, the yeast specific lysine permease, we identified key residues that are involved in the recognition of the main and side chains of arginine. We first showed that the network of interactions of arginine in Can1 is similar to that of AdiC, and that the selective recognition of arginine is mediated by two residues: Asn 176 and Thr 456. Substituting these residues by their corresponding residues in Lyp1 converted Can1 into a specific lysine permease. In the second part of this work, we studied the regulation of two permeases, Can1 and the yeast general amino acid permease, Gap1. In the presence of their substrates, Gap1 and Can1 undergo ubiquitin-dependent endocytosis and targeting to the vacuolar lumen for degradation. We showed that this downregulation is not due to intracellular accumulation of the transported amino acids but to transport catalysis itself. By permease structural modeling, mutagenesis, and kinetic parameter analysis, we showed that Gap1 and Can1 need to switch to an intermediary conformational state and persist a minimal time in this state after binding the substrate to trigger their endocytosis. This down-regulation depends on the Rsp5 ubiquitin ligase and involves the recruitment of arrestin-like adaptors, resulting in the ubiquitylation and endocytosis of the permease.<p><p>Our work shows the importance of the structural analysis of yAAPs to get further insight into the different aspects of their function and regulation. We validate the use of a bacterial APC transporter, AdiC, to construct three-dimensional models of yAAPs that can be used to guide experimental analyses and to provide a molecular framework for data interpretation. Our results contribute to a better understating of the recognition mode of amino acids by their permeases, and the regulation of this transport in response to substrate binding. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

SH2-containing inositol polyphosphate 5-phosphatases, SHIP2, has been established as a regulator of the insulin cascade, of cell adhesion and spreading, actin structures, remodelling and cytoskeletal organization. However, the molecular mechanisms underlying these processes still needed additional investigations. Among different regulatory mechanisms, protein-protein interaction play an essential role. To better understand the molecular mechanism of SHIP2 in signalling pathway as well as to reveal novel roles of SHIP2, a two-hybrid was performed to search for SHIP2 protein interactors. JNK-interacting protein 1 (JIP1) and intersectin 1 (ITSN1) were two of the newly identified protein partners of SHIP2. In this thesis, we characterized the associations of SHIP2 with JIP1 and ITSN1 in different aspects as identifying the interacting domain involved, biochemical function regulations and cellular biological roles.<p><p>The JIP scaffold family of proteins associate with MAPK, MAPKK and MAPKKK creating functional signaling modules to control the specificity of signal transduction. JIP1 is characterized as a scaffold protein assembling JNK, MAPK kinase 7 (MKK7), mixed lineage kinase (MLK), dual leucine zipper-bearing kinase (DLK). It thus enhances the selectivity and effectiveness of kinase activation during JNK signaling. In this thesis, the SHIP2-JIP1 interaction has been confirmed both in overexpression system in COS-7 and CHO-IR cells, and in native cells of COS-7. Both the proline-rich (PR) domain (residues 359-487) and PTB domain of JIP1 participated in this interaction. Overexpression of SHIP2 in COS-7 cells up-regulated JIP1-mediated JNK activation and the tyrosine phosphorylations of both JIP1 and MLK3. These effects were independent of SHIP2 catalytic activity. By the use of kinase inhibitors, we showed that Abl and Src family tyrosine kinases might be implicated in the regulation of JIP1 tyrosine phosphorylation. The residue Y270 of JIP1, a potential target of Abl tyrosine kinase, was shown to be involved in SHIP2-increased JIP1 tyrosine phosphorylation. In an in vitro assay, JIP1 negatively regulated the catalytic activity of SHIP2. In addition, upon the stimulation of okadaic acid, the overexpression of SHIP2 caused less viability of COS-7 cells. These data provide a new molecular link between SHIP2 and JIP1-mediated JNK pathway, and may help explain the biochemical mechanisms of SHIP2 in cellular apoptosis, as well as in insulin pathway.<p> <p>Another protein partner, ITSN1, is a multi-domain protein which plays a role in endocytosis, MAPK signalling and actin cytoskeleton. The interaction between SHIP2 and ITSN1 was confirmed in overexpression systems in COS-7 cells, as well as at the physiological concentration with the endogenously expressed proteins in C2C12 and COS-7 cells. EGF stimulation did not modulate the association of SHIP2 and ITSN1. ITSN1-SH3D, A, C and E domains interacted with the C-terminal part of SHIP2 with the binding affinity as SH3D>SH3A>SH3C>SH3E. Upon the stimulation of EGF, the expression of SHIP2 may recruit ITSN1 short form (ITSN1-S) to cell membrane. The ITSN-mediated ERK1/2 and JNK activations in response to EGF were not modulated when SHIP2 or catalytic mutant of SHIP2 or TSHIP2 was overexpressed. The link between SHIP2 and ITSN may provide one of the molecular mechanisms used by SHIP2 to participate in receptor endocytosis regulation.<p><p>In conclusion, our data of the associations of SHIP2 with JIP1 and ITSN1 provide evidence for potential novel biochemical mechanisms of SHIP2 to be implicated in JNK pathway as well as EGF receptor endocytosis. JIP1 and ITSN1, which are both implicated in the JNK pathway, may also have a link through the common protein partner SHIP2, giving rise to potential interesting study goal. <p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Degradation of membrane proteins in the vacuole/lysosome is dependent on their prior sorting into the multivesicular body (MVB) pathway. This sorting process involves incorporation of proteins into vesicles that are formed by budding of the limiting membrane of the endosome into the lumen of the organelle. The MVB sorting process on the whole is highly conserved from yeast to human, and depends on the Vps27/Hrs, ESCRT-I, -II, and -III protein complexes functioning sequentially on the endosomal membrane, as well as on additional factors, such as the ubiquitinating enzyme Rsp5/Nedd4. It has now been established that ubiquitin serves as a sorting signal for many cargoes into the MVB pathway. <p>In this thesis work, we provide evidence that Bro1 is not required for protein ubiquitination or early steps of endocytosis, but functions at the late endosome level as an integral component of the MVB pathway. Similarly to its human homologue Alix, Bro1 interacts with components of the ESCRT-I and ESCRT-III complexes. The putative role of Bro1/Alix in bridging an interaction between ESCRT-I and –III might be important to strengthen an association of these protein complexes to allow efficient sorting of cargo proteins. Deficiency in Bro1 results in recycling of the endocytosed Gap1 permease back to the plasma membrane, a process coupled to deubiquitination of the permease. This recycling is a non-classical phenotype for cells impaired in MVB pathway thus suggesting Bro1 to have a particular role in this sorting process. Furthermore, the conserved C-terminal proline-rich domain (PRD) of Bro1 is specifically important for MVB sorting of cargo proteins that are subject to ubiquitination. We show Bro1 (via its PRD) to play a highly important role in recruitment of the deubiquitinating enzyme Doa4 to the endosome. Consistent with this, Bro1 is required for deubiquitination of cargo proteins, a step occurring just before cargo incorporation into the endosomal vesicles, and similarly to Doa4, for ubiquitin recycling. In contrast to previous interpretations, we show that Doa4 has a direct role in sorting of ubiquitinated cargo proteins into the MVB pathway. We propose that Doa4 – via its association to Bro1 - achieves this role by catalyzing deubiquitination of cargo proteins and/or some components of the MVB sorting machinery. We further show Bro1 to interact with the ubiquitin ligase Rsp5, which, in addition to being required for cargo protein ubiquitination at the plasma membrane, apparently contributes to multiple steps of endocytosis and MVB sorting. Also the Bro1-Rsp5 interaction is dependent on the C-terminal PRD region of Bro1. We propose that this interaction is conserved. <p>A role for ubiquitin in regulation of the MVB sorting machinery is emerging: the function of factors recognizing and sorting ubiquitinated cargo proteins in the MVB pathway is suggested to be coupled to their cycling between ubiquitinated and deubiquitinated stages. A growing body of evidence indicates that ubiquitin ligases of the Rsp5/Nedd4 family play a central role in this regulation. We speculate the Bro1/Alix protein, through its ability to simultaneously interact with factors of the MVB sorting machinery and with ubiquitinating and deubiquitinating enzymes to play a central role in the successive rounds of ubiquitination and deubiquitination of specific factors along the MVB pathway. <p> / Doctorat en sciences, Spécialisation biologie moléculaire / info:eu-repo/semantics/nonPublished

Fibroblast growth factor (Fgf) signaling plays multiple inductive roles during development of vertebrates (Itoh 2007). Some Fgfs, such as Fgf8, are locally secreted and signal over a long range to provide positional information in the target tissue (Scholpp and Brand 2004). Fgf ligands signal in a receptor-dependent manner via tyrosine kinase receptors, four of which have been so far identified. Fgf8 signaling was shown to depend both on receptor activation as well as endocytosis. The specificity of Fgf ligands and receptors as well as the function of receptors in the control of the Fgf signaling range have been, however, largely unclear. In this study, we show that the putative Fgf8 receptor Fgfr1 is duplicated in zebrafish and that it acts redundantly in the formation of the posterior mesoderm. Also, in overexpression studies we confirm the notion that receptor endocytosis influences Fgf8 signaling range. Through TILLING mutant recovery and morpholino knockdown studies we also show that Fgfr2 is required for growth and skeletal development in zebrafish, whereas Fgfr4 is required for pectoral fin specification and growth.

Endocytosis is essential for the pathogenic development of Ustilago maydis. It has been shown that the initiation of pathogenicity relies upon the ability of the cell to recognize pheromone (a1 or a2) released from its mating partner and subsequently to form conjugated hyphae. The actin and microtubule cytoskeleton plays an essential role in all aspects of cell growth. A component of the actin cytoskeleton, the filamentous actin is required for cell-cell fusion, whereas the molecular motors, kinesin and dynein, move along microtubules and provide the long distance transport of many proteins and they are important in cell growth and pathogenicity. In this thesis, we investigated the role of the cytoskeleton in endocytosis and a1 pheromone transport, using a fluorescently labelled derivative of the a1 pheromone. We confirmed that uptake of the a1 pheromone is also receptormediated. In addition, we have shown that pheromone transport towards the cellular vacuole requires the actin and microtubule cytoskeletons. Furthermore, we revealed that the microtubule-dependent motors kinesin-1 and kinesin-3 and dynein were shown to be essential in the delivery of the pheromone to vacuoles. Moreover, a mutation in the early endosomal protein Yup1 gene causes a stop in delivery of the synthetic pheromone to the vacuole. This suggests that it travels with early endosomes. Within the actin cytoskeleton, we analysed the dynamics of actin patches in the presence of the synthetic pheromone and found that the dynamics of the patches increased significantly. Additionally, in the presence of an over-expression of the tail domain of the molecular motor myosin-5, the dynamics of the patches were significantly reduced and their intensity diminished.

The clathrin-independent endosomal system is required for cellular homeostasis and specialized modifications of the plasma membrane such as cell spreading and polarization. Clathrin-independent endocytosis (CIE) has been demonstrated in adherent cells including fibroblasts and epithelial cells. However, non-adherent cells also have highly dynamic clathrin-independent pathways, which have not been well described. Here, I have characterized Arf6-associated clathrin-independent endocytosis (CIE) in the human T cell line Jurkat and identified it's importance in immunological synapse formation. Our findings indicate that the CIE pathway is similar in Jurkat cells as compared to other cell types including rates of endocytosis and sorting after internalization. Two GTPases, Arf6 and Rab22, have been shown to regulate the clathrin-independent endosomal system and play a role in cell spreading. We found that wild type and constitutively active Arf6 co-localized with CIE cargo in resting T cells. Arf6 constitutively active mutant inhibited CIE cargo internalization but not internalization of CME cargo. Rab22 co-localized with CIE cargo at the endocytic-recycling compartment. Expression of the dominant negative Rab22 mutant also inhibited internalization of MHCI indicating it plays a direct role in CIE cargo internalization. T cells must modify their membranes to specifically interact with antigen presenting cells. To establish the role of CIE in this process, we then examined the role of Arf6 and Rab22 in T cell/antigen presenting cell conjugate formation. Both expression of dominant negative or constitutively active mutants of Arf6 reduced T cell conjugate formation while expression of only the Rab22 dominant negative mutant inhibited T cell/APC conjugate formation. Furthermore, T cells expressing the dominant negative mutant of Rab22 were not able to spread on antibody-coated coverslips that normally cause T cell activation. These results indicate that the clathrin independent endosomal system is required for membrane remodeling events necessary for T cell conjugate formation and T cell spreading during activation. I also conducted a proteomics screen to identify binding partners of CIE cargo proteins. I identified multiple proteins that could possibly play a role in CIE internalization and discovered a subset of proteins that specifically interact with A cargo proteins and not B cargo proteins. It is possible they could play a role in cargo retention at the plasma membrane or sorting after internalization. Three proteins of interest that interact with A cargo include NHERF-1 and ezrin, which participate in actin arrangements, and Dlg-1, a known scaffolding protein for synaptic vesicles. Ezrin and Dlg-1 co-localize with the CIE cargo protein CD98 in HeLa cells indicating that they could be interacting in cells.

No description available.

Le placenta assure les échanges materno-fœtaux et possède une fonction endocrine autonome. Les hormones placentaire lactogène (hPL) et chorionique gonadotrope (hCG) sont synthétisées par le syncytiotrophoblaste. A ce jour, les mécanismes impliqués dans le contrôle de la sécrétion de ces deux hormones ne sont pas connus. In vitro, l’influx d’ions Ca2+ entraîne une augmentation immédiate et soutenue de la libération d’hPL et d’hCG à partir d’explants de placentas à terme. En outre, l’élévation de la concentration extracellulaire en albumine, principale protéine maternelle circulante en contact direct avec le trophoblaste, stimule de manière immédiate et transitoire la libération d’hPL et d’hCG. L’objectif de nos travaux a été de vérifier la spécificité de l’activité sécrétagogue de l’albumine au niveau du placenta, de caractériser les messagers cellulaires potentiellement impliqués dans la libération d’hPL et d’hCG, et de définir l’interaction entre l’albumine et le trophoblaste, en utilisant des explants provenant de placentas humains à terme. Nos travaux démontrent que la riposte sécrétoire à l’albumine (5%, m/v) est largement mimée par d’autres agents colloïdaux (dextran et polygéline). Cette stimulation colloïdale de la libération d’hPL et d’hCG impliquerait une mobilisation de Ca2+ à partir de réserves intracellulaires. L’intervention de 3 messagers cellulaires a été envisagée: les IPs/DAG, l’AMPc, et le GMPc. Le fluorure de sodium, la forskoline, ou le nitroprussiate sodique, activateurs connus de la production respective des IPs, de l’AMPc, et du GMPc, augmentent de manière significative les taux placentaires de chacun de ces messagers, sans toutefois affecter la libération d’hPL ou d’hCG. De plus, l’élévation de la concentration extracellulaire en albumine (5%, m/v) ne modifie pas les taux des IPs, de l’AMPc et du GMPc dans les explants placentaires, tandis qu’elle stimule la sécrétion hormonale. Ces systèmes de signalisation, bien que fonctionnels au niveau du trophoblaste, ne joueraient donc pas un rôle majeur dans la régulation de la libération d’hPL et d’hCG. Nos résultats mettent en évidence une internalisation rapide d’albumine marquée, avec de l’125I ou de la fluorescéïne, dans le syncytiotrophoblaste. Une large fraction de cette albumine est recyclée, intacte, vers la circulation maternelle selon un processus sensible à l’abaissement de la température et indépendant du cytosquelette. L’albumine marquée restant dans les explants placentaires est partiellement dégradée. Trois mécanismes ont été envisagés pour expliquer ces mouvements d’entrée et de sortie de l’albumine au sein du placenta humain: l’endocytose médiée par l’albondine via les caveolae, le système des coated pits clathrine-dépendant, et l’endocytose médiée par la mégaline. Par immunohistochimie, nous avons montré que, dans le tissu placentaire, la caveoline-1, protéine caractéristique des caveolae, est localisée uniquement dans l’endothelium des capillaires fœtaux. La clathrine, au niveau des coated pits, et la mégaline se trouvent au contraire dans le syncytiotrophoblaste. La méthyl-b-cyclodextrine et l’hydrochlorure de chlorpromazine, inhibiteurs d’une endocytose dépendant de la clathrine, réduisent significativement l’internalisation placentaire de l’albumine marquée. Par contre, le DIDS ou le NPPB, susceptibles de perturber l’endocytose médiée par la mégaline, n’affectent pas la captation d’albumine marquée par les explants placentaires. L’albumine pénétrerait donc dans le syncytiotrophoblaste principalement par un processus clathrine-dépendant. La mégaline ne jouerait ici qu’un rôle mineur dans l’entrée de la protéine. Un tel processus de recyclage de l’albumine pourrait être similaire à celui décrit pour les immunoglobulines G au niveau du syncytiotrophoblaste. Ces mouvement d’entrée et de sortie de l’albumine ne semblent pas associés à la stimulation de la libération d’hPL et d’hCG par l’albumine. Ils pourraient par contre participer significativement, étant donné leur ampleur, à la nutrition fœtale. L’albumine est en effet un transporteur notoire d’ions et d’acides gras, molécules qui pourraient être acheminées au fœtus via le phénomène de recyclage placentaire de l’albumine mis en évidence par ce travail. / The human placenta is the site of all maternal-fetal exchanges, and is also an active endocrine organ. Placental lactogen (hPL) and chorionic gonadotrophin (hCG) hormones are synthesized by the syncytiotrophoblast. So far, the mechanisms involved in the regulation of both hormones secretion remain elusive. In vitro, calcium inflow causes an immediate and sustained rise in the hPL and hCG releases from human term placenta explants. Moreover, increasing the extracellular concentration of albumin, the major maternal plasma protein in direct contact with the human trophoblast, stimulates the hPL and hCG releases in an immediate and transient way. Our study have aimed to check the specificity of this secretory effect of albumin, to investigate the potential cellular messengers involved in the hPL and hCG releases, and to define the interaction between albumin and the throphoblast layer, using human term placenta explants. Our results indicate that the triggering effect of albumin (5%, w/v) is largely mimicked by two other colloidal agents (dextran and polygelin). This “colloidal” stimulation of the hPL and hCG releases would involve the mobilization of calcium from intracellular pools. Three cellular messengers have been considered to mediate this process: the IPs/DAG, the cAMP, and the cGMP. Sodium fluoride, forskolin, or sodium nitroprusside, known activators of respectively the IPs, cAMP, and cGMP production, significantly increase the placental content of each of those messengers, without modifying the hPL and hCG releases. In addition, raising the extracellular concentration of albumin does not cause any change in the placental level of IPs, cAMP, and cGMP, while stimulating the hormonal release. These three signaling pathways are thus functional in human term trophoblast but do not appear to significantly modulate the hPL and hCG secretions. Our findings show that albumin, labeled with 125I or with fluorescein, is rapidly internalized into the syncytiotrophoblast. Thereafter, the intact protein is largely recycled to the maternal circulation, through a temperature-sensitive and cytoskeleton-independent process. The labeled albumin remaining in placental explants is partially degraded. Three different mechanisms could participate to the albumin entry into the human placenta: the albondin-mediated endocytosis via the caveolae, the clathrin-dependent coated pits system, and the megalin-mediated endocytosis. Using immunohistochemistry, caveolin-1, marker of the caveolae, is localized in the endothelium of the fetal capillaries and not in the syncytiotrophoblast. By contrast, clathrin and megalin are observed only in the syncytiotrophoblast. Methyl-b-cyclodextrin, and chlorpromazine hydrochloride, known inhibitors of the clathrin-dependent endocytotic process, significantly reduce the placental uptake of labeled albumin. On the other hand, DIDS or NPPB, able to perturb the megalin-mediated endocytosis, do not affect the labeled albumin uptake. Thus, albumin seems to be internalized into the syncytiotrophoblast mainly through a clathrin-dependent mechanism. Megalin would only play a minor role in this process. Such movements of albumin in the human placenta may be similar to the recycling process reported for IgG at that site. The placental apical recycling of albumin is not associated to the albumin triggering effect on the hPL and hCG releases. This quantitatively significant internalization process may participate to the fetus’ nutrition. Indeed, Albumin carries ions and fatty acid, which could be brought to the fetus via the protein recycling evidenced by our study.

The wide use of nanoscale materials in food and health care products raises the concern of their possible uptake across the gastrointestinal tract, but very limited data are available on their uptake kinetics, and the potential hazards for humans. In this study, the uptake mechanism of titanium dioxide (TiO2) across the isolated perfused fish intestine and human intestinal Caco-2 cells were evaluated. The in vitro preparation of the whole gut sac and the isolated perfused intestine of rainbow trout were performed using both bulk and nano TiO2 in a concentration of 1 mg l-1 for up to 4 h. The results showed that the Ti from both bulk and TiO2 NPs were mainly accumulated in the mid and hind intestine, with 80% or more of the accumulation in the mucosa rather than the underlying muscularis. Perfused intestines showed a saturable, time-dependent accumulation of the Ti from TiO2 and the uptake of Ti from exposure to NPs was faster than that of the bulk form. The uptake of Ti from exposure to TiO2 NPs increases 10 fold when the CO2 in the gas mixture was lowered to 0.5%. Subsequently, further investigation on the mechanisms of uptake of TiO2 was applied using different kinds of inhibitors. Adding 10 mmol l-1 cyanide did not stop Ti uptake from TiO2 exposures, and 100 µmol l-1 vanadate (ATPase inhibitor) caused a 2.8 fold reduction in the net uptake rate of Ti for the TiO2 NP exposure. Luminal additions of 120 IU ml-1 nystatin (endocytosis inhibitor) blocked the uptake of Ti from both bulk and TiO2 NPs treatments. The results indicate that Ti accumulation from TiO2 exposures was sensitive to both nystatin and vanadate; the former suggesting that there is an endocytosis involvement in the uptake of TiO2 across the intestinal epithelium. Human intestinal Caco-2 cell showed a steady, saturable and time-dependent accumulation of Ti over 24 h exposures to 1 mg l-1 TiO2 (for all forms of TiO2). A scanning electron microscope study indicated the appearance of the particles underneath the cells, increasing the evidence of the Ti uptake from different forms of TiO2 by Caco-2 cells. Both nystatin and vanadate increase the accumulation of TiO2 which suggests interference of these drugs with endocytic pathways. All the data in the thesis demonstrates Ti uptake across the intestinal epithelium from TiO2 exposures involving CO2-dependent and nystatin-sensitive mechanisms. The results in this thesis have contributed to some understanding on the behaviour, uptake and effects of the TiO2 NPs across the intestine; and highlight the possible dietary hazard of the NPs to human health.

Le fer est un élément essentiel pour les plantes, mais toxique lorsqu'il est accumulé en excès. Chez Arabidopsis thaliana, le transporteur IRT1 joue un rôle essentiel dans l'acquisition du Fe depuis la solution du sol, en conditions limitantes en cet élément. Le gène IRT1 est régulé transcriptionnellement par le fer conduisant à une accumulation des transcrits IRT1 dans l'épiderme des racines carencées en fer. Par homologie avec les mécanismes décrits pour le transporteur de zinc ZRT1 de levure, une régulation post-traductionnelle d'IRT1, contrôlant la stabilité de celui-ci en présence de fer a été envisagée. IRT1 a donc été utilisé comme modèle pour caractériser le système endocytique des plantes. Nos travaux révèlent que la protéine IRT1 est localisée au niveau des endosomes précoces (TGN/EE) des cellules de poils racinaires. Des approches pharmacologiques ont permis de révéler un cyclage d'IRT1 entre la membrane plasmique et le TGN/EE ainsi qu'une dégradation vacuolaire. Nous avons également pu montrer que l'internalisation et la dégradation d'IRT1 ne sont pas affectées par la disponibilité en fer et sont sous le contrôle de la monoubiquitination de résidus lysines présents dans les parties cytosoliques de la protéine IRT1. Nos travaux suggèrent un modèle où l'internalisation d'IRT1 depuis la membrane plasmique, contrôlée par monoubiquitination, permet aux plantes de se prémunir contre la toxicité des métaux transportés par IRT1. Enfin, nous avons réalisé un crible double hybride en utilisant la boucle cytosolique d'IRT1 afin d'identifier des protéines contrôlant son trafic et/ou sa dégradation. Ce crible a permis notamment l'identification d'une protéine à domaine FYVE, localisée aux endosomes et dont la caractérisation fonctionnelle a été initiée / Iron is an essential element for plants but toxic when present in excess. IRT1 is the major root iron transporter responsible for iron uptake from the soil under iron limitation in Arabidopsis thaliana. IRT1 is transcriptionally regulated by iron, resulting in a high IRT1 expression in iron-starved root epidermal cells. In addition, IRT1 was suggested to be controlled at the post-translational level, with iron affecting IRT1 protein stability, in a similar fashion with the yeast ZRT1 zinc transporter. To shed light on two poorly-understood phenomena in plants, endocytosis and degradation of plasma membrane proteins, we studied the proposed post-translational regulation of IRT1 in Arabidopsis thaliana. Interestingly IRT1 protein is found in early endosomes of root hair cells. Pharmacological approaches reveal that IRT1 cycles back and forth with the plasma membrane to perform iron uptake, and is sent to the vacuole for proper turnover. We also demonstrate that iron nutrition have no effect on the levels and the subcellular localization of IRT1 protein. The internalization of IRT1 is dependent on the monoubiquitination of several cytosol-exposed lysine residues. Together, these data suggest a model where monoubiquitin-dependent endocytosis/recycling of IRT1 keeps the plasma membrane pool of IRT1 low, to better deal with metal uptake. Finally, in order to indentify genes involves in IRT1 endocytosis/recycling and turnover, we perform a yeast two-hybrid screen with IRT1 cytosolic loop. This screen allows the identification of a FYVE domain-containing protein localized in endocytic compartment which functional characterization was initiated.