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Neuhaus, Jean-Marc

Nom
Neuhaus, Jean-Marc
Affiliation principale
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Professeur ordinaire
Email
jean-marc.neuhaus@unine.ch
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https://libra.unine.ch/handle/123456789/300

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  • Publication
    Accès libre
    The functions of RMR proteins in the "Physcomitrella patens" secretory pathway
    (2017)
    Fahr, Noémie
    ;
    Neuhaus, Jean-Marc 
    Chez les plantes, les vacuoles occupent un grand nombre de fonctions, allant du maintien de la pression de turgescence et de la rigidité cellulaire en passant par le stockage ou la dégradation de diverses molécules. Deux types de vacuoles ayant un pH distinct peuvent coexister au sein d’une même cellule. Les vacuoles acides sont considérées comme étant des homologues aux lysosomes présents dans les cellules animales, tandis que les vacuoles neutres sont impliquées dans le stockage de protéines et de métabolites secondaires. L’adressage des protéines à la vacuole lytique a été largement étudié et les récepteurs vacuolaires impliqués, les VSRs, sont des protéines bien caractérisées. À l’opposé, les connaissances sur l’adressage des protéines à la vacuole neutre ou de stockage sont moindres. Les protéines RMR sont très probablement les récepteurs vacuolaires impliqués, bien que la délétion des cinq gènes RMR chez Physcomitrella patens n’ait conduit à aucun phénotype visible. Ce travail a donc pour objectif l’élucidation du rôle des protéines RMR chez la mousse.
    Dans le premier chapitre de cette thèse nous avons regroupé les principales données concernant le système sécrétoire et endomembranaire chez les plantes, et nous avons également documenté comment les protéines sont adressées aux vacuoles.
    Dans le second chapitre, nous avons étudié le système sécrétoire de la mousse en développant une bibliothèque de marqueurs fluorescents. Différents mécanismes cellulaires semblent conservés entre les mousses et les plantes à fleurs.
    Dans le troisième chapitre, nous nous sommes intéressés à la caractérisation des simples et quintuple knock-out mutants RMR. Nous avons finalement obtenu un phénotype de tri vacuolaire: un défaut d’adressage est observé avec le marqueur fluorescent Citrine-Card dans les simples, triple et quintuple KO mutants. Le signal fluorescent a été détecté dans le réticulum endoplasmique chez les mutants, tandis que la fluorescence est observée dans la vacuole centrale chez le WT. Cela montre que l’adressage à la vacuole d’une protéine comportant ce ctVSD est dépendant des RMRs.
    Dans la dernière partie de ce travail, nous avons identifié des partenaires interagissant très probablement avec la partie cytosolique de PpRMR2 par des analyses de GST pull-down et de spectrométrie de masse., Plant vacuoles play a wide range of functions within the cell, from the maintenance of turgor pressure and rigidity, to the storage or degradation of various molecules. Two types of vacuoles with distinct pH can coexist in the same single cell. Acidic vacuoles can be considered as homologues to animal lysosomes while neutral vacuoles are involved in proteins and secondary metabolites storage. Targeting of proteins to the lytic vacuole has been extensively studied and the vacuolar receptors involved, the VSRs, are now well characterized in higher plants. However, less is known about the traffic of proteins to the neutral/storage vacuole. RMR proteins are thought to be vacuolar receptors for the neutral/storage vacuole. However, the complete deletion of the five RMR genes in Physcomitrella patens did not lead to any developmental phenotype. This work aimed to investigate the role of RMR proteins in the moss.
    In the first chapter, we review the plant secretory pathway system and how proteins are targeted to vacuoles.
    In the second chapter, we studied the moss secretory pathway by developing a fluorescent reporter library. Several mechanisms seem to be conserved between the moss and the flowering plants.
    In the third chapter, we focused on the characterization of the single and quintuple knock-out RMR mutants. We finally obtained a trafficking phenotype: the fluorescent reporter Citrine-Card was mistargeted in the single, triple and quintuple KO mutants. Fluorescent signal was detected in endoplasmic reticulum in the mutants, while it was observed in the central vacuole in WT. Trafficking to vacuole of a protein carrying this ctVSD was RMR-dependent.
    In the last part of this thesis, we identified some putative binding partners of the cytosolic part of PpRMR2 by GST pull-down assay and mass spectrometry analysis.
  • Publication
    Accès libre
    Study of moss vacuoles and functional characterization of the putative vacuolar receptors: the RMR proteins
    (2012)
    Ayachi, Sanaa
    ;
    Neuhaus, Jean-Marc 
    The vacuolar system of plants is a key element of plant growth and development, it fulfils many other functions. Plant cell can have more than two different vacuolar sorting systems: the lytic and the (seed) protein storage or vegetative storage vacuoles. Soluble vacuolar proteins are sorted through the secretory pathway to these vacuoles by three different routes, depending on different types of Vacuolar Sorting Determinants (VSD) and involving several types of receptors and vesicles. The AtRMR proteins has been identified in cellular structures associated with the seed storage vacuole pathway (Jiang et al. 2000). Based on its localisation and homology to a known vacuolar receptor, it has been hypothesised to be a receptor protein for the C-terminal type of VSD (ct-VSD) involved in sorting to the storage vacuole. The genome of Physcomitrella patens contains five genes coding for RMR proteins.
    My work hypothesis is that the vacuolar system of higher plants has evolved from simple ancestors, which might have been preserved in lower plants. This evolution is reflected in the gene families involved in vacuole biogenesis. In a first part, we established the moss P. patens as a model system for the study of the secretory pathways. In a second part, we performed a comparative study of the plant-specific aspects of the vacuolar system. And finally in a third part, we tried to establish the functional role of PpRMR genes by the analysis of the complete RMR deletion mutants. Several strategies were considered to investigate a putative disorder due to RMRs loss of function. So far, no phenotype was detected in the mutants. Nevertheless the absence of the RMR family gene seems not to be necessary for moss development.
  • Publication
    Accès libre
    Comparison of proteolytic system secreted in dermatophytes and 'Aspergillus fumigatus', used as a reference
    (2011)
    Sriranganadane, Dev
    ;
    Neuhaus, Jean-Marc 
    Les dermatophytes sont des champignons pathogènes qui se développent dans le stratum corneum de la peau, les ongles et les cheveux et sont la cause du plus grand nombre des mycoses cutanées. En culture dans un milieu ne contenant que de la kératine, ces champignons sécrètent de nombreuses protéases pour digérer cette source de protéine en acides aminés et petits peptides qui sont utilisés comme nutriments. Les protéases sécrétées par les dermatophytes sont similaires à celles sécrétées par les espèces du genre Aspergillus. C’est pourquoi, le champignon Aspergillus fumigatus a été utilisé dans ce travail pour étudier les différentes étapes de dégradation des protéines par des champignons tels que les dermatophytes à pH acide et à pH neutre.

    Lors de la première étape de ce travail, nous avons montré que deux différents ensembles de protéases étaient sécrétés par Aspergillus fumigatus à pH 4.0 et à pH 7.0. A pH 7.0, cet ensemble comprend une subtilisine et une métalloprotéase qui sont des endoprotéases, des aminopeptidases non spécifiques (Laps pour leucine aminopeptidases) et une dipeptidylpeptidase IV (DppIV) qui est une X-prolyl peptidase. Il était connu que des peptides générés par une activité endoprotéolytique sur des grandes protéines pouvaient être digérés ensuite synergiquement par les Laps et la DppIV. Lors de ce processus les Laps ôtent les acides aminés un par un depuis l’extrémité N-terminale d’un peptide jusqu’à une séquence X-Pro où les laps s’arrêtent. Toutefois, les séquences X-Pro sont enlevées par la DppIV qui génère ainsi un nouveau substrat pour les Laps. A pH 4.0, l’ensemble des protéases sécrétées par Aspergillus fumigatus comprenait une pepsine, une protéase inconnue de la classe des glutamique-protéases (appelée ici AfuGprA), des sédolisines (SED) qui avaient été caractérisées comme étant des tripeptidyl-peptidases non spécifiques, et une nouvelle protéase de la famille S28 (appelée ici AfuS28). Il a été montré dans ce travail que des grands peptides pouvaient être digérés à pH acide par les activités synergiques des Seds et AfuS28. Lors de ce processus les Seds ôtent les acides aminés trois par trois depuis l’extrémité N-terminale d’un peptide jusqu’à une proline en position 3 ou 4. Toutefois, les séquences d’arrêt X-XX-Pro et X-X-XX-Pro sont enlevées par l’activité d’AfuS28 qui génère ainsi un substrat dégradable par les Seds. En conclusion, chacun des eux ensembles des protéases sécrétées par Aspergillus fumigatus comprenait des aminopeptidases non spécifiques butant sur des résidus Pro, et une prolyl peptidase. L’activité de cette dernière enzyme génère un nouveau substrat pour les aminopeptidases non spécifiques.

    La deuxième étape a consisté en l’étude d’AfuGprA et son importance dans l’activité endoprotéolytique d’Aspergillus fumigatus. Nous avons montré que soit Pep soit AfuGprA était nécessaire pour la croissance du champignon à pH acide dans un milieu contenant des protéines non dégradées comme seul nutriment.

    Et enfin, la dernière partie de cette thèse s’est focalisée sur l’identification des protéases sécrétées par deux espèces de dermatophytes, Microsporum canis et Arthroderma benhamiae dans un milieu ne contenant que des protéines. Comme chez Aspergillus fumigatus, ces deux dermatophytes sécrètent à pH 4.0 et à pH 7.0 un ensemble particulier de protéases. Nombres de ces protéases ne sont pas connues chez ces dernières, mais sont des orthologues probables de protéases caractérisées chez Aspergillus fumigatus. C’est la première fois que des protéases acides sont identifiées à pH acide chez ces champignons. Ces investigations suggèrent des mécanismes communs de dégradation des protéines chez les Aspergillus et chez les dermatophytes., Dermatophytes are highly specialized pathogenic fungi which grow exclusively in the stratum corneum, nails or hair and are the most common agents of superficial mycoses. In a medium containing keratin as the sole nitrogen source they secrete a set of endo- and exoproteases able to digest keratin into amino acids and short peptides to be assimilated. Proteases secreted by dermatophytes are similar to those of Aspergillus spp. Therefore, Aspergillus fumigatus was used as a model to investigate the different steps of protein degradation in acidic and neutral environments by fungi such as dermatohytes.

    During growth in a protein medium at neutral pH, Aspergillus fumigatus secretes neutral and alkaline endoproteases, an X-prolyl peptidase (DppIV) and leucine aminopeptidases (Laps) which are non-specific monoaminopeptidases. Laps cannot remove any amino acids from a peptide with a N-terminal X-Pro sequence. However, large peptides generated from protein digestion by endoproteolysis can be further digested into amino acids and X-pro dipeptides by the synergistic action of Laps and DppIV. We have shown that A. fumigatus secretes a distinct set of proteases at acidic pH which includes an aspartic endoprotease of the pepsin family (Pep1), a novel glutamic protease, AfuGprA, homologous to Aspergillus niger aspergillopepsin II, tripeptidyl-peptidases of the sedolisin family (SedB and SedD) and a novel prolylpeptidase, AfuS28.

    The importance of AfuGprA in protein digestion was evaluated by deletion of its encoding gene in A. fumigatus wild type D141 and in a pepΔ mutant. We have shown that either A. fumigatus Pep or AfuGprA is necessary for fungal growth in protein medium at acidic pHIn conclusion, Pep and AfuGprA constitute a pair of endoproteases active at acidic pH in analogy to A. fumigatus alkaline protease (Alp) and metalloprotease I (Mep), where at least one of these enzymes is necessary for fungal growth in protein medium at neutral pH.

    We have shown that Seds and AfuS28 synergistically digest large peptides generated by exoprotease activity into amino acids, di- and tripeptides. Seds degrade peptides from their N-terminus into tripeptides, however Pro in P1 and P’1 position acts as a stop sequence. In a complementary manner, X-X-Pro and X-X-X-Pro sequences can be removed by AfuS28 thus allowing Seds further sequential proteolysis. In conclusion, both alkaline and acidic sets of proteases contain exoprotease activity capable of cleaving after proline residues not bypassed by other exoproteases.

    In a third part of this thesis we have tested the ability of two dermatophyte species, Microsporum cani and Arthroderma benhamiae, to grow in a protein medium that promotes secretion of proteases. We have shown that at neutral and acidic pH, dermatophytes secreted different proteases. Our investigation revealed new dermatophyte secreted proteases homologous to those secreted by A. fumigatus and suggests common basic mechanisms for extracellular protein digestion in dermatophytes and in Aspergillus spp. at acidic and neutral pH.
  • Publication
    Accès libre
    The role of palmitoylation in the secretoy pathway of plants
    (2011)
    Stigliano, Egidio
    ;
    Neuhaus, Jean-Marc 
    This study aimed to study an important eukaryotic post-translational modification, the S-palmitoylation. Until now, there was no study of palmitoylation in plant cell biology. In the first part of this study, we wanted to study the palmitoylation of the vacuolar receptor AtRMR1, as predicted in silico. We used a very innovative technique, the Biotin Switch Assay, which does not use radioactive palmitate, is much less time-consuming as it is possible to obtain results after three to four day. Another advantage for cell biology is that it allows the characterization of entire palmitoyl-proteomes. Yeast and neuronal palmitoyl-proteomes have indeed been recently characterized. The study of RMR1’s palmitoylation revealed the first palmitoylated plant transmembrane protein. The palmitoylation of a small fraction of RMR1 at a higher molecular weight deserves further discussion.
    In the second part of this thesis, I addressed the more general role of palmitoylation in the secretory pathway through the use of a potent palmitoylation inhibitor: 2BP. This study showed a specific action of the drug in TGN/post-TGN compartments. The drug affected the structural maintenance of macrovesicles of secretion. The macrovesicles of secretion have recently been characterized by cryofixation and by electron tomography. They are structures reminiscent of a bunch of grapes, where each grape is a secretory vesicle. They are associated with the building with TGN-rich secretory vesicles with a diameter of few tens of nanometres. These vesicles are particularly visible in tissues with a high growth rate, such as pollen tubes. 2BP drastically changed the state of aggregation of macrovesicles of secretion. In an imaged way, each grape was released and a diffuse fluorescence was observed. Palmitoylation is therefore important in the formation or stability of this important secretory structure. A possible extension of this work would be the isolation of palmitoylated proteins involved in this stabilization. In addition, palmitoylated Rabs were detected for the first time in a plant. The three plant Rabs for which I detected the effect of 2BP are located in post-Golgi compartments.
    In a last part of the thesis, I decided to investigate the route of secretion of GFP-Chi, a vacuolar markerwidely used in the lab that can be followed along the route of secretion. Apparent contradictions have been reported: a vacuolar sorting of this marker by the Golgi-TGN-PVC pathway or an independent-COPII trafficking that can bypass the classic route. Unexpectedly, when GFP-Chi was co-expressed with NtSar1H74L (a dominant-negative mutant blocking the ER-Golgi trafficking by preventing the formation of COPII vesicles), the reporter reached the vacuole. This suggests that the alternative pathway bypassing the Golgi can take place. I also detected the presence of a possible GFP-Chi dimer associated with the membrane fraction upon ultracentrifugation. The nature of this dimer of a soluble protein remains to be investigated although several cases of aggregation of soluble proteins have been reported in the literature (like β-amyloids presents in the of Alzheimer disease). Another matter of great interest is whether the dimer reaches the vacuole or is only transient intermediate during the transport to the vacuole.
  • Publication
    Accès libre
    Localization and interaction of AtRMR receptors in the plant secretory pathway
    (2011)
    Occhialini, Alessandro
    ;
    Neuhaus, Jean-Marc 
    Durant les dernières années, il a été démontré que de nombreuses protéines vacuolaires sont triés vers leur destination finale par des récepteurs vacuolaires. Par conséquent, dans cette étude je me suis concentré sur les protéines RMR (récepteur membranaire Ring-H2), une nouvelle famille de récepteurs putatifs, composées de six gènes chez Arabidopsis thaliana (AtRMR), probablement impliquées dans le transport des protéines vers les vacuoles (Jiang et al., 2000 ; Park et al., 2005; Park et al., 2007; Hinz et al., 2007). Ces récepteurs ont été identifiés grâce à leur homologie avec le domaine PA (domaine associé aux protéases) présents dans les récepteurs vacuolaires VSR qui sont bien connus pour jouer un rôle de triage de certaines protéines vacuolaires (Paris et al. , 2002).
    On toutefois, les RMRs sont beaucoup moins connus que les VSRs. Dans la présente étude, je me suis concentré sur la localisation de différents RMRs présents dans les cellules végétales. Par ailleurs, j'ai étudié la possible dimérisation (homo-dimérisation et hétéro-dimérisation) entre les différents types de récepteurs AtRMR.
    Pour la localisation, j'ai généré différents vecteurs d'expression pour les plantes portant différentes protéines fluorescentes fusionnées à différent types de AtRMR. Par la suite, ces protéines de fusion ont été localisées dans les cellules en utilisant le microscope confocal. La localisation a été effectuée chez des plantes d'Arabidopsis thaliana transgéniques et chez des feuilles de Nicotiana benthamiana transformées par agroinfiltration. Dans ces expériences, AtRMR1 et AtRMR2 ont montré des localisations subcellulaires différentes. AtRMR1 est localisé dans TGN, tandis que AtRMR2 est localisé dans la membrane du Reticulum Endoplasmique (ER). Cette différente localisation est due à la présence d'un signal putatif de localisation présent dans la séquence linker de AtRMR1. En fait, quand cette séquence est placée sur AtRMR2, elle est capable de relocaliser la protéine dans le Réseau Trans-Golgien (TGN).
    Enfin, pour tester l'éventuelle dimérisation entre différents types de AtRMR, j'ai développé une technique de Complémentation Bimoléculaire Fluorescente (BiFC). En utilisant cette technique, j'ai démontré que AtRMR1 peut former des homo-dimères et peut interagir avec AtRMR2 en formant des hétéro-dimères. De plus, homo-et hétéro-dimères montrent la même localisation dans le TGN. Ce résultat a démontré que AtRMR2 peut quitter l’ER sous forme d’hétéro-dimère grâce à la présence du signal de localisation dans la séquence linker de AtRMR1. Par ailleurs, en utilisant des mutants de délétion de différents domaines, j'ai démontré que le domaine transmembranaire et la séquence linker sont probablement les domaines impliqués dans l'interaction protéine-protéine., In the last few years, it was demonstrated that many vacuolar proteins are sorted to their final destination by cargo receptors. Therefore in this study I focused on RMR proteins (Receptor Membrane Ring-H2), a new family of putative receptors, composed of six genes in Arabidopsis thaliana (AtRMR), probably involved in protein transport to vacuoles (Jiang et al.,, 2000; Park et al., 2005; Park et al., 2007; Hinz et al., 2007). These receptors were identified by their homology to the PA domain (Protease Associated Domain) present in the Vacuolar Sorting Receptors (VSR) that are well known to bind and sort vacuolar proteins (Paris et al., 2002).
    Much less is known about these proteins than about VSRs. In the present study I focused on the localization of the different members present in plant cells. Moreover I studied the possible dimerization (homo end/or hetero) between the different types of AtRMR receptors.
    For the localization, I have generated different plant expression vectors carrying different fluorescent protein reporters fused to AtRMRs to use in a confocal microscope experiment. The localization was performed in Arabidopsis thaliana transgenic plants and Nicotiana benthamiana leaves transformed by agro-infiltration. In these experiments AtRMR1 and AtRMR2 showed different subcellular localizations. AtRMR1 localizes in TGN while AtRMR2 localizes in the membrane of ER. This different localization is due by the presence of a putative localization signal present in the sequence linker of AtRMR1. In fact this sequence, when is placed on AtRMR2, is able to relocate the protein in the TGN.
    To test the possible AtRMR-AtRMR dimerization I developed Bimolecular Fluorescence Complementation (BiFC) reporters. Using this technique I demonstrated that AtRMR1 can make homodimers and can interact with AtRMR2 making heterodimers. Moreover homo- and heterodimers showed the same localization in the TGN. This result demonstrated that AtRMR2 can exit from the ER as a heterodimer thanks to the presence of the localization signal in the sequence linker of AtRMR1. Moreover using AtRMR deletion mutants I demonstrated that the transmembrane domain and the sequence linker are probably the domains involved in protein-protein interaction.
  • Publication
    Accès libre
    Functional characterisation of AtRMR proteins in "Arabidopsis thaliana"
    (2007)
    Zava, Olivier
    ;
    Neuhaus, Jean-Marc 
    Plant cells contain two or even three types of vacuoles: the lytic, the (seed) protein storage and vegetative storage vacuoles. Soluble vacuolar proteins are sorted through the secretory pathway to these vacuoles by three different routes, depending on different types of Vacuolar Sorting Determinants (VSD) and involving several types of receptors and vesicles. The AtRMR1 protein has been identified in cellular structures associated with the seed storage vacuole pathway (Jiang et al. 2000). Based on its localisation and homology to a known vacuolar receptor, it has been hypothesised to be a receptor protein for the C-terminal type of VSD (CtVSD) involved in sorting to the storage vacuole. The genome of Arabidopsis thaliana contains 5 genes homologous to AtRMR1. The main goal of this study was to test the involvement of AtRMR1 in vacuolar sorting and to test the specificity of the different AtRMR proteins for different known CtVSDs. To test the involvement of AtRMR proteins in vacuolar sorting we studied the effects of manipulations of these genes on targeting of vacuolar reporter proteins. I used two different models: A. thaliana leaf protoplasts and whole plants of insertional mutants from the SALK Institute collection. The protoplast model allowed me to study in vivo the effects on vacuolar sorting of versions of AtRMR1 with loss of function deletions or modified functions. I obtained interesting results with two of these constructs: one lacking the luminal VSD-binding domain (RMRΔlum) and one consisting of this soluble luminal domain retained in the ER by the addition of a HDEL peptide signal (RMRlumER). When overexpressed with GFP fused to the ssVSD of barley aleurain, the RMRΔlum construct showed a different pattern of fluorescence compared with the control: in RMRΔlum, a significant proportion of protoplasts showed a dot-like fluorescent pattern, whereas the fluorescence was more often found in the central vacuole or ER in the control. When I used either the CtVSD of tobacco chitinase or a short version of the barley aleurain ssVSD, I didn’t see a different fluorescent pattern with or without the dominant negative construct. To better estimate the effects of the dominant negative constructs, the level of secretion of enzymatic reporters was investigated. The reporters were α-amylase fused either to the CtVSD of tobacco chitinase, the CtVSD of barley lectin or the ssVSD of sweet potato sporamin. For these three different reporters, overexpression of RMRΔlum induced an increased secretion of the reporter, whereas a simple fractionation showed that the RMRlumER construct provoked their accumulation in microsomal compartments, presumably ER. Two other constructs, where either the luminal domain of AtRMR1 was redirected to the PM (RMRlumPM) or the soluble cytosolic domain was overexpressed (RMRcyt) didn’t have any effect on the sorting of enzymatic reporters, suggesting that the transmembrane and cytosolic domains are both important for the VSD binding and that the cytosolic domain alone is not able to interfere with the vacuolar sorting machinery. I completed these results with a study of vacuolar sorting in gene knockout (KO) mutants. I carried out observations on whole plants containing single KO mutations for AtRMR1, AtRMR3 and AtRMR4 genes, where the fluorescent reporters GFPchi or Aleu143GFP had been introduced by crossing or by agro-infiltration. In these plants, the fluorescence pattern of vacuolar reporters showed a striking difference compared with reporter plants: they mostly appeared in punctate, peripheral structures and tended to accumulate in the corners of the cells. Taken together these results give new insights in the receptor-mediated protein vacuolar sorting: (1) AtRMR1 is important for both the CtVSD and ssVSD pathways, (2) three single KO for three AtRMR genes show similar impairment in vacuolar sorting. There are at least two possible explanations that help to define a new model for RMR function: first, RMR could be a “general purpose” receptor that discriminates as early as in the ER/Golgi the proteins to be sorted to plant vacuoles (CtVSD and ssVSD proteins), whereas receptors of the VSR family would act more specifically in a later intermediate sorting compartment; second, AtRMR action could be regulated through the formation of receptor complexes, as at least three of them seem to be needed simultaneously for a proper sorting of vacuolar reporters.
  • Publication
    Accès libre
    Functional study of vacular sorting receptors in transgenic "Arabidopsis thaliana" plants
    (2006)
    Okmeni Nguemelieu, Jeannine
    ;
    Neuhaus, Jean-Marc 
    Fusion of the Green Fluorescence protein (GFP) to propeptides of different vacuolar proteins like barley aleurain and tobacco chitinase allowed to visualize two different vacuolar compartments with different sizes in different tissues. These propeptides contain vacuolar sorting determinant (VSD) of two different types: sequence-specific (aleurain) and C-terminal (chitinase). These VSDs are supposed to be recognized by receptors such as VSRs and RMRs. VSRs are supposed to mediate protein sorting to lytic vacuoles, while RMRs are supposed to mediate protein sorting to storage vacuoles. Partial cDNA sequences for these vacuolar sorting receptors were cloned into a geminivirus silencing vector, and introduced by biolistics into transgenic Arabidopsis plants expressing either Aleu-GFP or GFP-chi to visualize effects of gene silencing. The inactivation of the subfamily AtVSR3 in Aleu-GFP transgenic plants caused the absence of the GFP in the large central vacuole in epidermal cells (which are lytic vacuoles) of rosette leaves, while GFP appeared in small compartments which can be ER or Prevacuolar compartments (PVC). Silencing of subfamilies AtVSR 1and 2 did not affect strongly GFP distribution in cells. Seeds from these plants were not able to germinate, and scanning electron micrographs showed that seed coat cells were no more hexagonal and miss their columella compared to Wild type seeds. Unexpectedly, silencing of RMRs in Aleu-GFP plants lead to the secretion of GFP from mesophyll cells. In GFP-chi plants, RMRs silencing also lead to the secretion of the GFP into the extracellular space in mesophyll cells .In these plants, silencing of the VSR subfamilies did not affect the GFP fluorescent in epidermal cell vacuoles. Therefore we confirmed that VSRs and specially the subfamily 3 is the best candidate for sorting of proteins with sequence- specific VSDs in leaves while RMRs seem to be involved in the sorting in both pathways. Also interesting is the used of reverse genetic to study RMRs. This technic was used because of symptoms obtained with germinivirus. Using in situ hybridization, I have detected VSRs receptors in leaves and in roots of Arabiopsis thaliana plants. These results showed that AtVSR 1 and 5 mRNA were the most transcribed in leaves and in root. Finally, it seems that direct interaction between VSRs and RMRs is necessary to sort proteins to lytic vacuoles.
  • Publication
    Accès libre
    Evaluation of a yeast system for studying the function of plant vacuolar sorting receptors (VSRs)
    (2005)
    Hodel Hernandez, Doramys
    ;
    Neuhaus, Jean-Marc 
    The yeast S.cerevisiae is not an efficient tool for in vivo studies of plant vacuolar sorting receptors Plant and yeast vacuoles, the equivalents of mammalian lysosomes, are acidic compartments involved in hydrolytic functions. They are also essential for metabolite storage and for maintaining cytosolic ion and pH homeostasis (Klionsky and Emr, 1990; Wink, 1993). The vacuoles mainly receive proteins and lipids from the biosynthetic and endocytic vesicular transport pathways. Newly synthesized vacuolar proteins transit through the early compartments of the secretory pathway and are actively sorted away from secreted proteins in the trans-Golgi network (TGN) before being delivered via prevacuolar compartments (PVC) to the vacuoles. In contrast to yeast, the plant vacuolar sorting machinery is extremely complex, since some plant cells may have up to three functionally distinct vacuoles: the lytic vacuole, the storage vacuole (Hoh et al., 1995; Paris et al., 1996) and the neutral vacuole (Di Sansebastiano et al, 1998). Transport analysis of soluble vacuolar proteins in plants identified three classes of vacuolar sorting determinants (VSDs), which likely interact with specific vacuolar sorting receptors (VSR). It is assumed that this interaction allows the VSRs to direct their specific ligands to the right vacuole. Indeed, protein transport to the lytic vacuole has been shown to depend on sequence-specific VSDs (ssVSD) and is mediated by clathrin-coated vesicles (CCVs). Transport to the neutral and (seed) storage vacuoles requires the C-terminal VSD (ctVSD) and the structural type VSD (psVSD), respectively (reviewed in Neuhaus and Rogers, 1998). VSRPS-1 (previously named BP-80) is the first vacuolar sorting receptor identified in plants. VSRPS-1 was originally isolated from pea CCVs by its ability to bind to the ssVSD from barley proaleurain in a pH-sensitive manner in an in vitro assay (Kirsch et al., 1994). Since then, several VSRs were cloned from different plant species (Ahmed et al., 1997; Paris et al., 1997; Shimada et al, 1997) and seven homologues (AtVSR1, 2, 2', 3-6) were identified in A. thaliana (Laval et al., 1999, reviewed in Hadlington and Denecke, 2000). The existence of several homologues in one plant species suggests that these VSRs could have different ligand specificities, and might therefore be involved in the different plant vacuolar pathways and/or function at different stages of plant development (Paris and Neuhaus, 2002). Immunogold electron microscopy showed that both pea and A. thaliana VSRs are predominantly localized in the PVCs as well as in the TGN (Paris et al., 1997; Sanderfoot et al., 1998; Hinz et al., 1999; Li et al., 2002). VSRs also partially colocalized with AtPep12p, an homologue of a yeast t-SNARE that resides on PVCs, in A. thaliana roots (da Silva Conceicao et al., 1997; Sanderfoot et al., 1998) as well as in tomato and tobacco cells (Li et al., 2002). In addition, several proteins, such as t-SNARE AtVAM3p (Sato et al., 1997) and the Sec1p-homologue AtVPS45p (Bassham and Raikhel, 1998) that are involved in vesicular transport to the lytic vacuole have been identified by yeast complementation assays. All these findings have suggested that VSRs travelling through the PVC could function like their yeast counterpart Vps10p, which mediates the transport from the TGN to the PVC of several soluble vacuolar hydrolases such as carboxypeptidase Y (CPY, (Johnson et al., 1987)), proteinase A (Klionsky and Emr, 1998) and several misfolded proteins (Hong et al., 1996). This similarity had been further corroborated by our previous work showing that expression of VSRPS-1 in yeast cells leads to an efficient transport to the vacuole of a GFP fused to the petunia aleurain VSD (Humair et al., 2001). This result demonstrated that plant VSRPS-1 is functional in yeast cells and is capable of interacting, like Vps10p, with the yeast trafficking machinery. In the present study, we further investigated the trafficking of plant VSRs in yeast to determine the ligand specificities of the A. thaliana VSR family. We show that the five tested AtVSRs fail to redirect either aleu-GFP or GFP-Chi to the yeast vacuole. Surprisingly, we were also unable to detect a significant accumulation of aleu-GFP in the vacuole in the presence of VSRPS-1, in contrast to our previous results. Further investigation clearly demonstrated that VSRPS-1 is in fact rapidly degraded and does not reach the PVC in ?vps10 cells. We conclude that plant VSRs do not properly traffic in yeast cells, and therefore, that the yeast trafficking machinery is not a suitable system to study plant vacuolar protein interactions.