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Neuhaus, Jean-Marc

Nom
Neuhaus, Jean-Marc
Affiliation principale
Fonction
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
    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.