La paroi de la levure Saccharomyces cerevisiae est une structure très dynamique composée de beta-glucanes, de mannanes et de chitine (polymère de N-acétylglucosamine). Elle peut s’adapter à l’état physiologique et aux changements morphologiques des cellules, ainsi qu’aux contraintes environnementales. Cette remarquable plasticité est assurée par l’intervention de différentes voies de régulation et de signalisation dont la voie CWI (Cell Wall Integrity) et la voie de la Calcineurine ou Protein Phosphatase 2B. La toxine killer K9 est une petite protéine sécrétée par la levure Hansenula mrakii. Cette toxine exerce son action létale sur les souches contrôles de S. cerevisiae mais pas sur des mutants du gène KNR4. Elle inhibe in vitro la beta-(1,3)-glucan syntase. Ce travail a dans un premier temps utilisé la Microscope à Force Atomique (AFM) et mis en évidence que la paroi de S. cerevisiae contrôle et mutant knr4 sont affectées de façon similaires par un traitement par la toxine K9. Dans un second temps, nous avons pu démontrer que la localisation cellulaire de Knr4 aux sites de croissance polarisée est nécéssaire pour l’action létale de la toxine K9 sur les cellules de S. cerevisiae.Knr4 fait partie d’une famille de protéines très conservées dans le domaine fongique, impliquées dans le contrôle de l’intégrité pariétale et la morphogenèse. Elle constitue un élément coordinateur pour la voie CWI et la voie de la Calcineurine. Notre travail a mis en évidence que la phosphorylation des résidus serine 200 et serine 203 de Knr4 joue un rôle dans ce mécanisme de coordination / The aim of my thesis was to study the fuction of Knr4 in the cell wall synthesis, morphogenesis, and related signaling pathways. The content of my thesis is mainly divided into three parts: The first part concerns our search to find out unknown partners of Knr4 and to investigate the cellular pathways required for localization of Knr4 protein. To that end, we decided to use a series of deletion mutants interrupted in genes related to morphogenesis and establishment of cellular polarity. We selected candidate genes from the Saccharomyces cerevisiae genome database (SGD, Stanford), using the keywords “Morphogenesis” and “Cell Polarity”. After selection and addition, 25 genes related to the morphogenesis and cell polarity were chosen for our Knr4 localization analysis. Through analysis of the results, we got 10 interesting mutants related to morphogenesis and polarity in which knr4 protein localization was affected: bem2Δ, pcl1Δ, pcl2Δ, rrd1Δ, spa2Δ, tpd3Δ, bem1Δ, bnI1Δ, yck1Δ and bud6Δ, and two additional mutants pph21Δ related to the tpd3Δ and cna1Δ involved in the calcinerin pathway. The second part deals with a mutational analysis of in vivo phosphorylated residues of Knr4 in the function and localization the protein, as well as in the modulation of calcineurin activity and CWI pathway. We found that S200S203 phosphorylation mutants cannot rescue viability of a double mutant bck1Δknr4Δ, while they can rescue slt2Δknr4Δ. In addition, S200S203 phosphorylation mutants behave as the absence of Knr4 towards suppression of lethality caused by an hyperactivated Mkk1 allele. Also we found that the knr4with KNR4S200AS203A mutant can results in hyperactivation of the Calcineurin pathway compared to control situation. So serin 200 and serin 203 may be involved in the cross-talking with the calcineurin pathway and CWI pathway. The third part is the study of K9 killer toxin’s strong cytocidal activity against sensitive yeast strains, including Saccharomyces cerevisiae. Treatment with this toxin results in the formation of pores at the surface of the cells, and more specifically at places where cell wall synthesis is the most active, namely at the tip of growing buds or mating projections. Yeast cells treated with K9 toxin then die by releasing cytoplasm and cellular materials from these pores. In the yeast S. cerevisiae, Knr4 protein localizes at the sites of polarized growth (bud tips, shmoo tips), which are also the sites where the toxin forms pores in the cell wall. Mutants defective in KNR4 gene are remarkably resistant to this toxin. In this study, we analyzed for the first time the biophysical effects of K9 on the yeast cell wall using Atomic Force Microscopy (AFM), a cutting edge technology that allows measuring the nanomechanical properties of living yeast cells, and their alterations by various drugs. To this end, we measured the effects of K9 toxin on the nanomechanical properties of the cell wall of S. cerevisiae wild-type cells and mutants deleted for KNR4 gene, at the short (2 h) and long term (20 h). Our results reveal an important cell wall remodeling occurring in wild-type cells already after 2 hours and only visible in knr4 mutant after 20 hours of treatment. Moreover, we investigated the role of Knr4 protein in the cells sensitivity towards the toxin. We were able to show that the presence of the N-terminal domain of Knr4 protein, which is required for its correct cellular localization at the bud tip during cell cycle, is essential for the toxin K9 wild-type sensitivity. In addition, a series of deletion mutants from the YKO collection in which the Knr4 cellular localization is also lost display a reduced sensitivity to the K9 toxin. Taken together, these results shed light on the importance of the proper localization of Knr4 protein at sites of intensive cell wall growth for the wild-type cells sensitivity to K9 killer toxin.
| Identifer | oai:union.ndltd.org:theses.fr/2015ISAT0050 |
| Date | 04 May 2015 |
| Creators | Liu, Ran |
| Contributors | Toulouse, INSA, Martin-Yken, Hélène |
| Source Sets | Dépôt national des thèses électroniques françaises |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation, Text |
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