Crosstalk of signaling pathways for cell wall integrity, cytokinesis and glucose metabolism in the milk yeast Kluyveromyces lactis

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Title: Crosstalk of signaling pathways for cell wall integrity, cytokinesis and glucose metabolism in the milk yeast Kluyveromyces lactis
Authors: Rippert, Dorthe
Thesis advisor: Prof. Dr. Jürgen J. Heinisch
Thesis referee: Prof. Dr. Hans Merzendorfer
Abstract: This thesis was dedicated to the investigation of the interplay between signaling pathways governing cell wall biosynthesis, central carbohydrate metabolism and cytokinesis in two different yeast species. The first part of the thesis addressed cell wall biosynthesis, which forms an essential structure and determines both shape and integrity of fungal cells and hyphae. It also serves as a first barrier against changing and adverse environmental conditions. Cell wall synthesis and remodeling is primarily mediated by the cell wall integrity pathway in both the budding yeast Saccharomyces cerevisiae and the milk yeast Kluyveromyces lactis. The latter is a Crabtree-negative yeast with a similar life cycle to that of S. cerevisiae. Yet, K. lactis did not undergo a whole genome duplication, presenting a lower degree of genetic redundancy, which often avoids the need for multiple gene deletions. In this work the SNF1 kinase complex was identified as playing a role in cell wall synthesis. It belongs to the family of AMP-activated protein kinases (AMPKs), whose primary function is thought to be the regulation of energy balance in different organisms. Mutants with defects in this complex have a thinner cell wall than wild type and are hypersensitive to cell wall stress agents such as Caspofungin, Calcofluor white and Congo red. Epistasis analyses with mutants affecting cell wall integrity signaling suggested a parallel action of CWI- and SNF1 signaling in the two yeast species. Further genetic analyses indicated a known downstream effector of the SNF1 kinase complex, the transcriptional repressor Mig1, to mediate the signaling function in cell wall synthesis, too. Further epistasis analyses indicated that the hypersensitivity of the SNF1 complex mutants to the stress agents can be suppressed by an additional defect in the upper part of glycolysis. This has been attributed to the accumulation of the glycolytic intermediates glucose-6-phosphate and fructose-6-phosphate, which serve as precursors of cell wall polysaccharides. A function of the SNF1 complex in yeast cell wall synthesis has not been described, until now. In order to study the relation to cytokinesis, i.e. the last step of cell division, first some tools to follow this process in K. lactis had to be established in the second part of the thesis. Cytokinesis is also an essential feature of life since it ensures cell proliferation. In yeast and mammalian cells the concluding abscission of the plasma membrane is initiated by the construction of an actomyosin ring (AMR), accompanied by the formation of a primary septum, and followed by the deposition of secondary septa from both mother and daughter cells. Cytokinesis is a highly coordinated process and regulation is not yet fully explained. Proteins important for cytokinesis in S. cerevisiae were identified in K. lactis by in silico analyses and then genetically investigated. In contrast to S. cerevisiae, analysis of deletion mutants showed that deletion of the gene encoding K. lactis myosin II (KlMYO1) as a key component of the AMR is viable but temperature-sensitives and therefore dispensable for cytokinesis in K. lactis under normal growth conditions. Also different from its S. cerevisiae ortholog, a Klcyk3 deletion is lethal, while inn1 deletions are not viable in either yeast species. In contrast to the other genes studied, expression of INN1 does not cross-complement between the two yeast species, which could be narrowed down to a species specificity of the C2 domain in the Inn1 protein, which activates the chitin synthase II in S. cerevisiae. Deletions lacking the K. lactis chitin synthase II gene (KlCHS2) are also not viable. Fluorescently tagged versions of all proteins show a similar spatiotemporal localization at the bud neck as their counterparts in S. cerevisiae. The second part of this work thus provides insights into the regulation of cytokinesis in K. lactis and indicates that AMR constriction and its regulation could be more important in K. lactis than in S. cerevisiae, while the overall sequence of morphological events in cytokinesis is quite similar.
Subject Keywords: Saccharomyces cerevisiae; Kluyveromyces lactis; Signalttransduktion; Zellwand; Zellteilung; Regulation
Issue Date: 4-Aug-2016
License name: Namensnennung 3.0 Unported
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Type of publication: Dissertation oder Habilitation [doctoralThesis]
Appears in Collections:FB05 - E-Dissertationen

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