The Erv14 protein works as a cargo receptor in the COPII vesicles. Many proteins, including Na+, K+/H+ antiporter Nha1, which participates in the maintenance of cell alkali-metal-cation homeostasis, need Erv14 for their trafficking from the ER. When Erv14 is missing, the Nha1 antiporter is partially retained in the ER and its overall transport activity is affected. Although Erv14 interacts with the antiporter through Nha1's transmembrane domains, a shortened version of Nha1 lacking its long hydrophilic C-terminus does not require Erv14 for its efficient trafficking to plasma membrane. This thesis contributes to the understanding of the role of the Erv14 protein in the maintenance of alkali-metal-cation homeostasis in S. cerevisiae cells. Two S. cerevisiae strains lacking ERV14 gene were prepared and the effect of this deletion on the growth and salt tolerance of cells was studied. Using heterologous expression of NHA antiporters from various yeast species with variable lengths of their hydrophilic C-termini we studied localisation and function of these antiporters in S. cerevisiae cells in the presence and in the absence of Erv14 protein. Our results suggested that the length of the antiporter's C-terminus might play a role in its requirement of Erv14 presence for its trafficking through the...

IST2 is known as a gene encoding in the model yeast Saccharomyces cerevisiae a membrane protein, that is studied thanks to a unique way of biogenesis and trafficking that apparently does not use classical secretory pathway. Although the gene was named more than ten years ago according to the phenotype of cells with its deletion (Increased Sodium Tolerance), the role of this protein in cell tolerance to toxic sodium has not been elucidated. Our searches in databases revealed that similar proteins are encoded in the genomes of other species of yeast, but none of them has been studied so far. In this work, four new strains lacking IST2 have been constructed in the genetic backgrounds differing by the presence of genes encoding transport systems for accumulation of potassium (Trk1, Trk2), for export of surplus potassium cations (Tok1, Ena1-5, Nha1) and for export of toxic cations lithium and sodium (Ena1-5, Nha1). Plasmid carrying the gene coding IST2 sequence has also been conctructed. The effect of IST2 deletion in different genetic backgrounds was studied by phenotypic tests on solid and liquid media. It was found that IST2 probably does not play a role in osmotolerance in general (absence of the phenotype of IST2 deletion on high concentrations of KCl), but its presence affects ability of the cells...

3 Abstract Yeast Saccharomyces cerevisiae belongs to important models for alkali-metal-cation homeostasis research. As other cells, certain intracellular content of K+ is necessary for S. cerevisiae, but Na+ or other alkali metal cations (Li+ , Rb+ ) are toxic for yeast cells. Uniporters Trk1 and Trk2 are responsible for K+ accumulation, while efflux of Na+ , Li+ , Rb+ and K+ is ensured by Ena ATPases, Na+ (K+ )/H+ antiporter Nha1 and K+ specific channel Tok1. Several regulators of K+ (Na+ ) transporters are already known, but reciprocal regulation between transporters and overall picture of the maintenance of alkali-metal-cation homeostasis is still unclear. In this work, K+ circulation (simultaneous uptake and export of K+ ) was shown to be important in alkali-metal-cation homeostasis maintenance. K+ circulation is maintained using reciprocal regulation and interactions between K+ exporters and importers. Though obtained results showed that the alkali-metal-cation homeostasis and associated physiological parameters (e.g. membrane potential, cell size, salt sensitivity) are strain specific, Nha1p was verified to be important for cell survival in ever-changing natural environment. Furthermore, two novel positive regulators of Nha1p activity were found, 14-3-3 proteins and Cka1 kinase. 14-3-3 proteins...

Die toxische Wirkung von Kationen ist verantwortlich für eine Reihe biologischer und pathologischer Erscheinungen. Zu den übergreifenden Zielen des Gesamtvorhabens wurden als wissenschaftliche Arbeiten i) die Analyse, graphische Darstellung und darauf basierende Gewichtung spezifischer genomischer Promotor-Regionen, ii) die Verarbeitung, Auswertung und genomweite Analyse von Mikro-Array Experimenten über die Auswirkung verschiedener Schwermetalle auf S. cerevisiae, iii) Mitarbeit an einer Simulations-Umgebung mit Modulen zur Digitalisierung, Präsentation, Analyse und mathematischer Modellierung der räumlichen Verteilung biologisch relevanter Moleküle sowie iv) ein Ansatz zur Modellierung der Kationen-Homöostase unter Verwendung der Theorie der Nichtgleichgewichts Thermodynamik beigetragen. Im Vordergrund der bioinformatorischen Arbeiten stand dabei der iterative Prozess, in dem verfügbare experimentelle Ergebnisse in aussagefähige Modelle oder Anwendungen übertragen wurden und die Resultate der Modellierung oder Vorhersage wiederum in neue entsprechende Experimente umgesetzt wurden. Die Anwendungsumgebung zur Promotoranalyse sowie die Simulationsumgebung zur räumlichen Verteilung biologisch relevanter Moleküle wie zum Beispiel markierte Signalmoleküle wurde bereits veröffentlicht und erfolgreich eingesetzt. Die Ergebnisse der genomweiten Analyse liefern Erkenntnisse über die individuellen Mechanismen und Strategien der Hefe auf verschiedene Metallionen in toxischer Konzentration zu reagieren. Der theoretische biophysikalisch-thermodynamische Ansatz liefert ein fundamentales Modell der Kationen-Homöostase der zahlenmäßig bedeutendsten Kationen: Kalium, Natrium und Protonen. Das Modell wurde an experimentellen Daten getest und konnte diese reproduzieren. Entsprechende Perspektiven für die Weiterentwicklung des Modells werden diskutiert. / Cationic toxicity is relevant for a number of qualitatively different biological and medical phenomena such as cationic surfactants, salt and heavy metal stress in plants and a number of pathological conditions which share similar critical metabolic processes (i.e. protein aggregation and oxidative stress). In line with the overall project goals the scientific work contributed to i) the analysis, graphical presentation and the respective assessment of specific genomic promoter-regions, ii) the conversion, evaluation and genome wide analysis of micro-array experiments on the effects of exposition of S. cerevisiae to heavy metals, iii) a simulation environment comprising modules for digitalization, presentation, analysis and mathematical modeling of the spatio-temporal distribution of biologically relevant molecules, and iv) a cation homeostasis modeling approach based on the non-linear thermodynamics theory. The bioinformatical work focused on an iterative process in which available experimental results were transferred into meaningful models or applications and results of modeling or prediction into corresponding new experimental designs. The software for the promoter-analysis and the simulation environment for integration of spatio-temporal distribution of biologically relevant molecules like labeled signal molecules have already been published and successfully implemented.The results of the genome-wide analysis - based on experiments of a project partner - provide insights in the individual mechanisms and strategies of the yeast cell upon exposition to various (heavy) metals in toxic concentrations. The theoretical biophysical-thermodynamic approach provides a fundamental model of cation homeostasis in S.cerevisiae of the major cations: potassium, sodium and protons. The model - confronted with experimental data - is capable to reproduce the observed uptake rates to a reasonable degree. Perspectives for further development of the model are discussed.