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Preparation of organelle markers in the yeast Saccharomyces cerevisiae
Dekha, Daniella ; Vopálenská, Irena (advisor) ; Papoušková, Klára (referee)
The aim of the work was to prepare a set of strains with fluorescently labelled organelles. The organism used is a haploid strain with a high frequency of homologous recombination Saccharomyces cerevisiae BY4742. The yeast S. cerevisiae is a unicellular organism and is one of the most studied experimental living systems. Its generation time tends to be 1.5-2 hours, which allows rapid monitoring of culture development on both solid and liquid media. For each organelle, specific proteins were selected, namely proteins localized on the vacuole membrane (Vph1, Vtc3), peroxisomes (Pex3), nucleus (Nvj1), endoplasmic reticulum (Sec63), mitochondria (Cox4) and in the vacuole lumen (Prc1). Selected proteins were labeled by attaching fluorescent proteins (yEGFP, yomCherry or yomRuby2) to their C-termini. The prepared strains were analyzed in liquid and solid media. Growth curves were constructed, fluorescence at different growth stages was compared and the effect of two different carbon sources on the expression of selected genes was determined. A total of 12 strains with fluorescent organelle markers for the vacuolar membrane, peroxisomes, nucleus, endoplasmic reticulum, mitochondria, and vacuolar lumen and 5 strains with a pair of fluorescent markers for vacuolar membrane and selected other organelle...
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Role of the Erv14 protein in the maintenance of alkali-metal-cation homeostasis in the yeast Saccharomyces cerevisiae
Hrášková, Michaela ; Papoušková, Klára (advisor) ; Vopálenská, Irena (referee)
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...
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The role of plasma membrane Na+/H+ antiporters in the physiology of yeast cells
Papoušková, Klára ; Sychrová, Hana (advisor) ; Hašek, Jiří (referee) ; Flachs, Pavel (referee)
) r Nhal-type antiporterswith differentsubstratespeciÍicity(or affinityto individualsubstrates) andfunctions.One of themis involvedin thedetoxificationof cells andtheothercouldplay an importantrole in the maintenanceof stableintracellularpotassiumcontent,cell volume andpH of thecýoplasmthanlsto its abilitytotransportK+. At this time, no yeast speciespossessingsolely a Na*/fI*antiporterwith narrow substratespecificity (for Na* and Li) is known;therefore,the physiologicalrole of Nhal- type antiportersseemsto be complexwith a participationboth in the eliminationof toxic cationsfromcells andin themaintenanceof stableintracellularK* concentration,cell volume andcýoplasmic pH. 4.Summary Themainresultsof thethesíscanbesummarizedas: o The studiesof sequencesof yeastalkali metalcation/Iťantiporters(Nhal, Nhxl andKhal families)in silico showedthatthe structureof all threegroupsof theseproteinsprobably contains 12 transmembranesegments,we found some conservedamino acid residuesor motifslikely to be importantforproperfunctioningof antiporters,andourphylogeneticstudy revealedthatNhxl proteinsare relatedto both plant and mammalianNa*/Iť antiporters, Khal antiportersare close to thebacterialrelativesandNhal-type proteinsform a soparate goup so far characteizedonly in yeastsor fungi. o The D. hanseniiNHAI geneencodesa...
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The role of Erv14 protein in cells of the yeast Saccharomyces cerevisiae
Hrášková, Michaela ; Papoušková, Klára (advisor) ; Macůrková, Marie (referee)
The aim of this bachelor thesis is to summarize all published findings about the role of the Erv14 protein in the cells of Saccharomyces cerevisiae. The Erv14 protein works as a cargo receptor in the endoplasmic reticulum (ER), allowing an incorporation of selected trans-membrane proteins to the COPII vesicles, by which their transport to the Golgi apparatus is allowed. Many proteins were identified in the cells of S. cerevisiae, their transport from the ER depends on the presence of the Erv14 protein. In cells lacking Erv14, these proteins are retained in the ER and are not able to fulfil their functions. One of those proteins is for example Axl2, necessary for axial budding, Sma2, which ensures the standard formation of the prospore membrane during sporulation, the catalytic subunit of the chitin synthase III, Chs3, important for chitin synthesis, and plasma membrane Na+ , K+ /H+ antiporter Nha1 participating in the maintenance of cell alkali-metal-cation homeostasis. The importance of the Erv14 protein's function is proven by the fact that its homologs were also found in other organisms, such as animals, including human, as well as in plants. Keywords: Erv14, cargo receptor, COPII vesicles, transport, Saccharomyces cerevisiae
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The study of mutual interaction between pathogenic yeasts of the genus Candida and bacterium Pseudomonas aeruginosa during cocultivation
Mynářová, Lenka ; Hodek, Petr (advisor) ; Papoušková, Klára (referee)
The genus Candida includes several opportunistically pathogenic species which are common causative agents of the yeast infections in humans. Although current medical research is focused mostly on cancer, AIDS or Alzheimer disease, the problem of systemic candidiases cannot be neglected. These infections represent a real threat to the immunocompromissed patients, they are connected with a high mortality rate and expensive medication with poor prognosis. Pseudomonas aeruginosa could be an inspiration in a way of how to eliminate the pathogenic yeasts. The bacterium can inhibit growth of the most common yeast species of the genus Candida, C. albicans. This effect is based on production of toxic substances by the bacterium and on interaction of the bacterium with the C. albicans cell wall, which leads to the lysis of the yeast cells and which is not fully understood. Nevertheless, coexistence of these microorganisms is also possible and their relationship is affected by various factors. Knowledge of these inter- microbial interactions was obtained from studies of diseases and pathologies, during which C. albicans + P. aeruginosa coinfections occur. In this thesis I studied mechanisms of interaction between pathogenic yeast C. albicans and bacterium P. aeruginosa by a) C. albicans gene expression...
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