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The role of transport systems for potassium cations in the physiology of Saccharomyces cerevisiae yeast
Marešová, Lydie ; Sychrová, Hana (advisor) ; Pácha, Jiří (referee) ; Janderová, Blanka (referee)
[n this work, we present the characteristics of kha 1 6,. strains in the background of various multiple mutations in genes encoding alkali-metal-cation transporters. Two main phenotype manifestations of the khal deletion were growth defect on high externa\ pH and hygromycin sensitivity. The correlation between these phenotypes and the kha 1 deletion was confirmed by plasmid complementation. Fluorescence microscopy of GFP-tagged Kha 1 p showed that this antiporter is localized preferentially intracellu\arly (in contrast to the plasma-membrane Na+/H+ antiporter Nha 1 p). Based on these findings, Kha 1 p is probably not localized in plasma membrane and do es not mediate efflux of alkali metal cations from cells (as published before in RammÍrez el aJ., 1998), but is important for the regulation of intracelular cation homeostasis and optima\ pH contro l, similarly as the Nhx l p. The khal deletion phenotypes were complemented by heterologous expression of a plant antiporter AtChx 17, showing that the proteins AfChx 17 and SeKhal could have similar ťunction and that S cerevisiae khal deletion mutants could serve for heterologous expression and characterization of some plant transporters in yeast, especially those localized intracellularly. We also showed that the presence of the Tokl channel strongly infl uences...
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Stoichiometry of transport mediated by bacterial membrane transport protein MntH of E. coli
Doležalová, Iva ; Chaloupka, Roman (advisor) ; Marešová, Lydie (referee)
The Nramp family (Natural Resistance-Associated Macrophage Protein) includes secondary active transport proteins transporting divalent metal ions together with protons into a cell. Divalent metal ions play a major role in the cell metabolism and participate in many intracellular processes. We have studied bacterial Nramp ortholog - MntH (Proton-dependent Manganese Transporter), its transport stoichiometry and other functionally important characteristics. We have found that the external pH affected the transport stoichiometry. The functional differences between the wild type and the protein with mutation N401G appears only in lower external pH. Moreover, uncoupled proton flux mediated by MntH under specific experimental conditions is inhibited by calcium, whereas coupled proton flux increases after the addition of calcium ions. This can be connected to the fact that calcium is also transported by MntH. Surprisingly, the transport stoichiometry of neither the wild type nor the protein with mutation N401G is affected by the addition of calcium or the change of the external medium.
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The role of transport systems for potassium cations in the physiology of Saccharomyces cerevisiae yeast
Marešová, Lydie ; Sychrová, Hana (advisor) ; Pácha, Jiří (referee) ; Janderová, Blanka (referee)
[n this work, we present the characteristics of kha 1 6,. strains in the background of various multiple mutations in genes encoding alkali-metal-cation transporters. Two main phenotype manifestations of the khal deletion were growth defect on high externa\ pH and hygromycin sensitivity. The correlation between these phenotypes and the kha 1 deletion was confirmed by plasmid complementation. Fluorescence microscopy of GFP-tagged Kha 1 p showed that this antiporter is localized preferentially intracellu\arly (in contrast to the plasma-membrane Na+/H+ antiporter Nha 1 p). Based on these findings, Kha 1 p is probably not localized in plasma membrane and do es not mediate efflux of alkali metal cations from cells (as published before in RammÍrez el aJ., 1998), but is important for the regulation of intracelular cation homeostasis and optima\ pH contro l, similarly as the Nhx l p. The khal deletion phenotypes were complemented by heterologous expression of a plant antiporter AtChx 17, showing that the proteins AfChx 17 and SeKhal could have similar ťunction and that S cerevisiae khal deletion mutants could serve for heterologous expression and characterization of some plant transporters in yeast, especially those localized intracellularly. We also showed that the presence of the Tokl channel strongly infl uences...
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