New applications of pHluorin—measuring intracellular pH of prototrophic yeasts and determining changes in the buffering capacity of strains with affected potassium homeostasis |
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Authors: | Lydie Mare?ová Barbora Ho?ková Eva Urbánková Roman Chaloupka Hana Sychrová |
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Affiliation: | 1. Department of Membrane Transport, Institute of Physiology, Academy of Sciences of the Czech Republic, v.v.i., Videnska 1083, 142 20 Prague 4, Czech Republic;2. Charles University in Prague, Faculty of Mathematics and Physics, Institute of Physics, Ke Karlovu 5, 121 16 Prague 2, Czech Republic |
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Abstract: | pHluorin is a pH‐sensitive variant of green fluorescent protein for measuring intracellular pH (pHin) in living cells. We constructed a new pHluorin plasmid with the dominant selection marker KanMX. This plasmid allows pH measurements in cells without auxotrophic mutations and/or grown in chemically indefinite media. We observed differing values of pHin for three prototrophic wild‐types. The new construct was also used to determine the pHin in strains differing in the activity of the plasma membrane Pma1 H+‐ATPase and the influence of glucose on pHin. We describe in detail pHluorin measurements performed in a microplate reader, which require much less hands‐on time and much lower cell culture volumes compared to standard cuvettes measurements. We also utilized pHluorin in a new method of measuring the buffering capacity of yeast cell cytosol in vivo, shown to be ca. 52 mM /pH for wild‐type yeast and moderately decreased in mutants with affected potassium transport. Copyright © 2010 John Wiley & Sons, Ltd. |
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Keywords: | alkali‐metal‐cation transporters Pma1 ATPase fluorescence measurements microplate reader |
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