New applications of pHluorin--measuring intracellular pH of prototrophic yeasts and determining changes in the buffering capacity of strains with affected potassium homeostasis.
2010
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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