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