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New applications of pHluorin—measuring intracellular pH of prototrophic yeasts and determining changes in the buffering capacity of strains with affected potassium homeostasis
Author(s) -
Marešová Lydie,
Hošková Barbora,
Urbánková Eva,
Chaloupka Roman,
Sychrová Hana
Publication year - 2010
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/yea.1755
Subject(s) - auxotrophy , biology , yeast , intracellular , mutant , potassium , biochemistry , intracellular ph , saccharomyces cerevisiae , cytosol , gene , enzyme , chemistry , organic chemistry
pHluorin is a pH‐sensitive variant of green fluorescent protein for measuring intracellular pH (pH in ) 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 pH in for three prototrophic wild‐types. The new construct was also used to determine the pH in in strains differing in the activity of the plasma membrane Pma1 H + ‐ATPase and the influence of glucose on pH in . 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 m M /pH for wild‐type yeast and moderately decreased in mutants with affected potassium transport. Copyright © 2010 John Wiley & Sons, Ltd.