New applications of pHluorin--measuring intracellular pH of prototrophic yeasts and determining changes in the buffering capacity of strains with affected potassium homeostasis.
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 mM/pH for wild-type yeast and moderately decreased in mutants with affected potassium transport.
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. lydie@biomed.cas.cz
Lydie
L
Maresová
Barbora
B
Hosková
Eva
E
Urbánková
Roman
R
Chaloupka
Hana
H
Sychrová
D002021:Buffers; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics; D006706:Homeostasis; D006863:Hydrogen-Ion Concentration; D010957:Plasmids; D011188:Potassium / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D013045:Species Specificity
D002021:Buffers; C485187:PHluorin; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D011188:Potassium