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.
Maresová
Lydie
L
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
Hosková
Barbora
B
Urbánková
Eva
E
Chaloupka
Roman
R
Sychrová
Hana
H
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