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

Citace poskytuje Crossref.org

The Hrk1 kinase is a determinant of acetic acid tolerance in yeast by modulating H+ and K+ homeostasis

The Toxic Effects of Ppz1 Overexpression Involve Nha1-Mediated Deregulation of K+ and H+ Homeostasis

Genomewide Elucidation of Drug Resistance Mechanisms for Systemically Used Antifungal Drugs Amphotericin B, Caspofungin, and Voriconazole in the Budding Yeast

Trk1, the sole potassium-specific transporter in Candida glabrata, contributes to the proper functioning of various cell processes

Four Saccharomyces species differ in their tolerance to various stresses though they have similar basic physiological parameters

H+ translocation by weak acid uncouplers is independent of H+ electrochemical gradient

A novel method for assessment of local pH in periplasmic space and of cell surface potential in yeast

Potassium Uptake Mediated by Trk1 Is Crucial for Candida glabrata Growth and Fitness

Changes in the Sterol Composition of the Plasma Membrane Affect Membrane Potential, Salt Tolerance and the Activity of Multidrug Resistance Pumps in Saccharomyces cerevisiae

Potassium supply and homeostasis in the osmotolerant non-conventional yeasts Zygosaccharomyces rouxii differ from Saccharomyces cerevisiae

In vivo determination of organellar pH using a universal wavelength-based confocal microscopy approach