title: A novel method for assessment of local pH in periplasmic space and of cell surface potential in yeast.
abstract: Yeast cells exhibit a negative surface potential due to negative charges at the cell membrane surface. Consequently, local concentrations of cations at the periplasmic membrane surface may be significantly increased compared to their bulk environment. However, in cell suspensions only bulk concentrations of cations can be measured directly. Here we present a novel method enabling the assessment of local pH at the periplasmic membrane surface which can be directly related to the underlying cell surface potential. In this proof of concept study using Saccharomyces cerevisiae cells with episomally expressed pH reporter, pHluorin, intracellular acidification induced by the addition of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) was measured using synchronously scanned fluorescence spectroscopy (SSF). The analysis of titration curves revealed that the pH at the periplasmic surface of S. cerevisiae cells was about two units lower than the pH of bulk medium. This pH difference was significantly decreased by increasing the ionic strength of the bulk medium. The cell surface potential was estimated to amount to -130 mV. Comparable results were obtained also with another protonophore, pentachlorophenol (PCP).
authors.affiliation: Faculty of Mathematics and Physics, Charles University, Ke Karlovu 3, 12116, Prague, Czech Republic. plasek@karlov.mff.cuni.cz.
authors.forename: Jaromír
authors.initials: J
authors.lastname: Plášek
authors.affiliation: Faculty of Mathematics and Physics, Charles University, Ke Karlovu 3, 12116, Prague, Czech Republic.
authors.forename: David
authors.initials: D
authors.lastname: Babuka
authors.affiliation: Faculty of Mathematics and Physics, Charles University, Ke Karlovu 3, 12116, Prague, Czech Republic.
authors.forename: Dana
authors.initials: D
authors.lastname: Gášková
authors.affiliation: Faculty of Mathematics and Physics, Charles University, Ke Karlovu 3, 12116, Prague, Czech Republic.
authors.forename: Iva
authors.initials: I
authors.lastname: Jančíková
authors.affiliation: Faculty of Mathematics and Physics, Charles University, Ke Karlovu 3, 12116, Prague, Czech Republic.
authors.forename: Jakub
authors.initials: J
authors.lastname: Zahumenský
authors.affiliation: Institute of Cellular and Molecular Botany, University of Bonn, Kirschallee 1, 53115, Bonn, Germany.
authors.forename: Milan
authors.initials: M
authors.lastname: Hoefer
mesh_terms: D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D049452:Green Fluorescent Proteins; D006863:Hydrogen-Ion Concentration; D008564:Membrane Potentials; D008722:Methods; D019897:Periplasm / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000166:cytology; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: C485187:PHluorin; D049452:Green Fluorescent Proteins; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone
keywords: Cell surface potential; Cytosolic pH; Periplasmic pH; Saccharomyces cerevisiae; Yeast; pHluorin