Evidence for a selective and electroneutral K+/H(+)-exchange in Saccharomyces cerevisiae using plasma membrane vesicles.
The existence of a K+/H+ transport system in plasma membrane vesicles from Saccharomyces cerevisiae is demonstrated using fluorimetric monitoring of proton fluxes across vesicles (ACMA fluorescence quenching). Plasma membrane vesicles used for this study were obtained by a purification/reconstitution protocol based on differential and discontinuous sucrose gradient centrifugations followed by an octylglucoside dilution/gel filtration procedure. This method produces a high percentage of tightly-sealed inside-out plasma membrane vesicles. In these vesicles, the K+/H+ transport system, which is able to catalyse both K+ influx and efflux, is mainly driven by the K+ transmembrane gradient and can function even if the plasma membrane H(+)-ATPase is not active. Using the anionic oxonol VI and the cationic DISC2(5) probes, it was shown that a membrane potential is not created during K+ fluxes. Such a dye response argues for the presence of a K+/H+ exchange system in S. cerevisiae plasma membrane and established the non-electrogenic character of the transport. The maximal rate of exchange is obtained at pH 6.8. This reversible transport system presents a high selectivity for K+ among other monovalent cations and a higher affinity for the K+ influx into the vesicles (exit from cells). The possible role of this K+/H+ exchange system in regulation of internal potassium concentration in S. cerevisiae is discussed.
Camarasa
C
C
Institut des Produits de la Vigne, Institut de la Recherche Agronomique, Montpellier, France.
Prieto
S
S
Ros
R
R
Salmon
J M
JM
Barre
P
P
D001693:Biological Transport, Active; D002462:Cell Membrane / Q000201:enzymology*; D002463:Cell Membrane Permeability; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D018699:Coated Vesicles / Q000201:enzymology*; D004225:Dithiazanine / Q000494:pharmacology; D003576:Electron Transport Complex IV / Q000032:analysis; D017506:H(+)-K(+)-Exchanging ATPase / Q000302:isolation & purification* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007555:Isoxazoles / Q000494:pharmacology; D008358:Mannose / Q000032:analysis; D008361:Mannosidases / Q000032:analysis; D008564:Membrane Potentials; D011188:Potassium / Q000493:pharmacokinetics; D011522:Protons; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D043323:alpha-Mannosidase
D007555:Isoxazoles; D011522:Protons; D004225:Dithiazanine; C031184:oxonol VI; D003576:Electron Transport Complex IV; D008361:Mannosidases; D043323:alpha-Mannosidase; D017506:H(+)-K(+)-Exchanging ATPase; D008358:Mannose; D011188:Potassium