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 monit oring of proton fluxes across vesicles (ACMA fluorescence quenching). Plasma membrane vesicles used for this study were obtained by a purifi cation/reconstitution protocol based on differential and discontinuous sucrose gradient centrifugations followed by an octylglucoside diluti on/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 efffux, is mainly driven by the K+ transmembrane gradient and can function even if the plasma membrane H+-ATPase is not active. Using t he anionic oxonol VI and the cationic DISC2(5) probes, it was shown th at a membrane potential is not created during K+ fluxes. Such a dye re sponse argues for the presence of a K+/H+ exchange system in S. cerevi siae plasma membrane and established the non-electrogenic character of the transport. The maximal rate of exchange is obtained at pH 6.8. Th is reversible transport system presents a high selectivity for K+ amon g other monovalent cations and a higher affinity for the K+ influx int o the vesicles (exit from cells). The possible role of this K+/H+ exch ange system in regulation of internal potassium concentration in S. ce revisiae is discussed.