Kinetics and pH-dependence of glycine-proton symport in Saccharomyces cerevisiae

Abstract Interactions between intercellular pH (pH i ) and H + -coupled transmembrane transport of glycine have been studied by means of 31 P-NMR, using both aerobic and ‘energy starved’ cells of the yeast Saccharomyces cerevisiae . The general features of glycine transport in the yeast strain used (NCYC 239) are similar to those already reported for Saccharomyces carlsbergensis and S. cerevisiae , there being two kinetically distinct glycine uptake systems, with pH-independent K 1 2 values near 14 and 0.4 mM, respectively, but pH-dependent maximal velocities. Glycine transport itself has no measurable effect on pH i in aerobic cells, and only a marginal effect in energy-starved cells, but changes of pH i , imposed by extracellular addition of butyric acid, strongly influence glycine transport. Indeed, the dependence of glycine influx (in energy-starved cells) upon cytoplasmic H + concentration appears to be third order, showing Hill slopes of 2.7–3.0. A crucial kinetic role for cytoplasmic pH in glycine transport is further indicated by a proportionality between the decline of flux and the decline of pH i produced by various metabolic inhibitors and uncouplers. Extracellular pH (pH o , by contrast, has only a weak effect on glycine influx, showing a Hill slope of 0.5. The major observations can be accommodated by a simple cyclic carrier scheme, in which 2 or more protons are transported along with glycine, but only one extracellular proton binding site dissociates in the testing range, with a p K near 5.5. The model requires a finite membrane potential, which must be somewhat sensitive to both pH i and pH o , and accomodates the discrepancy between measured net proton flux (one per glycine) and the kinetically required proton flux (two or more per glycine) by shunting through other proton-conducting pathways in the yeast membrane.