Proton stoichiometry of the overexpressed uracil symport of the yeast Saccharomyces cerevisiae

Abstract

Information about the molecular biology of the uracil carrier of Saccharomyces cerevisiae is now available but its properties as a symport are unexplored. We have now studied its proton stoichiometry at pH 6.5, 4.8 and 4.2, in a yeast strain overexpressing the symport and unable to metabolize uracil. After the depletion of cellular ATP, uracil uptake followed an approximately exponential time course to reach a plateau. Proton uptake was then indistinguishable from the basal rate shown in controls without added uracil. It was concluded that more than 87% of the uracil flux through the system was coupled to symported protons. Because the basal rate was a significant fraction of the total rate of proton uptake at pH 4.2 or 4.8, the ratio of the proton and uracil flows could not be defined uniquely during the initial near-linear phase of uptake. However, the average rate of proton uptake during increasing time intervals up to 120 s was shown to be a linear function of the corresponding average rate of uracil uptake. This relationship, defining approx. 90% of the eventual uracil uptake, was used to deduce the mean proton stoichiometry as approx. 3 at pH 4.2, falling to near 2 at pH 6.5. The rate of uracil uptake increased 4-fold when the negative proton gradient (ΔµH) acting across the plasma membrane increased from 60 to 200 mV. The rate fell markedly after depolarization by KCl. The maximum uracil gradient (ΔµS) was less affected by depolarization. The ratio ΔµS/ΔµH fell from approx. 2 to near 1 as ΔµH increased in the above range. In contrast with the starved yeast, uracil accumulation during energy metabolism followed a pump-leak model.