Demonstration of high-affinity Mn2+ uptake in Saccharomyces cerevisiae: specificity and kinetics

Abstract

The existence of multiple transport systems for Mn2+ in Saccharomyces cerevisiae has been demonstrated in this study. Mn2+ (supplied as MnCI2) was accumulated by S. cerevisiae at all Mn2+ concentrations examined (25 nM-1 mM) but a log-log plot of uptake rates and total amounts accumulated revealed the existence of at least two Mn2+ concentration-dependent transport systems. Over a low Mn2+ concentration range (25-1000 nM), high-affinity Mn2+ uptake occurred with a K m value of 0.3 μM, while transformation of kinetic data obtained over the concentration range 5-200 μM revealed another system with a K m of 62 μM. Meaningful kinetic analyses were not possible at higher Mn2+ concentrations because of toxicity: only about 30% of cells remained viable after 30 min incubation with 1000 μM MnCI2. Release of K+ accompanied Mn2+ accumulation and this increased with increasing Mn2+ concentration. However, even in non-toxic Mn2+ concentrations, the ratio of Mn2+ uptake to K+ release greatly exceeded electroneutral stoichiometric exchange. In 50 μM MnCI2, the ratio was 1: 123 and this increased to 1:2670 in 1000 μM MnCI2, a toxic concentration. External Mg2+ was found to decrease Mn2+ accumulation at all concentrations examined, but to differing extents. Over the low Mn2+ concentration range (5-200 μM), Mg2+ competitively inhibited Mn2+ uptake with a half-maximal inhibitory concentration, K i, of 5.5 μM Mg2+. However, even in the presence of a 50-fold excess of Mg2+, inhibition of Mn2+ uptake was of the order of 72% and it appears that the cellular requirement for Mn2+ could be maintained even in the presence of such a large excess of Mg2+. Over the high Mn2+ concentration range (5-200 μM), the K i for Mg2+ was 25.2 μM. At low Mn2+ concentrations, Zn2+ and Co2+, but not Cd2+, inhibited Mn2+ uptake, which indicated that the high-affinity Mn2+ uptake system was of low specificity, while at higher Mn2+ concentrations, where the lower-affinity Mn2+ transport system operated, inhibition was less marked. However, competition studies with potentially toxic metal cations were complicated due to toxic effects, particularly noticeable at 50 μM Co2+ and Cd2+.