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

Department of Biological Sciences, University of Dundee, Dundee DD1 4HN, UK

²Author for correspondence: Geoffrey M. Gadd. Tel: +44 1382 344266. Fax: +44 1382 322318. e-mail: g.m.gadd@dundee.ac.uk

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⁺.