The kinetics of ⁷⁵[Se]-selenite uptake by Saccharomyces cerevisiae and the vacuolization response to high concentrations

Abstract

Uptake of ⁷⁵[Se]-selenite by Saccharomyces cerevisiae has been characterized. At a 0.5 mM selenite, ~0.14 nmol Se (10⁶ cells)⁻¹ was rapidly accumulated by the cells at a rate of ~56 pmol.min⁻¹ (10⁶ cells)⁻¹ which was independent of temperature and glucose. This rapid phase was followed by a slower uptake phase which was sensitive to glucose, temperature and metabolic inhibitors [2,4-dinitrophenol (DNP), carbonyl cyanide m-chlorophenyl hydrazone (CCCP), potassium cyanide (KCN) and sodium azide (NaN₃)] and therefore presumed to be metabolism-dependent. Two transport systems appeared to be involved in selenite uptake. At the low range of selenite concentrations used (0.025–0.1 mM), a high affinity transport system occurred with apparent K_m and V_max values of 54.0 μM and 3.14 pmol.min⁻¹ (10⁶ cells)⁻¹ respectively. A low affinity system was present at higher concentrations (0.1–1.0 mM) with apparent kinetic parameters of K_m=435 μM and V_max=11.6 pmol.min⁻¹ (10⁶ cells)⁻¹. Elevated sulphate concentrations (up to 2.5 mM) did not affect the accumulation of selenite. However, the transport rate from 0.5 mM selenite was stimulated by sulphite, with the maximum effect occurring at 0.5 mM sulphite. Methionine had a detectable inhibitory action on selenite uptake whereas cystine and cysteine completely inhibited active transport of selenite. Transmission electron microscopy of 5 mM selenite-grown cells revealed the presence of abundant small cytoplasmic vesicles containing electron-dense granules which could represent an intracellular selenium-detoxification mechanism.