Energy-Dependent Transport of Manganese into Yeast Cells and Distribution of Accumulated Ions

Abstract

Manganese transport into yeast cells is energy-dependent. It is dependent on endogenous sources of energy and is inhibited by olygomycin (12.5 -25 kg/ml), 2,4-dinitrophenol (1 mM), 2-deoxyglucose (1 -50 mM) and sodium azide (1 -10 mM), but is stimulated by cyanide and glucose. The stimulating effect of glucose is eliminated by N-ethylmaleimide and iodoacetate, which apparently inhibit the transport of glucose itself. About 75% of the manganese accumulated in the presence of glucose is found in yeast protoplasts and nearly 25% in the cell walls. A major portion of the accumulated manganese is found in vacuoles. The concentration of osmotically free manganese in the cytosol did not exceed 2 m M , but the concentration in vacuoles was up to 14mM. The tonoplast is assumed to have a transport system for divalent cations, thereby regulating their concentration in the cytosol.The transport of bivalent manganese ions has long attracted the attention of investigators [l -41.Data have been obtained which support the hypothesis that a M 2 +-dependent plasmolemma ATPase provides energy for this process; this enzyme uses ATP only of glycolytic origin [4-61. The state of the accumulated ions (free and bound manganese) and also their distribution inside the yeast cells have not been studied yet.Recently using a cytochemical method of analysis we found that magnesium ions are localized in yeast vacuoles and that the accumulated manganese is also concentrated in these organelles [7]. An independent biochemical approach supported the idea that a considerable portion of the total magnesium (up to 40%) is localized in yeast vacuoles [8,9]. In this work we have used this approach in order to study the distribution of manganese in the cells of Saccharomyces carhbergensis. In addition, the energy-dependent character of manganese accumulation by yeast was demonstrated. Microorgan ismsAll the experiments were carried out on the yeasts Saccharomyces cereuisiae (strain IBPM-355) and Succharomyces carlsbergensis (strain IBPM-366). Yeasts were grown in flasks in Reader medium at 29 "C. Inoculation was carried out with 24-h innocula; after 5 h the cells were collected by centrifugation, and were washed with distilled water. Then they were incubated for 60 min in a medium containing potassium phosphate (0.33 M) and glucose (0.1 M) at 30 "C or 37 "C under the same shaking conditions as during growth. Then the cells were washed with distilled water and were used for experiments to investigate the accumulation of manganese ions. The accumulation of manganese was carried out for 45 min at 30 "C, pH 5.5 and under continuous shaking. The concentration of manganese sulphate was 3 mM and glucose (if added) 100 mM [3].Usually cells were separated from medium by centrifugation followed by washing with water.In the experiments on the influence of different inhibitors on manganese uptake, this process was stopped at specific times by the addition of La3+ ions to a final concentration 0.3 mM. Then the cells were separated by centrifugation and washed with...

A Novel Intracellular K+/H+ Antiporter Related to Na+/H+ Antiporters Is Important for K+ Ion Homeostasis in Plants

“…Determination of Vacuolar and Cytoplasmic Ion Content-The vacuolar and cytoplasmic Na ϩ and K ϩ content was determined by treating the cells with cytochrome c that selectively permeabilizes the plasma membrane (35,36). Cells were grown and washed as above and resuspended in 50 l of 2% cytochrome c, 18 g/ml antimycin, 1 mM HEPES, 10 mM MgSO 4 , 10 mM CaCl 2 and 5 mM 2-deoxy D-glucose.…”

“…Intracellular ions were extracted by addition of HCl to a final concentration of 0.4% and incubation for 20 min at 95°C. After removal of cell debris by centrifugation, potassium and sodium ion content was determined with an atomic absorption spectrometer.Determination of Vacuolar and Cytoplasmic Ion Content-The vacuolar and cytoplasmic Na ϩ and K ϩ content was determined by treating the cells with cytochrome c that selectively permeabilizes the plasma membrane (35,36). Cells were grown and washed as above and resuspended in 50 l of 2% cytochrome c, 18 g/ml antimycin, 1 mM HEPES, 10 mM MgSO 4 , 10 mM CaCl 2 and 5 mM 2-deoxy D-glucose.…”

Interactions of fungi with toxic metals

“…Mutants of S. cerevisiae defective in vacuolar function were sensitive to Ca^"^ and other toxic metals (Kitamoto et al, 1988). Polyphospbates, the only inorganic macromolecular anion found in the vacuole, have an important role in maintaining ionic compartmentation and their biosynthesis accompanies vacuolar accumulation of Mg^^ or Mn^" ( Okorokov et al, 1977, Okorokov, Licbko & Kulaev, 1980Uchkoetal, 1982;Okorokov e^ a/., 1983a, 6;Miller, 1984;Kibn et al, 1988). The significance of polypbosphate granules as a store for divalent cations has long been appreciated (Doonan et al, 1979;Roomans, 1980).…”

Adenylate cyclase activity in permeabilised yeast

“…However, glucose did not directly inhibit AC in situ under conditions similar to those used [6] to demonstrate the control of Escherichia coli AC by glucose. The apparent absolute dependence of yeast AC on Mn 2÷ both in situ (see above) and in vitro (where rates with 5 mM MgCI: alone are <3% of those with Mn 2+ [5]) is a puzzle, because it is unlikely that significant amounts of Mn 2÷ are available to AC in yeast ceils, although the vacuole can accumulate Mn 2÷ [14]. Mn 2÷ may short-circuit a yet unknown regulatory mechanism for yeast AC, as they appear to do for mammalian ACs [ 15].…”