Separate compartments of the yeast cell possess their own exopolyphosphatases differing from each other in their properties and dependence on culture conditions. The low-molecular-mass exopolyphosphatases of the cytosol, cell envelope, and mitochondrial matrix are encoded by the PPX1 gene, while the high-molecular-mass exopolyphosphatase of the cytosol and those of the vacuoles, mitochondrial membranes, and nuclei are presumably encoded by their own genes. Based on recent works, a preliminary classification of the yeast exopolyphosphatases is proposed.
Purified fractions of cytosol, vacuoles, nuclei, and mitochondria of Saccharomyces cerevisiae possessed inorganic polyphosphates with chain lengths characteristic of each individual compartment. The most part (80-90%) of the total polyphosphate level was found in the cytosol fractions. Inactivation of a PPX1 gene encoding ~40-kDa exopolyphosphatase substantially decreased exopolyphosphatase activities only in the cytosol and soluble mitochondrial fraction, the compartments where PPX1 activity was localized. This inactivation slightly increased the levels of polyphosphates in the cytosol and vacuoles and had no effect on polyphosphate chain lengths in all compartments. Exopolyphosphatase activities in all yeast compartments under study critically depended on the PPN1 gene encoding an endopolyphosphatase. In the single PPN1 mutant, a considerable decrease of exopolyphosphatase activity was observed in all the compartments under study. Inactivation of PPN1 decreased the polyphosphate level in the cytosol 1.4-fold and increased it 2- and 2.5-fold in mitochondria and vacuoles, respectively. This inactivation was accompanied by polyphosphate chain elongation. In nuclei, this mutation had no effect on polyphosphate level and chain length as compared with the parent strain CRY. In the double mutant of PPX1 and PPN1, no exopolyphosphatase activity was detected in the cytosol, nuclei, and mitochondria and further elongation of polyphosphates was observed in all compartments.
The secondary transport systems of the yeast vacuolar membrane have been investigated by (i) the method of radioactive isotopes ([Wlarginine);(ii) activation of H+-ATPase by cations (Cat+), when the enzyme is under H+ control and (iii) measurement of changes in the proton gradient (ApH) and membrane potential (E,,,) due to the supposed substrates of the transporters. The main mechanism of cation transport across the yeast tonoplast is probably H+/Cat+ antiport. The apparent K,,, of antiporters for Ca2+, Mg2+, MnZ+, ZnZf and P, are 0.06,0.3,0.8,0.0554.17 and 1.5 mM, respectively. H+-ATPase
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...
The uneven distribution of Mg2", K+, and phosphate in Saccharomyces carlsbergensis was demonstrated by the differential extraction of ions. Their concentrations were 5, 60, and 1 mM in the cytoplasm and 73, 470, and 110 mM in vacuoles, respectively. The intracellular gradients of these ions were 1:15, 1:8, and 1:110, respectively, across the tonoplast. The determination of free Mg2" (1.35 mM in the cytosol and 20 mM in vacuoles) showed that the ion accumulation in vacuoles could not be explained by the higher degree of ion complexing in these organelles.
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