Evidence for a selective and electroneutral K+/H+-exchange in Saccharomyces cerevisiae using plasma membrane vesicles

The existence of a K⁺/H⁺ transport system in plasma membrane vesicles from Saccharomyces cerevisiae is demonstrated using fluorimetric monitoring of proton fluxes across vesicles (ACMA fluorescence quenching). Plasma membrane vesicles used for this study were obtained by a purification/reconstitution protocol based on differential and discontinuous sucrose gradient centrifugations followed by an octylglucoside dilution/gel filtration procedure. This method produces a high percentage of tightly-sealed inside-out plasma membrane vesicles. In these vesicles, the K⁺/H⁺ transport system, which is able to catalyse both K⁺ influx and efflux, is mainly driven by the K⁺ transmembrane gradient and can function even if the plasma membrane H⁺-ATPase is not active. Using the anionic oxonol VI and the cationic DISC₂(5) probes, it was shown that a membrane potential is not created during K⁺ fluxes. Such a dye response argues for the presence of a K⁺/H⁺ exchange system in S. cerevisiae plasma membrane and established the non-electrogenic character of the transport. The maximal rate of exchange is obtained at pH 6·8. This reversible transport system presents a high selectivity for K⁺ among other monovalent cations and a higher affinity for the K⁺ influx into the vesicles (exit from cells). The possible role of this K⁺/H⁺ exchange system in regulation of internal potassium concentration in S. cerevisiae is discussed.     

Heterologous expression reveals unique properties of Trk K+ importers from nonconventional biotechnologically relevant yeast species together with their potential to support Saccharomyces cerevisiae growth

In the model yeast Saccharomyces cerevisiae, Trk1 is the main K⁺ importer. It is involved in many important physiological processes, such as the maintenance of ion homeostasis, cell volume, intracellular pH, and plasma-membrane potential. The ScTrk1 protein can be of great interest to industry, as it was shown that changes in its activity influence ethanol production and tolerance in S. cerevisiae and also cell performance in the presence of organic acids or high ammonium under low K⁺ conditions. Nonconventional yeast species are attracting attention due to their unique properties and as a potential source of genes that encode proteins with unusual characteristics. In this work, we aimed to study and compare Trk proteins from Debaryomyces hansenii, Hortaea werneckii, Kluyveromyces marxianus, and Yarrowia lipolytica, four biotechnologically relevant yeasts that tolerate various extreme environments. Heterologous expression in S. cerevisiae cells lacking the endogenous Trk importers revealed differences in the studied Trk proteins' abilities to support the growth of cells under various cultivation conditions such as low K⁺ or the presence of toxic cations, to reduce plasma-membrane potential or to take up Rb⁺. Examination of the potential of Trks to support the stress resistance of S. cerevisiae wild-type strains showed that Y. lipolytica Trk1 is a promising tool for improving cell tolerance to both low K⁺ and high salt and that the overproduction of S. cerevisiae's own Trk1 was the most efficient at improving the growth of cells in the presence of highly toxic Li⁺ ions.     

The role of K+ and H+ transport systems during glucose‐ and H2O2‐induced cell death in Saccharomyces cerevisiae

Glucose, in the absence of additional nutrients, induces programmed cell death in yeast. This phenomenon is independent of yeast metacaspase (Mca1/Yca1) and of calcineurin, requires ROS production and it is concomitant with loss of cellular K⁺ and vacuolar collapse. K⁺ is a key nutrient protecting the cells and this effect depends on the Trk1 uptake system and is associated with reduced ROS production. Mutants with decreased activity of plasma membrane H⁺‐ATPase are more tolerant to glucose‐induced cell death and exhibit less ROS production. A triple mutant ena1‐4 tok1 nha1, devoid of K⁺ efflux systems, is more tolerant to both glucose‐ and H₂O₂‐induced cell death. We hypothesize that ROS production, activated by glucose and H⁺‐ATPase and inhibited by K⁺ uptake, triggers leakage of K⁺, a process favoured by K⁺ efflux systems. Loss of cytosolic K⁺ probably causes osmotic lysis of vacuoles. The nature of the ROS‐producing system sensitive to K⁺ and H⁺ transport is unknown. Copyright © 2010 John Wiley & Sons, Ltd.     

Activity and Activity Coefficient of Counter Monovalent Ions in Aqueous Polyelectrolyte Solutions from Carboxyalkylated Starch

Activity and activity coefficient were determined and compared for counter H⁺, Li⁺, Na⁺, K⁺ and Ag⁺ ions in polyelectrolyte solutions prepared from the condensation products of starch and sodium salts of mono-, di-, and trichloroacetic acids. Na-glass electrode was used for measuring the activity of Na⁺ ions; Ag-electrode for the measurement of the activity of Ag⁺ ions, and ion-exchange permselective membrane were also used in concentration cells to determine the activities of H⁺, Na⁺, and K⁺ ions. For salt-free polyelectrolyte solutions, the activity coefficient of each ion, at equal concentrations, increases in the above mentioned order of the polymer used. In all cases the activity coefficient increases in the following order of the studied monovalent cations: Ag⁺ < K⁺ < Na⁺ < Li⁺ < H⁺.     

Potassium uptake systems of Candida krusei

Candida krusei is a pathogenic yeast species that is phylogenetically outside both of the well-studied yeast groups, whole genome duplication and CUG. Like all other yeast species, it needs to accumulate high amounts of potassium cations, which are needed for proliferation and many other cell functions. A search in the sequenced genomes of nine C. krusei strains revealed the existence of two highly conserved genes encoding putative potassium uptake systems. Both of them belong to the TRK family, whose members have been found in all the sequenced genomes of species from the Saccharomycetales subclade. Analysis and comparison of the two C. krusei Trk sequences revealed all the typical features of yeast Trk proteins but also an unusual extension of the CkTrk2 hydrophilic N-terminus. The expression of both putative CkTRK genes in Saccharomyces cerevisiae lacking its own potassium importers showed that only CkTrk1 is able to complement the absence of S. cerevisiae's own transporters and provide cells with a sufficient amount of potassium. Interestingly, a portion of the CkTrk1 molecules were localized to the vacuolar membrane. The presence of CkTrk2 had no evident phenotype, due to the fact that this protein was not correctly targeted to the S. cerevisiae plasma membrane. Thus, CkTrk2 is the first studied yeast Trk protein to date that was not properly recognized and targeted to the plasma membrane upon heterologous expression in S. cerevisiae.     

Sol-gel transition temperatures of high acyl gellan with monovalent and divalent cations from rheological measurements

The sol–gel transition temperatures of 0.1–1.0% high acyl gellan (HAG) with 0–200 mM NaCl or KCl and 0–20 mM CaCl₂ or MgCl₂ were determined using rheological measurements. Transition temperatures for monovalent cations, Na⁺ and K⁺, in the range of 50–80 °C were not significantly different (p > 0.5). Absence of thermal hysteresis was the salient feature. However, thermal hysteresis (∼4.4 °C) was observed for 0.1% HAG without added salt, but disappeared on increasing HAG and counterion concentrations. Few concentrations of HAG and added monovalent and divalent cations showed thermal hysteresis not higher than 2.5 °C. Transition temperatures for divalent cations were similar to those for monovalent cations although for considerably lower concentrations of Ca²⁺ or Mg²⁺. Increasing concentrations of monovalent and divalent counterions give rise to higher transition temperatures but not to higher storage moduli. This was interpreted as a lack of cross-link formation in the three-dimensional network structure of the gels. A single sol–gel transition diagram for monovalent cations is proposed, in which different zones associated with the presence of ordered and disordered conformations serve to identify the conditions in which HAG can exist in aqueous media.     

Human NKCC2 cation–Cl– co‐transporter complements lack of Vhc1 transporter in yeast vacuolar membranes

Cation–chloride co‐transporters serve to transport Cl^– and alkali metal cations. Whereas a large family of these exists in higher eukaryotes, yeasts only possess one cation–chloride co‐transporter, Vhc1, localized to the vacuolar membrane. In this study, the human cation–chloride co‐transporter NKCC2 complemented the phenotype of VHC1 deletion in Saccharomyces cerevisiae and its activity controlled the growth of salt‐sensitive yeast cells in the presence of high KCl, NaCl and LiCl. A S. cerevisiae mutant lacking plasma‐membrane alkali–metal cation exporters Nha1 and Ena1‐5 and the vacuolar cation–chloride co‐transporter Vhc1 is highly sensitive to increased concentrations of alkali–metal cations, and it proved to be a suitable model for characterizing the substrate specificity and transport activity of human wild‐type and mutated cation–chloride co‐transporters. Copyright © 2013 John Wiley & Sons, Ltd.     

Deletion of the N-terminal domain of the yeast vacuolar (Na+,K+)/H+ antiporter Vnx1p improves salt tolerance in yeast and transgenic Arabidopsis

Cation/proton antiporters play a major role in the control of cytosolic ion concentrations in prokaryotes and eukaryotes organisms. In yeast, we previously demonstrated that Vnx1p is a vacuolar monovalent cation/H⁺ exchanger showing Na⁺/H⁺ and K⁺/H⁺ antiporter activity. We have also shown that disruption of VNX1 results in an almost complete abolishment of vacuolar Na⁺/H⁺ exchange, but yeast cells overexpressing the complete protein do not show improved salinity tolerance. In this study, we have identified an autoinhibitory N-terminal domain and have engineered a constitutively activated version of Vnx1p, by removing this domain. Contrary to the wild type protein, the activated protein has a pronounced effect on yeast salt tolerance and vacuolar pH. Expression of this truncated VNX1 gene also improves Arabidopsis salt tolerance and increases Na⁺ and K⁺ accumulation of salt grown plants thus suggesting a biotechnological potential of activated Vnx1p to improve salt tolerance of crop plants.     

Overexpressed maltose transporters in laboratory and lager yeasts: Localization and competition with endogenous transporters

Plain and fluorescently tagged versions of Agt1, Mtt1 and Malx1 maltose transporters were overexpressed in two laboratory yeasts and one lager yeast. The plain and tagged versions of each transporter supported similar transport activities, indicating that they are similarly trafficked and have similar catalytic activities. When they were expressed under the control of the strong constitutive PGK1 promoter only minor proportions of the fluorescent transporters were associated with the plasma membrane, the rest being found in intracellular structures. Transport activity of each tagged transporter in each host was roughly proportional to the plasma membrane‐associated fluorescence. All three transporters were subject to glucose‐triggered inactivation when the medium glucose concentration was abruptly raised. Results also suggest competition between endogenous and overexpressed transporters for access to the plasma membrane.     

Characterization of plasma membrane H+-ATPase from salt-tolerant yeast Candida versatilis

Plasma membrane was isolated from the salt-tolerant yeast Candida versatilis and the ATPase in plasma membrane was characterized. The ATPase was a typical H⁺-ATPase with similar properties to the Saccharomyces cerevisiae and Zygosaccharomyces rouxii enzymes. It was reacted with antibody (IgG) raised against S. cerevisiae plasma membrane H⁺-ATPase. The ATPase activity was not changed by adding NaCl and KCl to the assay solutions, but was increased by NH, especially by ammonium sulfate. In vivo stimulation of ATPase activity was observed by the addition of NaCl into the culture medium, as observed in Z. rouxii. No in vivo activation of H⁺-ATPase by glucose metabolism was observed in C. versatilis cells and the activity was independent of the growth phase, like Z. rouxii and unlike S. cerevisiae cells.     

Mechanisms of Salt Tolerance Conferred by Overexpression of the HAL1 Gene in Saccharomyces cerevisiae

Overexpression of the HAL1 gene improves the tolerance of Saccharomyces cerevisiae to NaCl by increasing intracellular K⁺ and decreasing intracellular Na⁺. The effect of HAL1 on intracellular Na⁺ was mediated by the PMR2/ENA1 gene, corresponding to a major Na⁺ efflux system. The expression level of ENA1 was dependent on the gene dosage of HAL1 and overexpression of HAL1 suppressed the salt sensitivity of null mutants in calcineurin and Hal3p, other known regulators of ENA1 expression. The effect of HAL1 on intracellular K⁺ was independent of the TRK1 and TOK1 genes, corresponding to a major K⁺ uptake system and to a K⁺ efflux system activated by depolarization, respectively. Overexpression of HAL1 reduces K⁺ loss from the cells upon salt stress, a phenomenon mediated by an unidentified K⁺ efflux system. Overexpression of HAL1 did not increase NaCl tolerance in galactose medium. NaCl poses two types of stress, osmotic and ionic, counteracted by glycerol synthesis and sodium extrusion, respectively. As compared to glucose, with galactose as carbon source glycerol synthesis is reduced and the expression of ENA1 is increased. As a consequence, osmotic adjustment through glycerolsynthesis, a process not affected by HAL1, is the limiting factor for growth on galactose under NaCl stress. © 1997 John Wiley & Sons, Ltd.     

The in vivo activation of Saccharomyces cerevisiae plasma membrane H+-ATPase by ethanol depends on the expression of the PMA1 gene,but not of the PMA2 gene

The expression of the PMA1 and PMA2 genes during Saccharomyces cerevisiae growth in medium with glucose plus increasing concentrations of ethanol was monitored by using PMA1-lacZ and PMA2-lacZ fusions and Northern blot hybridizations of total RNA probed with PMA1 gene. The presence of sub-lethal concentrations of ethanol enhanced the expression of PMA2 whereas it reduced the expression of PMA1. The inhibition of PMA1 expression by ethanol corresponded to a decrease in the content of plasma membrane ATPase as quantified by immunoassays. Although an apparent correspondence could exist between the increase of plasma membrane ATPase activity and the level of PMA2 expression, the maximal level of PMA2 expression remained about 200 times lower than PMA1. On the other hand, ethanol activated the plasma membrane H⁺-ATPase activity from a strain expressing only the PMA1 ATPase but did not activate that from a strain expressing only the PMA2 ATPase. These results provide evidence that in the presence of ethanol it is the PMA1 ATPase which is activated, probably by a post-translational mechanism and that the PMA2 ATPase is not involved.     

Gelling Properties of Gellan Solutions Containing Monovalent and Divalent Cations

Gelling temperatures of gellan solutions with the addition of Na⁺ and K⁺ ranging from 15 to 450 mM or Ca⁺⁺ and Mg⁺⁺ from 2 to 40 mM were determined by dynamic rheological testing at four polymer concentrations between 0.4 and 2.0% (w/w). Gelling temperatures were much higher for gellan solutions containing divalent cations than for those containing the same amount of monovalent cations. Solutions containing K⁺ gelled at higher temperatures than those containing Na⁺. Effects of Ca⁺⁺ and Mg⁺⁺ on gelling temperatures were not significantly different. A general model was developed to predict the gelling temperature of gellan solutions as functions of cation and polymer concentrations.     

Monovalent cation fluxes and physiological changes of Debaryomyces hansenii grown at high concentrations of KCl and NaCl

Debaryomyces hansenii showed an increased growth in the presence of either 1m KCl or 1m NaCl and a low acidification of the medium, higher for the cells grown in the presence of NaCl. These cells accumulated high concentrations of the cations, and showed a very fast capacity to exchange either Na⁺ or K⁺ for the opposite cation. They showed a rapid uptake of ⁸⁶ Rb⁺ and ²² Na⁺ . ⁸⁶ Rb⁺ transport was saturable, with K_m and V_max values higher for cells grown in 1m NaCl. ²² Na⁺ uptake showed a diffusion component, also higher for the cells grown with NaCl. Changes depended on growth conditions, and not on further incubation, which changed the internal ion concentration. K⁺ stimulated proton pumping produced a rapid extrusion of protons, and also a decrease of the membrane potential. Cells grown in 1m KCl showed a higher fermentation rate, but significantly lower respiratory capacity. ATP levels were higher in cells grown in the presence of NaCl; upon incubation with glucose, those grown in the presence of KCl reached values similar to the ones grown in the presence of NaCl. In both, the addition of KCl produced a transient decrease of the ATP levels. As to ion transport mechanisms, D. hansenii appears to have (a) an ATPase functioning as a proton pump, generating a membrane potential difference which drives K⁺ through a uniporter; (b) a K⁺ /H⁺ exchange system; and (c) a rapid cation/cation exchange system. Most interesting is that cells grown in different ionic environments change their studied capacities, which are not dependent on the cation content, but on differences in their genetic expression during growth. © 1998 John Wiley & Sons, Ltd.     

Skimmed milk demineralization by electrodialysis: Conventional versus selective membranes

Electrodialysis (ED) is a membrane technique that is useful in the demineralization of dairy products. In this work, experimental results obtained in batch electrodialysis of skimmed milk are presented. Two kinds of membranes were used: the first kind were conventional membranes in PVC and the second were monovalent ion selective membranes which are now available on the market. This latter type of membrane permits the transport of monovalent ions but limits the passage of large ions which have a high valency. The limiting current was measured as a function of feed flow into the stack. The results obtained when using both types of membranes were compared for elimination rate of K⁺, Na⁺, Cl⁻¹, Ca²⁺, Mg²⁺ and PO₄³⁻ ions as well as for current efficiency and electric power consumption in the separation process.     

Characterization of glycolytic metabolism and ion transport of Candida albicans

The main energetic pathways, fermentation and respiration, and the general ion transport properties of Candida albicans were studied. Compared to Saccharomyces cerevisiae, we found that in C. albicans: (a) the cell mass yield when grown in YPD was significantly larger; (b) it required longer times to be starved of endogenous substrates; (c) ethanol production was lower but significant; (d) respiration was also lower; (e) it showed a small activity of an alternative oxidase; (f) fermentation and oxidative phosphorylation seemed to compete for both ADP and NADH; and (g) NADH levels were lower. Regarding ion transport and compared to S. cerevisiae: (a) the general mechanism was similar, with a plasma membrane H⁺‐ATPase that generates both a plasma membrane ΔpH and a ΔΨ, the latter being responsible for driving K⁺ inside; (b) its acidification capacity is slightly smaller and less sensitive to activation by high pH; and (c) the presence of K⁺ results in a large activation of both respiration and fermentation, most probably due to the energy required in the process. ADP produced by H⁺‐ATPase stimulation by high pH or the addition of K⁺ at low pH results in the increase of both respiration and fermentation. Copyright © 2012 John Wiley & Sons, Ltd.     

Softening Effects of Monovalent Cations in Acidified Cucumber Mesocarp Tissue

Due to the importance of salt as an ingredient in cucumber pickle products, the effect of salt concentration on first-order softening rates during acid storage was determined. Softening rates of unfermented tissue increased as the NaCl increased from 0 to 1.5M, whether or not the tissue was blanched. For unheated, fermented tissue, softening rates increased between 0 and 0.2M NaCl but did not increase above 0.2M NaCl. Changes in the degree of pectin methylation were not highly correlated with changes in softening rates. The ions Li⁺, K⁺, Rb⁺, and Cs⁺ had softening effects similar to Na⁺. An enthalpy of activation of 145 kJ/mole was determined for softening of blanched tissue stored in 1.5M NaCl. This is the first demonstration of a softening effect by monovalent cations in cucumber tissue.     

EFFECT OF SOME MONOVALENT AND DIVALENT METAL IONS ON THE FLOCCULATION OF BREWERS YEAST STRAINS*

In agreement with previous reports, it has been found that both Mg⁺⁺ and Mn⁺⁺ ions can imitate Ca⁺⁺ as inducers of flocculation, though the intensity of the flocculation is considerably reduced. This reduction is not dependent upon the ionic concentration and a 10-fold increase in Mg⁺⁺ or Mn⁺⁺ from the normal concentration of 10 mg ion/litre fails to increase the flocculation intensity. Low concentrations (1–10 mg ion/litre) of Na⁺ or K⁺ induce flocculation in those strains displaying intense flocculation with Ca⁺⁺, but high concentrations of either Na⁺ or K⁺ (50–100 mg ion/litre) antagonize floc formation. It is suggested that divalent ions act by bridging cells through negative charges on the cell surface, whereas monovalent ions induce flocculation via a “counter ion” effect where the repellent forces of the negative charges on the cell surface are neutralized, thus allowing some floc formation due to hydrogen bonding or other types of non-ionic bonding between cells. The antagonism of high concentrations of monovalent ions towards flocculation may be due to the fact that all available cell surface charges are neutralized, resulting in insulation of the cells and thus preventing cell-to-cell hydrogen bonding.     

Antimicrobial Activity of Malting Barley Grain Thaumatin‐Like Protein Isoforms,S and R

Two basic proteins were isolated to homogeneity from malting barley (Hordeum vulgare L.) grain. Proteins were identified as members of a Thaumatin‐Like Protein (TLP) family, by Western blot. Isoforms, assigned as TLP‐S and TLP‐R, have slightly different mobility at about 22 and 27 kDa in nonreducing and reducing conditions, and pI values of 9.5 and 9.4, respectively. The antifungal potency of malting barley grain TLPs isoforms was examined on Micrococcus lysodeikticus, Saccharomyces cerevisiae, Candida albicans and plant pathogen Fusarium sporotrichioides growth in vitro. It was found that that IC₅₀ value for TLP‐S was two fold higher. Antibacterial and antifungal activities of both isoforms were completely abolished by divalent (Ca²⁺, Mn²⁺, Mg²⁺) and monovalent (K⁺) cations, at concentrations approximating physiological ionic strength and higher. Glucanase activity was not observed; neither TLP‐S nor TLP‐R digested glucan. On the basis of these results, the importance of TLP for barley grain protection against fungal diseases has been discussed together with the mechanism of antimicrobial action.     

The plasma membrane H+-ATPase,a simple polypeptide with a long history

The plasma membrane H⁺-ATPase of fungi and plants is a single polypeptide of fewer than 1,000 residues that extrudes protons from the cell against a large electric and concentration gradient. The minimalist structure of this nanomachine is in stark contrast to that of the large multi-subunit F_OF₁ ATPase of mitochondria, which is also a proton pump, but under physiological conditions runs in the reverse direction to act as an ATP synthase. The plasma membrane H⁺-ATPase is a P-type ATPase, defined by having an obligatory phosphorylated reaction cycle intermediate, like cation pumps of animal membranes, and thus, this pump has a completely different mechanism to that of F_OF₁ ATPases, which operates by rotary catalysis. The work that led to these insights in plasma membrane H⁺-ATPases of fungi and plants has a long history, which is briefly summarized in this review.