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.     

Characterization of the recombinant succinic semi‐aldehyde dehydrogenase from Saccharomyces cerevisiae

The yeast succinic semi‐aldehyde dehydrogenase gene (SSADH; EC 1.2.1.16) was cloned and overexpressed in Escherichia coli. Based on SDS–PAGE, the molecular mass of the subunit was around 54 kDa, and the purified recombinant enzyme had a tetrameric molecular mass of ca. 200 kDa. The specific activity of the recombinant enzyme was 1.90 µm NADH formed/min/mg, and showed maximal activity at pH 8.4. The recombinant protein was highly specific for succinate semi‐aldehyde (K_m = 15.48 ± 0.14 µm ) and could use both NAD⁺ and NADP⁺ as co‐factors, with K_m values of 579.06 ± 30.1 µm and 1.017 ± 0.46 mm, respectively. Initial velocity studies showed that NADH was a competitive inhibitor with respect to NAD⁺ (K_i = 129.5 µm ) but a non‐competitive inhibitor with respect to succinate semi‐aldehyde. Adenine nucleotides of AMP, ADP and ATP inhibited yeast SSADH activity with K_i = 1.13–10.2 mm, and showed competitive inhibition with respect to NAD⁺ and mixed‐competitive, non‐competitive and non‐competitive inhibition, respectively, with respect to succinate semi‐aldehyde. The kinetic data suggest a 'ping‐pong' mechanism. Copyright © 2014 John Wiley & Sons, Ltd.     

Potassium concentration and pH inter‐relationships in grape juice and wine of Chardonnay and Shiraz from a range of rootstocks in different environments

Background and Aims: pH adjustment during winemaking is a significant cost to the Australian wine industry. This study addresses potassium (K⁺) concentration and pH inter‐relationships in grape juice and wine of Chardonnay and Shiraz. Methods and Results: Chardonnay and Shiraz on own roots, and on Ramsey, 1103 Paulsen, 140 Ruggeri, K51‐40, Schwarzmann, 101‐14, Rupestris St. George and 1202 Couderc were compared at Koorlong and Merbein (Victoria), and Padthaway, Nuriootpa and Rowland Flat (South Australia). Petiole K⁺ concentrations at flowering were a poor indicator of grape juice and wine K⁺ concentrations. The concentration of H⁺ ions in grape juice and wine decreased as K⁺ concentrations increased resulting in increased pH. The relationship between H⁺ and K⁺ concentrations was linear for Chardonnay but exponential for Shiraz, where K⁺ concentrations were higher. Wine K⁺ and grape juice K⁺ concentrations exhibited a positive linear relationship, with slope for Chardonnay about half that for Shiraz, indicating a net loss of K⁺ between grape juice and wine of 58% for Chardonnay and 13% for Shiraz. Conclusions: The study has linked higher wine pH to both higher juice soluble solids and K⁺, and to poorer wine colour hue. Loss of K⁺ during fermentation and cold stabilisation appeared higher for Chardonnay than for Shiraz. Significance of the Study: Rootstocks that lead to lower K⁺ concentrations and pH in grape juice and wine are identified. Differences in the K⁺ concentration dynamics between grape juice and wine of Chardonnay and Shiraz are described and quantified.     

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.     

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.     

Quantitation of NAD+ biosynthesis from the salvage pathway in Saccharomyces cerevisiae

Nicotinamide adenine dinucleotide (NAD⁺) is synthesized via two major pathways in prokaryotic and eukaryotic systems: the de novo biosynthesis pathway from tryptophan precursors, or the salvage biosynthesis pathway from either extracellular nicotinic acid or various intracellular NAD⁺ decomposition products. NAD⁺ biosynthesis via the salvage pathway has been linked to an increase in yeast replicative lifespan under calorie restriction (CR). However, the relative contribution of each pathway to NAD⁺ biosynthesis under both normal and CR conditions is not known. Here, we have performed lifespan, NAD⁺ and NADH (the reduced form of NAD⁺) analyses on BY4742 wild‐type, NAD⁺ salvage pathway knockout (npt1Δ) and NAD⁺ de novo pathway knockout (qpt1Δ) yeast strains cultured in media containing either 2% glucose (normal growth) or 0.5% glucose (CR). We have utilized ¹⁴C labelled nicotinic acid in the culture media combined with HPLC speciation and both UV and ¹⁴C detection to quantitate the total amounts of NAD⁺ and NADH and the amounts derived from the salvage pathway. We observed that wild‐type and qpt1Δ yeast exclusively utilized extracellular nicotinic acid for NAD⁺ and NADH biosynthesis under both the 2% and 0.5% glucose growth conditions, suggesting that the de novo pathway plays little role if a functional salvage pathway is present. We also observed that NAD⁺ concentrations decreased in all three strains under CR. However, unlike the wild‐type strain, NADH concentrations did not decrease and NAD⁺: NADH ratios did not increase under CR for either knockout strain. Lifespan analyses revealed that CR resulted in a lifespan increase of approximately 25% for the wild‐type and qpt1Δ strains, while no increase in lifespan was observed for the npt1Δ strain. In combination, these data suggest that having a functional salvage pathway is required for lifespan extension under CR. Copyright © 2009 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.     

Effects of Cation Properties on Sol-gel Transition and Gel Properties of κ-carrageenan

The phase transition temperatures, rheological properties and gel‐network characteristics for gelation of κ‐carrageenan‐salt (NaCl, KCl and CaCl₂) solutions and their aged gels were investigated. The effectiveness of increasing gelling and gel‐melting temperatures at the salt concentrations examined followed the sequence of K⁺ > Ca2⁺ > Na⁺. This sequence was also true for the gel strength and the melting enthalpy (DH) of the most crosslinked junction zones of aged gels at low salt concentrations. Nonetheless, a different order (Ca⁺⁺ > K⁺ and Na⁺) was found for increasing storage modulus and gelation rate during early‐stage gelation, thermal hysteresis and the DH of aged gels in some salt‐carrageenan systems.     

Mdm31 protein mediates sensitivity to potassium ionophores but does not regulate mitochondrial morphology or phospholipid trafficking in Schizosaccharomyces pombe

Mdm31p is an inner mitochondrial membrane (IMM) protein with unknown function in Saccharomyces cerevisiae. Mutants lacking Mdm31p contain only a few giant spherical mitochondria with disorganized internal structure, altered phospholipid composition and disturbed ion homeostasis, accompanied by increased resistance to the electroneutral K⁺/H⁺ ionophore nigericin. These phenotypes are interpreted as resulting from diverse roles of Mdm31p, presumably in linking mitochondrial DNA (mtDNA) to the machinery involved in segregation of mitochondria, in mediating cation transport across IMM and in phospholipid shuttling between mitochondrial membranes. To investigate which of the roles of Mdm31p are conserved in ascomycetous yeasts, we analysed the Mdm31p orthologue in Schizosaccharomyces pombe. Our results demonstrate that, similarly to its S. cerevisiae counterpart, SpMdm31 is a mitochondrial protein and its absence results in increased resistance to nigericin. However, in contrast to S. cerevisiae, Sz. pombe cells lacking SpMdm31 are also less sensitive to the electrogenic K⁺ ionophore valinomycin. Moreover, mitochondria of the fission yeast mdm31Δ mutant display no changes in morphology or phospholipid composition. Therefore, in terms of function, the two orthologous proteins appear to have considerably diverged between these two evolutionarily distant yeast species, possibly sharing only their participation in ion homeostasis. Copyright © 2015 John Wiley & Sons, Ltd.     

Use of ade1 and ade2 mutations for development of a versatile red/white colour assay of amyloid‐induced oxidative stress in saccharomyces cerevisiae

Mutations in adenine biosynthesis pathway genes ADE1 and ADE2 have been conventionally used to score for prion [PSI⁺] in yeast. If ade1‐14 mutant allele is present, which contains a premature stop codon, [psi^?] yeast appear red on YPD medium owing to accumulation of a red intermediate compound in vacuoles. In [PSI⁺] yeast, partial inactivation of the translation termination factor, Sup35 protein, owing to its amyloid aggregation allows for read‐through of the ade1‐14 stop codon and the yeast appears white as the red intermediate pigment is not accumulated. The red colour development in ade1 and ade2 mutant yeast requires reduced‐glutathione, which helps in transport of the intermediate metabolite P‐ribosylaminoimidazole carboxylate into vacuoles, which develops the red colour. Here, we hypothesize that amyloid‐induced oxidative stress would deplete reduced‐glutathione levels and thus thwart the development of red colour in ade1 or ade2 yeast. Indeed, when we overexpressed amyloid‐forming human proteins TDP‐43, Aβ‐42 and Poly‐Gln‐103 and the yeast prion protein Rnq1, the otherwise red ade1 yeast yielded some white colonies. Further, the white colour eventually reverted back to red upon turning off the amyloid protein's expression. Also, the aggregate‐bearing yeast have increased oxidative stress and white phenotype yeast revert to red when grown on media with reducing agent. Furthermore, the red/white assay could also be emulated in ade2‐1, ade2Δ, and ade1Δ mutant yeast and also in an ade1‐14 mutant with erg6 gene deletion that increases cell‐wall permeability. This model would be useful tool for drug‐screening against general amyloid‐induced oxidative stress and toxicity. Copyright © 2016 John Wiley & Sons, Ltd.     

Comparative study of physiological adaptation to salt stress in the genome shuffled Candida versatilis and a wild-type salt-tolerant yeast strain

Candida versatilis is a yeast with a complex salt-tolerant system. It can maintain normal physiological activities and metabolic fermentation under a high-salt environment. The cellular mechanisms of adaptation to salt stress in strains of a wild type of C. versatilis (WT) and S3–5, genome shuffling strains of C. versatilis with improved tolerance to salt, were investigated. The content of intra- and extra-cellular glycerol, intra-cellular Na⁺, as well as membrane fluidity and permeability, were determined under salt-stressed yeast growth conditions. The results showed that Na⁺/H⁺-antiporter played a primary role in Na⁺ extrusion and H⁺-ATPase has been associated with yeast survival under salt stress. Considerable amounts of glycerol were produced and secreted by the yeast to outside the cell under this salt stress. Changes in the portion of membrane saturated and unsaturated fatty acid composition of C. versatilis in response to osmotic stress lead to membrane permeability and fluidity decreases. They could restrict the influx of Na⁺, enhance H⁺-ATPase activity, and prevent leakage of glycerol across the cell membrane under osmotic stress. The salt tolerance of genome shuffled strain S3–5 was higher than WT. It could be correlated with a higher level of intra-cellular accumulation of glycerol and sodium ions in cells of S3–5 than WT as well as a higher portion of oleic fatty acid (C18: 1) and a lower level of linoleic acid (C18: 2) in cell membranes of the studied yeast mutant. It can be concluded that S3–5 improved physiological regulatory mechanisms of response to salt stress, such as decreased membrane fluidity and a permeability that rapidly adjusted to osmotic stress.     

Melatonin improves survival and respiratory activity of yeast cells challenged by alpha‐synuclein and menadione

One of the hallmarks of Parkinson disease is α‐synuclein aggregate deposition that leads to endoplasmic reticulum stress, Golgi fragmentation and impaired energy metabolism with consequent redox imbalance. In the last decade, many studies have used Saccharomyces cerevisiae as a model in order to explore the intracellular consequences of α‐synuclein overexpression. In this study we propose to evaluate the respiratory outcome of yeast cells expressing α‐synuclein. Cell viability or growth on selective media for respiratory activity was mainly affected in the α‐synuclein‐expressing cells if they were also treated with menadione, which stimulates reactive oxygen species production. We also tested whether melatonin, a natural antioxidant, would counteract the deleterious effects of α‐synuclein and menadione. In fact, melatonin addition improved the respiratory growth of α‐synuclein/menadione‐challenged cells, presented a general improvement in the enzymatic activity of the respiratory complexes and finally elevated the rate of mitophagy, an important cellular process necessary for the clearance of damaged mitochondria. Altogether, our data confirms that α‐synuclein impairs respiration in yeast, which can be rescued by melatonin addition.     

Salt-induced modulation in inorganic nutrients, antioxidant enzymes, proline content and seed oil composition in safflower (Carthamus tinctorius L.)

BACKGROUND: Safflower (Carthamus tinctorius L.) has gained considerable ground as a potential oil‐seed crop. However, its yield and oil production are adversely affected under saline conditions. The present study was conducted to appraise the influence of salt (NaCl) stress on yield, accumulation of different inorganic elements, free proline and activities of some key antioxidant enzymes in plant tissues as well as seed oil components in safflower. Two safflower accessions differing in salt tolerance (Safflower‐33 (salt sensitive) and Safflower‐39 (salt tolerant)) were grown under saline (150 mmol L^?1) conditions and salt‐induced changes in the earlier‐mentioned physiological attributes were determined. RESULTS: Salt stress enhanced leaf and root Na⁺, Cl^? and proline accumulation and activities of leaf superoxide dismutase, catalase and peroxidase, while it decreased K⁺, Ca²⁺ and K⁺/Ca²⁺ and Ca²⁺/Na⁺ ratios and seed yield, 100‐seed weight, number of seeds, as well as capitula, seed oil contents and oil palmitic acid. No significant effect of salt stress was observed on seed oil α‐tocopherols, stearic acid, oleic acid or linoleic acid contents. Of the two safflower lines, salt‐sensitive Safflower‐33 was higher in leaf and root Na⁺ and Cl^?, while Safflower‐39 was higher in leaf and root K⁺, K⁺/Ca²⁺ and Ca²⁺/Na⁺ and seed yield, 100‐seed weight, catalase activity, seed oil contents, seed oil α‐tocopherol and palmitic acid. Other attributes remained almost unaffected in both accessions. CONCLUSION: Overall, high salt tolerance of Safflower‐39 could be attributed to Na⁺ and Cl^? exclusion, high accumulation of K⁺ and free proline, enhanced CAT activity, seed oil α‐tocopherols and palmitic acid contents. Copyright © 2011 Society of Chemical Industry     

Factors that determine the fracture properties and microstructure of globular protein gels

Protein gel matrices are responsible for the texture of many foods. Therefore an understanding of the chemical reactions and physical processes associated with fracture properties of gels provides a fundamental understanding of select mechanical properties associated with texture. Globular proteins form thermally induced gels that are classified as fine-stranded, mixed or particulate, based on the protein network appearance. The fundamental properties of true shear stress and true shear strain at fracture, used to describe the physical properties of gels, depend on the gel network. Type and amount of mineral salt in whey protein and β-lactoglobulin protein dispersions determines the type of thermally induced gel matrix that forms, and thus its fracture properties. A fine-stranded matrix is formed when protein suspensions contain monovalent cation (Li⁺, K⁺, Rb⁺, Cs⁺) chlorides, sodium sulfate or sodium phosphate at ionic strengths ≤0.1 mol/dm³. This matrix has a well-defined network structure, and varies in stress and strain at fracture at different salt concentrations. At ionic strengths >0.1 mol/dm³ the matrix becomes mixed. This network appears as a combination of fine strands and spherical aggregates, and has high stress values and minimum strain values at fracture. Higher concentrations of monovalent cation salts cause the formation of particulate gels, which are high in stress and strain at fracture. The salt concentration required to change microstructure depends on the salt's position in the Hofmeister series. The formation of a particulate matrix also occurs when protein suspensions contain low concentrations (10–20 mol/dm³) of divalent cation (Ca²⁺, Mg²⁺, Ba²⁺) chloride salts or di-cationic 1,6-hexanediamine at pH 7.0. The divalent cation effect on β-lactoglobulin gelation is associated with minor changes in tertiary structure involving amide—amide interproton connectivities (determined by ¹H NMR) at 40–45°C, increasing hydrophobicity and intermolecular aggregation. The type of matrix formed appears to be related to the dispersed or aggregated state of proteins prior to denaturation. Mixed and particulate matrices result from conditions which favor aggregation at temperatures (25–45°C) which are much lower than the denaturation temperature (~65°C). Therefore, general (e.g. Hofmeister series) and protein-specific factors can affect the dispersibility of proteins and thereby determine the microstructure and fracture properties of globular protein gels.     

Effect of Salts and Sugars on Pressure‐induced Gelatinisation of Wheat,Tapioca, and Potato Starches

The impact of a variety of chlorides, sodium salts, potassium salts and low‐molecular sugars on pressure‐induced gelatinisation of wheat, tapioca and potato starches has been investigated. Just as thermal gelatinisation, pressure‐induced gelatinisation is influenced by solutes in the starch suspensions. In general the effects of salts and sugars on starch gelatinisation were comparable for thermal and pressure treatments. For example, the gelatinisation pressure was reduced by sugars whereas the degree of gelatinisation was linearly correlated with the number of equatorial hydroxyl groups. The influence of salts on the gelatinisation pressure varied and the extent of effect on the gelatinisation pressure did not only depend on the solute added but also on the type of starch. At high chloride concentrations (> 2 M) the impact of the salts on starch gelatinisation augmentation followed the order Na⁺<K⁺<Li⁺<Ca²⁺, which corresponds to the order of the lyotropic series. At concentrations above 1 M the effect of potassium salts on starch gelatinisation upon pressurisation also followed the order of the Hofmeister series (Cl⁻<Br⁻<I⁻<SCN⁻).     

Overexpression of vacuolar H+‐ATPase‐related genes in bottom‐fermenting yeast enhances ethanol tolerance and fermentation rates during high‐gravity fermentation

Vacuolar H⁺‐ATPase (V‐ATPase) is thought to play a role in stress tolerance. In this study it was found that bottom‐fermenting yeast strains, in which the V‐ATPase‐related genes DBF2, VMA41/CYS4/NHS5 and RAV2 were overexpressed, exhibited stronger ethanol tolerance than the parent strain and showed increased fermentation rates in a high‐sugar medium simulating high‐gravity fermentation. Among the strains examined, the DBF2‐overexpressing bottom‐fermenting yeast strain exhibited the highest ethanol tolerance and fermentation rate in YPM20 medium. Using this strain, high‐gravity fermentation was performed by adding sugar to the wort, which led to increased fermentation rates and yeast viability compared with the parent strain. These findings indicate that V‐ATPase is a stress target in high‐gravity fermentation and suggests that enhancing the V‐ATPase activity increases the ethanol tolerance of bottom‐fermenting yeast, thereby improving the fermentation rate and cell viability under high‐gravity conditions. Copyright © 2012 The Institute of Brewing & Distilling     

Resveratrol Preserves Mitochondrial Function,Stimulates Mitochondrial Biogenesis,and Attenuates Oxidative Stress in Regulatory T Cells of Mice Fed a High‐Fat Diet

Consumption of high‐fat diet (HFD) is related with increased oxidative stress and dysfunctional mitochondria in many organs. The effects of resveratrol (trans‐3,5,4′‐trihydroxystilbene) that can protect T lymphocytes in various disease conditions on the HFD‐induced apoptosis of CD4⁺CD25⁺CD127^low/? regulatory T cells (Tregs) were studied, and the possible mechanism was postulated. Resveratrol significantly decreased Tregs death induced by 20‐wk HFD, being associated with the reduction of reactive oxygen species production and the alleviation of HFD‐induced loss of mitochondrial membrane potential (Δψm) in Tregs. Furthermore, resveratrol increased the expression of factors that regulated mitochondrial biogenesis in Tregs. Finally, resveratrol recovered the HFD‐induced activation of apoptotic markers in Tregs. Resveratrol protected Tregs against HFD‐induced apoptosis by reducing oxidative stress, restoring mitochondrial functional activities, and stimulating mitochondrial biogenesis.     

Monovalent cation transporters at the plasma membrane in yeasts

Maintenance of proper intracellular concentrations of monovalent cations, mainly sodium and potassium, is a requirement for survival of any cell. In the budding yeast Saccharomyces cerevisiae, monovalent cation homeostasis is determined by the active extrusion of protons through the Pma1 H⁺-ATPase (reviewed in another chapter of this issue), the influx and efflux of these cations through the plasma membrane transporters (reviewed in this chapter), and the sequestration of toxic cations into the vacuoles. Here, we will describe the structure, function, and regulation of the plasma membrane transporters Trk1, Trk2, Tok1, Nha1, and Ena1, which play a key role in maintaining physiological intracellular concentrations of Na⁺, K⁺, and H⁺, both under normal growth conditions and in response to stress.     

Most,but not all,yeast strains in the deletion library contain the [PIN+] prion

The yeast deletion library is a collection of over 5100 single gene deletions that has been widely used by the yeast community. The presence of a non‐Mendelian element, such as a prion, within this library could affect the outcome of many large‐scale genomic studies. We previously showed that the deletion library parent strain contained the [PIN⁺] prion. [PIN⁺] is the misfolded infectious prion form of the Rnq1 protein that displays distinct fluorescent foci in the presence of RNQ1–GFP and exists in different physical conformations, called variants. Here, we show that over 97% of the library deletion strains are [PIN⁺]. Of the 141 remaining strains that have completely (58) or partially (83) lost [PIN⁺], 139 deletions were able to efficiently maintain three different [PIN⁺] variants despite extensive growth and storage at 4 °C. One strain, cue2Δ, displayed an alteration in the RNQ1–GFP fluorescent shape, but the Rnq1p prion aggregate shows no biochemical differences from the wild‐type. Only strains containing a deletion of either HSP104 or RNQ1 are unable to maintain [PIN⁺], indicating that 5153 non‐essential genes are not required for [PIN⁺] propagation. Copyright © 2009 John Wiley & Sons, Ltd.     

Use of a Milk Assay to Evaluate the Effects of Potassium on Commercial Yeast Lactases

A reliable enzyme assay was developed that could routinely be used to accurately reflect lactase activity. The assay was performed with a nonfat dry milk substrate at 37°C. This procedure was then utilized to evaluate the effects of potassium ions as a stabilizer and activator on four yeast lactase enzymes. Low levels of potassium ions (K⁺), incorporated as potassium chloride (KCl), increased enzyme units by 20% and 30% in two liquid lactases derived from Saccharomyces lactis. Levels as high as 9.4% KCl increased the activity of one liquid lactase as much as 160%. The K⁺ activated and stabilized a powdered form of lactase isolated from Kluyveromyces fragilis. The activity of the fourth lactase tested, also derived from S. Zactis, was slightly enhanced.