Influence of prior Cd(2+) exposure on the uptake of Cd(2+) and other elements in the phytochelatin-deficient mutant, cad1-3, of Arabidopsis thaliana

In order to test the potential effect of prior exposure to different Cd concentrations on Cd uptake and accumulation, plants of Arabidopsis thaliana, including a phytochelatin-deficient mutant, cad1-3, and the wild type, were compared. For Cd uptake experiments, plants were grown for 1 week in nutrient solution containing different Cd concentrations (0, 0.05, 0.1, 0.25, 0.5, and 1.0 microM Cd(NO(3))(2)). Thereafter they were subjected to 0.5 microM Cd labelled with (109)Cd for 2 h. Uptake experiments with (109)Cd showed that the phytochelatin-deficient mutant cad1-3, accumulated less Cd than the wild type. Both a lower proportion and lower total amount of absorbed Cd were translocated to the shoot in cad1-3 plants compared to wild-type plants. Cadmium exposure also influenced the amounts of nutrients found, whereby after exposure to high Cd concentrations (0.5, 1.0 microM) during growth, cad1-3 roots contained less Fe, K, Mg, P, and S compared to roots of the wild type. In cad1-3 these elements decreased with increasing Cd concentration. The total Cd content in roots and shoots increased significantly with increasing Cd concentration during growth, although the increase was much less in cad1-3 plants. In time-dependent experiments of Cd uptake carried out between 15 and 120 min on plants not previously exposed to Cd, no significant difference in Cd accumulation between the mutant and wild type were found, although a smaller amount of Cd was translocated to the shoot in cad1-3 plants. The possibility that the differences in Cd accumulation in mutant and wild-type lines may be due to the cytosolic Cd regulation, which is inhibited by the complexation of Cd by phytochelatins, is discussed.     

Fructose 2,6-bisphosphate and fructose-6-P 2-kinase in Saccharomyces cerevisiae in relation to metabolic state in wild type and fructose-6-P 1-kinase mutant strains

In wild type Saccharomyces cerevisiae, fructose-6-P is known to be in much lower amounts than needed to saturate fructose-6-P 1-kinase in vitro, and the same is true for a mutant with reduced affinity for fructose-6-P, even though its in vivo fructose-6-P concentration is much higher than normal. Both the wild type and mutant fructose-6-P 1-kinases were activated in vitro by fructose-2,6-P2 in the 0.1 microM concentration range, and the effector was present in more than adequate amounts. Hence, it is likely to be necessary for sufficient flux through the fructose-6-P 1-kinase reaction in vivo, and the data also fit with fructose-2,6-P2 acting at different sites on the enzyme from fructose-6-P. In growth on glucose, a variety of wild type strains contained 5-10 microM fructose-2,6-P2, and various fructose-6-P 1-kinase mutant strains had levels of up to 150 microM in the presence of glucose. Fructose-2,6-P2 was also found (0.5-10 microM) in derepressed cultures after glucose exhaustion and in growth on pyruvate. Activities of fructose-6-P 2-kinase in the various strains and situations are also presented. The data generally indicate a correlation between levels of fructose-2,6-P2 and fructose-6-P and suggest that fructose-2,6-P2 is not rapidly degraded after glucose exhaustion.     

Glucose toxicity and inability of Bacteroides ruminicola to regulate glucose transport and utilization.

Ammonia-limited (3.5 mM ammonia) cultures of Bacteroides ruminicola B(1)4 had a high number of viable cells (greater than 10(9)/ml), but only when the concentration of glucose was not too high (10 mM or less). When the glucose concentration was increased from 10 to 50 mM, there was a marked decrease in viability (10(5)-fold or greater). Because there was little decline in pH and only a small increase in succinate and acetate as the glucose concentration was increased, it did not appear that end products were killing the cells. This conclusion was supported by the observation that reinoculated cultures grew in the spent medium which had been supplemented with ammonia. Unlabeled rhamnose did not inhibit [14C]-glucose uptake, and cultures which were selected with a low concentration of rhamnose tolerated high concentrations of glucose (50 mM). The glucose-resistant mutant transported glucose at a lower rate than the wild type, and the Vmax of glucose transport was fourfold lower. The wild type stored much more polysaccharide than the glucose-resistant mutant, but it is not clear if polysaccharide accumulation per se is responsible for the glucose toxicity. These results indicated that B. ruminicola B(1)4 is unable to regulate glucose transport and utilization when growth is limited by ammonia.     

Glucose toxicity and inability of Bacteroides ruminicola to regulate glucose transport and utilization.

Ammonia-limited (3.5 mM ammonia) cultures of Bacteroides ruminicola B(1)4 had a high number of viable cells (greater than 10(9)/ml), but only when the concentration of glucose was not too high (10 mM or less). When the glucose concentration was increased from 10 to 50 mM, there was a marked decrease in viability (10(5)-fold or greater). Because there was little decline in pH and only a small increase in succinate and acetate as the glucose concentration was increased, it did not appear that end products were killing the cells. This conclusion was supported by the observation that reinoculated cultures grew in the spent medium which had been supplemented with ammonia. Unlabeled rhamnose did not inhibit [14C]-glucose uptake, and cultures which were selected with a low concentration of rhamnose tolerated high concentrations of glucose (50 mM). The glucose-resistant mutant transported glucose at a lower rate than the wild type, and the Vmax of glucose transport was fourfold lower. The wild type stored much more polysaccharide than the glucose-resistant mutant, but it is not clear if polysaccharide accumulation per se is responsible for the glucose toxicity. These results indicated that B. ruminicola B(1)4 is unable to regulate glucose transport and utilization when growth is limited by ammonia.     

Enhanced conversion of soluble starch to trehalose by a mutant of Saccharomycopsis fibuligera sdu

In our previous studies, it was found that Saccharomycopsis fibuligera sdu cells could accumulate 18.0% (gg(-1)) trehalose from soluble starch in SSY medium. However, the yeast strain contained high activities of acid and neutral trehalases, which were reported to mobilize trehalose accumulated by the cells during fermentation. In order to enhance the yield of trehalose, it is necessary to remove the trehalase activities from the cells. By mutagenesis of ethylmethanesulfonate, one mutant that assimilated trehalose very slowly, but grew on other carbon sources as fast as its parent strain, was isolated. In Biostat B2 2-1 fermentation, trehalose accumulation of the mutant was much higher than that of the wild type when grown in YPS medium containing starch. The activities of acid and neutral trehalases of this mutant were much lower than those of the wild type, respectively. We think the reduction of acid and neutral trehalase activities is considered to be responsible for the increased yield of trehalose accumulated by the mutant.     

Mechanism of nitrogenase switch-off by oxygen.

Oxygen caused a reversible inhibition (switch-off) of nitrogenase activity in whole cells of four strains of diazotrophs, the facultative anaerobe Klebsiella pneumoniae and three strains of photosynthetic bacteria (Rhodopseudomonas sphaeroides f. sp. denitrificans and Rhodopseudomonas capsulata strains AD2 and BK5). In K. pneumoniae 50% inhibition of acetylene reduction was attained at an O2 concentration of 0.37 microM. Cyanide (90 microM), which did not affect acetylene reduction but inhibited whole-cell respiration by 60 to 70%, shifted the O2 concentration that caused 50% inhibition of nitrogenase activity to 2.9 microM. A mutant strain of K. pneumoniae, strain AH11, has a respiration rate that is 65 to 75% higher than that of the wild type, but its nitrogenase activity is similar to wild-type activity. Acetylene reduction by whole cells of this mutant was inhibited 50% by 0.20 microM O2. Inhibition by CN- of 40 to 50% of the O2 uptake in the mutant shifted the O2 concentration that caused 50% inhibition of nitrogenase to 1.58 microM. Thus, when the respiration rates were lower, higher oxygen concentrations were required to inhibit nitrogenase. Reversible inhibition of nitrogenase activity in vivo was caused under anaerobic conditions by other electron acceptors. Addition of 2 mM sulfite to cell suspensions of R. capsulata B10 and R. sphaeroides inhibited nitrogenase activity. Nitrite also inhibited acetylene reduction in whole cells of the photodenitrifier R. sphaeroides but not in R. capsulata B10, which is not capable of enzymatic reduction of NO2-. Lower concentrations of NO2- were required to inhibit the activity in NO3- -grown cells, which have higher activities of nitrite reductase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Carbonic Anhydrase-Deficient Mutant of Chlamydomonas reinhardii Requires Elevated Carbon Dioxide Concentration for Photoautotrophic Growth

A mendelian mutant of the unicellular green alga Chlamydomonas reinhardii has been isolated which is deficient in carbonic anhydrase (EC 4.2.1.1) activity. This mutant strain, designated ca-1-12-1C (gene locus ca-1), was selected on the basis of a high CO₂ requirement for photoautotrophic growth. Photosynthesis by the mutant at atmospheric CO₂ concentration was very much reduced compared to wild type and, unlike wild type, was strongly inhibited by O₂. In contrast to a CO₂ compensation concentration of near zero in wild type at all O₂ concentrations examined, the mutant exhibited a high, O₂-stimulated CO₂ compensation concentration. Evidence of photorespiratory activity in the mutant but not in wild type was obtained from the analysis of photosynthetic products in the presence of ¹⁴CO₂. At air levels of CO₂ and O₂, the mutant synthesized large amounts of glycolate, while little glycolate was synthesized by wild type under identical conditions. Both mutant and wild type strains formed only small amounts of glycolate at saturating CO₂ concentration. At ambient CO₂, wild type accumulated inorganic carbon to a concentration several-fold higher than that in the suspension medium. The mutant cells accumulated inorganic carbon internally to a concentration 6-fold greater than found in wild type, yet photosynthesis was CO₂ limited. The mutant phenotype was mimicked by wild type cells treated with ethoxyzolamide, an inhibitor of carbonic anhydrase activity. These observations indicate a requirement for carbonic anhydrase-catalyzed dehydration of bicarbonate in maintaining high internal CO₂ concentrations and high photosynthesis rates. Thus, in wild type cells, carbonic anhydrase rapidly converts the bicarbonate taken up to CO₂, creating a high internal CO₂ concentration which stimulates photosynthesis and suppresses photorespiration. In mutant cells, bicarbonate is taken up rapidly but, because of a carbonic anhydrase deficiency, is not dehydrated at a rate sufficiently rapid to maintain a high internal CO₂ concentration.     

Low ascorbic acid in the vtc-1 mutant of Arabidopsis is associated with decreased growth and intracellular redistribution of the antioxidant system

Ascorbic acid has numerous and diverse roles in plant metabolism. We have used the vtc-1 mutant of Arabidopsis, which is deficient in ascorbate biosynthesis, to investigate the role of ascorbate concentration in growth, regulation of photosynthesis, and control of the partitioning of antioxidative enyzmes. The mutant possessed 70% less ascorbate in the leaves compared with the wild type. This lesion was associated with a slight increase in total glutathione but no change in the redox state of either ascorbate or glutathione. In vtc-1, total ascorbate in the apoplast was decreased to 23% of the wild-type value. The mutant displayed much slower shoot growth than the wild type when grown in air or at high CO(2) (3 mL L(-1)), where oxidative stress is diminished. Leaves were smaller, and shoot fresh weight and dry weight were lower in the mutant. No significant differences in the light saturation curves for CO(2) assimilation were found in air or at high CO(2), suggesting that the effect on growth was not due to decreased photosynthetic capacity in the mutant. Analysis of chlorophyll a fluorescence quenching revealed only a slight effect on non-photochemical energy dissipation. Hydrogen peroxide contents were similar in the leaves of the vtc-1 mutant and the wild type. Total leaf peroxidase activity was increased in the mutant and compartment-specific differences in ascorbate peroxidase (APX) activity were observed. In agreement with the measurements of enzyme activity, the expression of cytosolic APX was increased, whereas that for chloroplast APX isoforms was either unchanged or slightly decreased. These data implicate ascorbate concentration in the regulation of the compartmentalization of the antioxidant system in Arabidopsis.     

Saccharomyces cerevisiae Bor1p is a boron exporter and a key determinant of boron tolerance

Boron is toxic to living organisms when present in excess. Saccharomyces cerevisiae Bor1p is a plasma membrane protein that decreases the intracellular concentration of boron and confers boron tolerance in yeasts. We investigated the detailed characteristics of boron transport by Bor1p and its roles in boron tolerance. Boron transport assays showed that the bor1 deletion mutant (bor1Delta) accumulates higher intracellular concentrations of boron and has a lower rate of boron export. The bor1Delta showed greater susceptibility to high concentrations of boron than the wild-type strain, and the growth rates of both strains were negatively correlated with the intracellular concentrations of boron. With normal to toxic levels of external boron, green fluorescent protein (GFP)-tagged Bor1p localized to the plasma membrane irrespective of the concentration of boron in the medium. Taken together, these results establish Bor1p as a plasma membrane boron exporter and a key determinant of boron tolerance.     

Characterization of the structural and functional defect in the Escherichia coli single-stranded DNA binding protein encoded by the ssb-1 mutant gene. Expression of the ssb-1 gene under lambda pL regulation

The ssb-1 gene encoding a mutant single-stranded DNA binding protein (SSB-1) has been cloned into a vector placing its expression under lambda pL regulation. This construction results in more than 100-fold increased expression of the mutant protein following temperature induction. Tryptic peptide analysis of the mutant protein by high-pressure liquid chromatography and solid-phase protein sequencing has shown that the ssb-1 mutation results in these substitution of tyrosine for histidine at residue 55 of SSB. This change could only occur in one step by a C----T transition in the DNA sequence which has been confirmed. Physicochemical studies of the homogeneous mutant protein have shown that in contrast to that of the wild-type SSB, the tetrameric structure of SSB-1 is unstable and gradually dissociates to monomer as the protein concentration is decreased from about 10 microM to less than 0.5 microM. The SSB-1 tetramer appears to be stable to elevated temperature (45 degrees C) but the monomer is not. We estimate the normal cellular concentration of SSB-1 (single chromosomal gene) to be 0.5-1 microM. Thus, there is a plausible physical explanation for our previous finding that increased expression of ssb-1 reverses the effects of a single gene (chromosomal) copy amount of SSB-1 (Chase, J.W., Murphy, J.B., Whittier, R.F., Lorensen, E., and Sninsky, J.J. (1983) J. Mol. Biol. 164, 193-211). However, even though the in vivo effects of ssb-1 and most of the in vitro defects of SSB-1 protein are reversed simply by increasing SSB-1 protein concentration, the mutant protein is not as effective a helix-destabilizing protein as wild-type SSB as measured by its ability to lower the thermal melting transition of poly[d-(A-T)].     

The role of a single N-linked glycosylation site for a functional epitope of herpes simplex virus type 1 envelope glycoprotein gC

A monoclonal antibody, B1C1, binding to an epitope of antigenic site II of the herpes simplex virus type 1 (HSV-1) glycoprotein gC-1, is a potent inhibitor of two important biological functions of gC-1: its binding to cell surface heparan sulfate and its binding to the receptor for complement factor C3b. Here, we have analyzed a B1C1-resistant HSV- 1 variant (HSV-12762/B1C1B4.2), obtained after passage of wild type HSV- 1 (HSV-12762) in the presence of high concentrations of B1C1. The transport of newly synthesized mutant gC-1 to the cell surface was comparable to that of wild type glycoprotein, but no binding of surface- associated mutant gC-1 to B1C1 was detected. However, mutant and wild type gC-1 bound equally well to other site II Mabs. Attachment of wild type but not mutant virus was inhibited by B1C1. Sequencing of the mutant gC-1 gene revealed only one nucleotide change, resulting in replacement of Thr150 by an Ile, in turn destroying an N-glycosylation site at Asn148. Loss of one complex type N-linked glycan was confirmed by endoglycosidase digestion and subsequent SDS-polyacrylamide gel electrophoresis. Circular dichroism analysis of purified gC-1 from cells infected with mutant or wild type virus did not reveal any difference in secondary structure between mutant and wild type gC-1. It was not possible to obtain a B1C1-resistant phenotype by nucleotide- directed mutagenesis of gC-1 where Asn148 was changed to a glutamine. These data demonstrated that the threonine of the glycosylation site and not the N-linked glycan in itself was essential for B1C1 binding      

Arsenic uptake, translocation and speciation in pho1 and pho2 mutants of Arabidopsis thaliana

Arsenate [As (V)] is taken up by phosphate [P (V)] transporters in the plasma membrane of roots cells, but the translocation of As from roots to shoots is not well understood. Two mutants of Arabidopsis thaliana (L.) [( pho1 , P deficient) and ( pho2 , P accumulator)], with defects in the regulation and translocation of P (V) from roots to shoots, were therefore used in this study to investigate uptake, translocation and speciation of As in roots and shoots of plants grown in soil or nutrient solution. The shoots of the pho2 mutant contained higher P concentrations, but similar or slightly higher As concentrations, in comparison with the wild type. In the pho1 mutant, the P concentration in the shoots was lower, and the As concentration was higher, in comparison with the wild type. Both pho2 and the wild type contained mainly As (III) in roots and shoot (67–90% of total As). Arsenic was likely to be translocated by a different pathway to P (V) in the pho2 and pho1 mutants . Therefore, it is suggested that As (III) is the main As species translocated from roots to shoots in Arabidopsis thaliana.     

Altered nucleoside transporters in mammalian cells selected for resistance to the physiological effects of inhibitors of nucleoside transport

From a mutagenized population of wild type S49 T lymphoma cells, clones were generated that were resistant to the physiological effects of the potent inhibitor of nucleoside transport, 4-nitrobenzyl-6-thioinosine (NBMPR). These cells were selected for their ability to survive in semisolid medium containing 0.5 mM hypoxanthine, 0.4 microM methotrexate, 30 microM thymidine, 30 microM deoxycytidine, in the presence of 30 microM NBMPR. NBMPR protected wild type cells from the effects of a spectrum of cytotoxic nucleosides, whereas two mutant clones, KAB1 and KAB5, were still sensitive to nucleoside-mediated cytotoxicity in the presence of NBMPR. Comparisons of the abilities of wild type cells and mutant cells to incorporate exogenous nucleoside to the corresponding nucleoside triphosphate indicated that the KAB1 and KAB5 mutant cells were refractory to normal inhibition by NBMPR. Moreover, rapid transport studies indicated that mutant cells, unlike wild type parental cells, had acquired a substantial NBMPR-insensitive nucleoside transport component. Binding studies with [3H]NBMPR indicated that KAB5 cells were 70-75% deficient in the number of NBMPR binding sites, whereas KAB1 cells possessed a wild type complement of NBMPR binding sites. These data suggest that the NBMPR binding site in wild type S49 cells is genetically distinguishable from the nucleoside carrier site.     

Site-directed mutagenesis of GLUT1 in helix 7 residue 282 results in perturbation of exofacial ligand binding.

The structure-function relationship of the HepG2/erythrocyte-type glucose transporter (GLUT1) has been studied by in vitro site-directed mutagenesis. Chinese hamster ovary clones in which glucose transporters were transfected were shown by Western blotting with a GLUT1 anti-COOH-terminal peptide antibody to have expression levels of Gln282----Leu, Asn288----Ile, and Asn317----Ile mutations that were comparable with the wild type. All three mutant GLUT1 clones had high 2-deoxy-D-glucose transport activity compared with a nontransfected clone, suggesting that these residues are not absolutely required for the transport function. We have examined the possibility that the inner and outer portions of the transport pathway are structurally separate by measuring the interaction of the mutant transporters with the inside site-specific ligand cytochalasin B and the outside site-specific ligand 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannos-4 -yloxy)-2- propylamine (ATB-BMPA). All three mutant GLUT1 clones showed high levels of cytochalasin B labeling, and the N288I and N317I mutants showed high levels of ATB-BMPA labeling. In contrast to the transport and cytochalasin B labeling results, the transmembrane helix 7 Gln282----Leu mutant was labeled by ATB-BMPA to a level that was only 5% of the level observed in the wild type. We have confirmed that this mutant was defective in the outer site by comparing the inhibition of wild-type and mutant 2-deoxy-D-glucose transport by the outside site-specific ligand 4,6-O-ethylidene-D-glucose. 4,6-O-Ethylidene-D-glucose inhibited wild-type transport with a Ki of approximately 12 mM, but this was increased to greater than 120 mM in the Gln282----Leu mutant. Thus, of the 3 residues mutated in this study, only glutamine 282 substitution causes a major perturbation in function, and this is a specific and striking reduction in the affinity for the outside site-specific ligands ATB-BMPA and 4,6-O-ethylidene-D-glucose.     

Membrane potential defect in hygromycin B-resistant pma1 mutants of Saccharomyces cerevisiae

The proton transport properties of hygromycin B-resistant pma1 mutants which show kinetic defects in the plasma membrane H+-ATPase were examined. It was found that net proton efflux, as measured by whole cell medium acidification in the presence of 25 mM KCl, was similar for normal and pma1 mutant cells. However, in the absence of added KCl, the extent of net proton efflux was considerably less in wild type than in pma1 mutant cells. The cellular membrane potential was implicated as an important factor in regulating net proton transport and was determined from [14C]tetraphenylphosphonium uptake studies to be considerably depolarized in the pma1 mutants. The growth of wild type cells, which is normally inhibited by hygromycin B at 200 micrograms/ml, was found to be resistant to the antibiotic by the addition of 50 mM KCl to the growth medium. These results suggest that the electrogenic behavior of proton transport by the H+-ATPase may be altered in pma1 mutants and that resistance to hygromycin B may be mediated via depolarization of the cellular membrane potential.     

Insertion of an elastase-binding loop into interleukin-1 beta

The protease-binding sequence EAIPMSIPPE from alpha 1-antitrypsin has been inserted into the cytokine interleukin-1 beta, replacing residues 50-53. The resulting mutant protein was cleaved specifically at a single site by elastase and chymotrypsin, but not by trypsin. The cleavage by elastase was shown to be between Met and Ser of the inserted loop. In contrast, wild-type interleukin is not susceptible to cleavage by any of these enzymes. The mutant protein acts as an inhibitor of elastase, with a KI of approximately 30 microM. The wild type displays no such inhibitory activity. The overall structure of the mutant, as demonstrated by CD, appears to be indistinguishable from that of the wild type. These results indicate that the protease-binding region of alpha 1-antitrypsin can be recognized and is active even within the context of an entirely different protein structure. Given that interleukin-1 beta binds to, and is internalized by, many types of cells, this hybrid protein also demonstrates the feasibility of using interleukin-1 beta as a delivery system for useful therapeutic agents.     

Schizosaccharomyces pombe Pmr1p is essential for cell wall integrity and is required for polarized cell growth and cytokinesis

The cps5-138 fission yeast mutant shows an abnormal lemon-like morphology at 28 degrees C in minimal medium and a lethal thermosensitive phenotype at 37 degrees C. Cell growth is completely inhibited at 28 degrees C in a Ca2+-free medium, in which the wild type is capable of growing normally. Under these conditions, actin patches become randomly distributed throughout the cell, and defects in septum formation and subsequent cytokinesis appear. The mutant cell is hypersensitive to the cell wall-digesting enzymatic complex Novozym234 even under permissive conditions. The gene SPBC31E1.02c, which complements all the mutant phenotypes described above, was cloned and codes for the Ca2+-ATPase homologue Pmr1p. The gene is not essential under optimal growth conditions but is required under conditions of low Ca2+ (<0.1 mM) or high temperature (>35 degrees C). The green fluorescent protein-tagged Cps5 proteins, which are expressed under physiological conditions (an integrated single copy with its own promoter in the cps5Delta strain), display a localization pattern typical of endoplasmic reticulum proteins. Biochemical analyses show that 1,3-beta-D-glucan synthase activity in the mutant is decreased to nearly half that of the wild type and that the mutant cell wall contains no detectable galactomannan when the cells are exposed to a Ca2+-free medium. The mutant acid phosphatase has an increased electrophoretic mobility, suggesting that incomplete protein glycosylation takes place in the mutant cells. These results indicate that S. pombe Pmr1p is essential for the maintenance of cell wall integrity and cytokinesis, possibly by allowing protein glycosylation and the polarized actin distribution to take place normally. Disruption and complementation analyses suggest that Pmr1p shares its function with a vacuolar Ca2+-ATPase homologue, Pmc1p (SPAPB2B4.04c), to prevent lethal activation of calcineurin for cell growth.     

Protein phosphatase type 2B (calcineurin)-mediated, FK506-sensitive regulation of intracellular ions in yeast is an important determinant for adaptation to high salt stress conditions.

To assess the physiological function of Ca(2+)-dependent protein phosphatase (PP2B) in the yeast Saccharomyces cerevisiae, the phenotypes of PP2B-deficient mutants were investigated. Although PP2B was dispensable for growth under normal conditions, the mutations did, however, cause growth inhibition under certain stress circumstances. The growth of the mutants was inhibited by NaCl and LiCl, but not by KCl, CaCl2, MgCl2 or nonspecific osmotic stresses. Upon shift to high NaCl medium, intracellular Na+ levels of both wild type yeast and the mutants initially increased at a comparable rate. However, internal Na+ in wild type cells started to decline more rapidly than the mutant cells during cultivation in high NaCl medium, indicating that PP2B is important in maintaining a gradient across the membrane. The protection against salt stress was achieved, at least in part, by the stimulation of Na+ export. The maintenance of a high level of internal K+ in high NaCl medium was also PP2B-dependent. In the presence of the immunosuppressant FK506, the growth behaviour and intracellular Na+ and K+ of wild type cells in high NaCl medium became very similar to those of the PP2B-deficient mutant in a manner dependent on the presence of the FK506 binding protein.     

The Saccharomyces cerevisiae Ca2+ channel Cch1pMid1p is essential for tolerance to cold stress and iron toxicity

Cch1p and Mid1p are components of a high-affinity Ca(2+)-permeable channel in the yeast plasma membrane. Here, we show that growth of mutants in the Cch1pMid1p channel is markedly hypersensitive to low temperature and to high iron concentration in the medium. Both phenotypes were suppressed by high Ca(2+) concentration. Iron stress elicited an increased Ca(2+) influx into both wild type and cch1Deltamid1Delta yeast. Inhibition of calcineurin strongly depressed growth of iron-stressed wild type yeast, indicating that calcineurin is a downstream element of the iron stress response. Iron hypersensitivity of the cch1Deltamid1Delta mutant was not associated with an increased iron uptake. An involvement of oxidative stress in the iron-hypersensitive phenotype was indicated by the findings that the antioxidants tocopheryl acetate and (ethyl)glutathione improved growth and viability of the iron-stressed mutant. Further, the degree of glutathione oxidation was increased in the presence of iron. The results indicate that iron stress leads to an increased oxidative poise and that Cch1pMid1p is essential to tolerate this condition.