[Show abstract][Hide abstract] ABSTRACT: Alkaline pH stress invokes a potent and fast transcriptional response in Saccharomyces cerevisiae that includes many genes repressed by glucose. Certain mutants in the glucose-sensing and -response pathways, such as those lacking the Snf1 kinase, are sensitive to alkalinization. In the present study we show that the addition of glucose to the medium improves the growth of wild-type cells at high pH, fully abolishes the snf1 alkali-sensitive phenotype and attenuates high pH-induced Snf1 phosphorylation at Thr(210). Lack of Elm1, one of the three upstream Snf1 kinases (Tos3, Elm1 and Sak1), markedly increases alkali sensitivity, whereas the phenotype of the triple mutant tos3 elm1 sak1 is even more pronounced than that of snf1 cells and is poorly rescued by glucose supplementation. DNA microarray analysis reveals that about 75% of the genes induced in the short term by high pH are also induced by glucose scarcity. Snf1 mediates, in full or in part, the activation of a significant subset (38%) of short-term alkali-induced genes, including those encoding high-affinity hexose transporters and phosphorylating enzymes. The induction of genes encoding enzymes involved in glycogen, but not trehalose, metabolism is largely dependent of the presence of Snf1. Therefore the function of Snf1 in adaptation to glucose scarcity appears crucial for alkaline pH tolerance. Incorporation of micromolar amounts of iron and copper to a glucose-supplemented medium resulted in an additive effect and allows near-normal growth at high pH, thus indicating that these three nutrients are key limiting factors for growth in an alkaline environment.
[Show abstract][Hide abstract] ABSTRACT: The phosphatase calcineurin and the kinases Hal4/Hal5 regulate high-affinity potassium uptake in Saccharomyces cerevisiae through the Trk1 transporter. We demonstrate that calcineurin is necessary for high-affinity potassium uptake even in the absence of Na(+) stress. HAL5 expression is induced in response to stress in a calcineurin-dependent manner through a newly identified functional CDRE (nt -195/-189). Lack of calcineurin decreases Hal5 protein levels, although with little effect on Trk1 amounts. However, the growth defect of cnb1 cells at K(+)-limiting conditions can be rescued in part by overexpression of HAL5, and this mutation further aggravates the potassium requirements of a hal4 strain. This suggests that the control exerted by calcineurin on Hal5 expression may be biologically relevant for Trk1 regulation.
[Show abstract][Hide abstract] ABSTRACT: Unlike most other organisms, the essential five-step coenzyme A biosynthetic pathway has not been fully resolved in yeast. Specifically, the genes encoding the phosphopantothenoylcysteine decarboxylase (PPCDC) activity still remain unidentified. Sequence homology analyses suggest three candidates-Ykl088w, Hal3 and Vhs3-as putative PPCDC enzymes in Saccharomyces cerevisiae. Notably, Hal3 and Vhs3 have been characterized as negative regulatory subunits of the Ppz1 protein phosphatase. Here we show that YKL088w does not encode a third Ppz1 regulatory subunit, and that the essential roles of Ykl088w and the Hal3 and Vhs3 pair are complementary, cannot be interchanged and can be attributed to PPCDC-related functions. We demonstrate that while known eukaryotic PPCDCs are homotrimers, the active yeast enzyme is a heterotrimer that consists of Ykl088w and Hal3/Vhs3 monomers that separately provides two essential catalytic residues. Our results unveil Hal3 and Vhs3 as moonlighting proteins involved in both CoA biosynthesis and protein phosphatase regulation.
Nature Chemical Biology 12/2009; 5(12):920-8. · 12.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Failure to use glucose as carbon source results in transcriptional activation of numerous genes whose expression is otherwise repressed. HXT2 encodes a yeast high affinity glucose transporter that is only expressed under conditions of glucose limitation. We show that HXT2 is rapidly and potently induced by environmental alkalinization, and this requires both the Snf1 and the calcineurin pathways. Regulation by calcineurin is mediated by the transcription factor Crz1, which rapidly translocates to the nucleus upon high pH stress, and acts through a previously unnoticed Crz1-binding element (calcineurin-dependent response element) in the HXT2 promoter (-507 GGGGCTG -501). We demonstrate that, in addition to HXT2, many other genes required for adaptation to glucose shortage, such as HXT7, MDH2, or ALD4, transcriptionally respond to calcium and high pH signaling through binding of Crz1 to their promoters. Therefore, calcineurin-dependent transcriptional regulation appears to be a common feature for many genes encoding carbohydrate-metabolizing enzymes. Remarkably, extracellular calcium allows growth of a snf1 mutant on low glucose in a calcineurin/Crz1-dependent manner, indicating that activation of calcineurin is sufficient to override a major deficiency in the glucose-repression pathway. We propose that alkalinization of the medium results in impaired glucose utilization and that activation of certain glucose-metabolizing genes by calcineurin contributes to yeast survival under this stress situation.
Journal of Biological Chemistry 06/2008; 283(20):13923-33. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Zinc is an essential metal that, when in excess, can be deleterious to the cell. Therefore, homeostatic mechanisms for this cation must be finely tuned. To better understand the response of yeast in front of an excess of zinc, we screened a systematic deletion mutant library for altered growth in the presence of 6 mM zinc. Eighty-nine mutants exhibited increased zinc sensitivity, including many genes involved in vacuolar assembling and biogenesis. Interestingly, a mutant lacking the Aft1 transcription factor, required for the transcriptional response to iron starvation, was found to be highly sensitive to zinc. Genome-wide transcriptional profiling revealed that exposure to 5 mM ZnCl(2) results in rapid increase in the expression of numerous chaperones required for proper protein folding or targeting to vacuole and mitochondria, as well as genes involved in stress response (mainly oxidative), sulphur metabolism and some components of the iron regulon. The effect of the lack of Aft1 both in the absence and in the presence of zinc overload was also investigated. Exposure to high zinc generated reactive oxygen species and markedly decreased glutathione content. Interestingly, zinc excess results in decreased intracellular iron content and aconitase and cytochrome c activities in stationary-phase cultures. These findings suggest that high zinc levels may alter the assembly and/or function of iron-sulphur-containing proteins, as well as the biosynthesis of haem groups, thus establishing a link between zinc, iron and sulphur metabolism.
[Show abstract][Hide abstract] ABSTRACT: Alkalinization of the external environment represents a stress situation for Saccharomyces cerevisiae. Adaptation to this circumstance involves the activation of diverse response mechanisms, the components of which are still largely unknown. We show here that mutation of members of the cell integrity Pkc1/Slt2 MAPK module, as well as upstream and downstream elements of the system, confers sensitivity to alkali. Alkalinization resulted in fast and transient activation of the Slt2 MAPK, which depended on the integrity of the kinase module and was largely abolished by sorbitol. Lack of Wsc1, removal of specific extracellular and intracellular domains, or substitution of Tyr(303) in this putative membrane stress sensor rendered cells sensitive to alkali and considerably decreased alkali-induced Slt2 activation. In contrast, constitutive activation of Slt2 by the bck1-20 allele increased pH tolerance in the wsc1 mutant. DNA microarray analysis revealed that several genes encoding cell wall proteins, such as GSC2/FKS2, DFG5, SKT5, and CRH1, were induced, at least in part, by high pH in an Slt2-dependent manner. We observed that dfg5, skt5, and particularly dfg5 skt5 cells were alkali-sensitive. Therefore, our results show that an alkaline environment imposes a stress condition on the yeast cell wall. We propose that the Slt2-mediated MAPK pathway plays an important role in the adaptive response to this insult and that Wsc1 participates as an essential cell-surface pH sensor. Moreover, these results provide a new example of the complexity of the response of budding yeast to the alkalinization of the environment.
Journal of Biological Chemistry 01/2007; 281(52):39785-95. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Adaptive response of the yeast Saccharomyces cerevisiae to environmental alkalinization results in remodeling of gene expression. A key target is the gene ENA1, encoding a Na(+)-ATPase, whose induction by alkaline pH has been shown to involve calcineurin and the Rim101/Nrg1 pathway. Previous functional analysis of the ENA1 promoter revealed a calcineurin-independent pH responsive region (ARR2, 83 nucleotides). We restrict here this response to a small (42 nucleotides) ARR2 5.-region, named MCIR (minimum calcineurin independent response), which contains a MIG element, able to bind Mig1,2 repressors. High pH-induced response driven from this region was largely abolished in snf1 cells and moderately reduced in a rim101 strain. Cells lacking Mig1 or Mig2 repressors had a near wild type response, but the double mutant presented a high level of expression upon alkaline stress. Deletion of NRG1 (but not of NRG2) resulted in increased expression. Induction from the MCIR region was marginal in a quadruple mutant lacking Nrg1,2 and Mig1,2 repressors. In vitro band shift experiments demonstrated binding of Nrg1 to the 5. end of the ARR2 region. Furthermore, we show that Nrg1 binds in vivo around the MCIR region under standard growth conditions, and that binding is largely abolished after high pH stress. Therefore, the calcineurin-independent response of the ENA1 gene is under the regulation of Rim101 (through Nrg1) and Snf1 (through Nrg1 and Mig2). Accordingly, induction by alkaline stress of the entire ENA1 promoter in a snf1 rim101 mutant in the presence of the calcineurin inhibitor FK506 is completely abolished. Thus, the transcriptional response to alkaline stress of the ENA1 gene integrates three different signaling pathways.
Journal of Biological Chemistry 01/2007; 281(48):36632-42. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Type 2C protein phosphatases are encoded in Saccharomyces cerevisiae by several related genes (PTC1-5 and PTC7). To gain insight into the functions attributable to specific members of this gene family, we have investigated the transcriptional profiles of ptc1-5 mutants. Two main patterns were obtained as follows: the one generated by the ptc1 mutation and the one resulting from the lack of Ptc2-5. ptc4 and ptc5 profiles were quite similar, whereas that of ptc2 was less related to this group. Mutation of PTC1 resulted in increased expression of numerous genes that are also induced by cell wall damage, such as YKL161c, SED1, or CRH1, as well as in higher amounts of active Slt2 mitogen-activated protein kinase, indicating that lack of the phosphatase activates the cell wall integrity pathway. ptc1 cells were even more sensitive than slt2 mutants to a number of cell wall-damaging agents, and both mutations had additive effects. The sensitivity of ptc1 cells was not dependent on Hog1. Besides these phenotypes, we observed that calcineurin was hyperactivated in ptc1 cells, which were also highly sensitive to calcium ions, heavy metals, and alkaline pH, and exhibited a random haploid budding pattern. Remarkably, many of these traits are found in certain mutants with impaired vacuolar function. As ptc1 cells also display fragmented vacuoles, we hypothesized that lack of Ptc1 would primarily cause vacuolar malfunction, from which other phenotypes would derive. In agreement with this scenario, overexpression of VPS73, a gene of unknown function involved in vacuolar protein sorting, largely rescues not only vacuolar fragmentation but also sensitivity to cell wall damage, high calcium, alkaline pH, as well as other ptc1-specific phenotypes.
Journal of Biological Chemistry 12/2006; 281(46):35057-69. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Exposure of the yeast Saccharomyces cerevisiae to alkaline stress resulted in adaptive changes that involved remodeling the gene expression. Recent evidence suggested that the calcium-activated protein phosphatase calcineurin could play a role in alkaline stress signaling. By using an aequorin luminescence reporter, we showed that alkaline stress resulted in a sharp and transient rise in cytoplasmic calcium. This increase was largely abolished by addition of EGTA to the medium or in cells lacking Mid1 or Cch1, components of the high affinity cell membrane calcium channel. Under these circumstances, the alkaline response of different calcineurin-sensitive transcriptional promoters was also blocked. Therefore, exposure to alkali resulted in entry of calcium from the external medium, and this triggered a calcineurin-mediated response. The involvement of calcineurin and Crz1/Tcn1, the transcription factor activated by the phosphatase, in the transcriptional response triggered by alkalinization has been globally assessed by DNA microarray analysis in a time course experiment using calcineurin-deficient (cnb1) and crz1 mutants. We found that exposure to pH 8.0 increased at least 2-fold the mRNA levels of 266 genes. In many cases (60%) the response was rather early (peak after 10 min). The transcriptional response of 27 induced genes (10%) was reduced or fully abolished in cnb1 cells. In general, the response of crz1 mutants was similar to that of calcineurin-deficient cells. By analysis of a systematic deletion library, we found 48 genes whose mutation resulted in increased sensitivity to the calcineurin inhibitor FK506. Twenty of these mutations (42%) also provoked alkaline pH sensitivity. In conclusion, our results demonstrated that calcium signaling and calcineurin activation represented a significant component of the yeast response to environmental alkalinization.
Journal of Biological Chemistry 11/2004; 279(42):43614-24. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The yeast gene VHS3 (YOR054c) has been recently identified as a multicopy suppressor of the G(1)/S cell cycle blockade of a conditional sit4 and hal3 mutant. Vhs3 is structurally related to Hal3, a negative regulatory subunit of the Ser/Thr protein phosphatase Ppz1 important for cell integrity, salt tolerance, and cell cycle control. Phenotypic analyses using vhs3 mutants and overexpressing strains clearly show that Vhs3 has functions reminiscent to those of Hal3 and contrary to those of Ppz1. Mutation of Vhs3 His(459), equivalent to the supposedly functionally relevant His(90) in the plant homolog AtHal3a, did not affect Vhs3 functions mentioned above. Similarly to Hal3, Vhs3 binds in vivo to the C-terminal catalytic moiety of Ppz1 and inhibits in vitro its phosphatase activity. Therefore, our results indicate that Vhs3 plays a role as an inhibitory subunit of Ppz1. We have found that the vhs3 and hal3 mutations are synthetically lethal. Remarkably, lethality is not suppressed by deletion of PPZ1, PPZ2, or both phosphatase genes, indicating that it is not because of an excess of Ppz phosphatase activity. Furthermore, a Vhs3 version carrying the H459A mutation did not rescue the synthetically lethal phenotype. A conditional vhs3 tetO:HAL3 double mutant displays, in the presence of doxycycline, a flocculation phenotype that is dependent on the presence of Flo8 and Flo11. These results indicate that, besides its role as Ppz1 inhibitory subunit, Vhs3 (and probably Hal3) might have important Ppz-independent functions.
Journal of Biological Chemistry 09/2004; 279(33):34421-30. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Exposure of the yeast Saccharomyces cerevisiae to an alkaline environment represents a stress situation that negatively affects growth and results in an adaptive transcriptional response. We screened a collection of 4825 haploid deletion mutants for their ability to grow at mild alkaline pH, and we identified 118 genes, involved in numerous cellular functions, whose absence results in reduced growth. The list includes several key genes in copper and iron homeostasis, such as CCC2, RCS1, FET3, LYS7, and CTR1. In contrast, a screen of high-copy number plasmid libraries for clones able to increase tolerance to alkaline pH revealed only two genes: FET4 (encoding a low affinity transporter for copper, iron, and zinc) and CTR1 (encoding a high affinity copper transporter). The beneficial effect of overexpression of CTR1 requires a functional high affinity iron transport system, as it was abolished by deletion of FET3, a component of the high affinity transport system, or CCC2, which is required for assembly of the transport system. The growth-promoting effect of FET4 was not modified in these mutants. These results suggest that the observed tolerance to alkaline pH is because of improved iron uptake and indicate that both iron and copper are limiting factors for growth under alkaline pH conditions. Addition to the medium of micromolar concentrations of copper or iron ions drastically improved growth at high pH. Supplementation with iron improved somewhat the tolerance of a fet3 strain but was ineffective in a ctr1 mutant, suggesting the existence of additional copper-requiring functions important for tolerance to an alkaline environment.
Journal of Biological Chemistry 06/2004; 279(19):19698-704. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An optimised Agrobacterium-mediated gene transfer protocol was developed in order to obtain watermelon transgenic plants [Citrullus lanatus (Thunb.) Matsun. & Nakai.]. Transformation efficiencies ranged from 2.8% to 5.3%, depending on the cultivar. The method was applied to obtain genetically engineered watermelon plants expressing the Saccharomyces cerevisiae HAL1 gene related to salt tolerance. In order to enhance its constitutive expression in plants, the HAL1 gene was cloned in a pBiN19 plasmid under control of the 35S promoter with a double enhancer sequence from the cauliflower mosaic virus and the RNA4 leader sequence of the alfalfa mosaic virus. This vector was introduced into Agrobacterium tumefaciens strain LBA4404 for further inoculation of watermelon half-cotyledon explants. The introduction of both the neomycin phosphotransferase II and HAL1 genes was assessed in primary transformants (TG1) by polymerase chain reaction analysis and Southern hybridisation. The expression of the HAL1 gene was determined by Northern analysis, and the diploid level of transgenic plants was confirmed by flow cytometry. The presence of the selectable marker gene in the expected Mendelian ratios was demonstrated in TG2 progenies. The TG2 kanamycin-resistant plantlets elongated better and produced new roots and leaves in culture media supplemented with NaCl compared with the control. Salt tolerance was confirmed in a semi-hydroponic system (EC=6 dS m(-1)) on the basis of the higher growth performance of homozygous TG3 lines with respect to their respective azygous control lines without the transgene. The halotolerance observed confirmed the inheritance of the trait and supports the potential usefulness of the HAL1 gene of S. cerevisiae as a molecular tool for genetic engineering of salt-stress protection in other crop species.
Theoretical and Applied Genetics 08/2003; 107(3):462-9. · 3.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The short-time transcriptional response of yeast cells to a mild increase in external pH (7.6) has been investigated using DNA microarrays. A total of 150 genes increased their mRNA level at least twofold within 45 min. Alkalinization resulted in the repression of 232 genes. The response of four upregulated genes, ENA1 (encoding a Na+-ATPase also induced by saline stress) and PHO84, PHO89 and PHO12 (encoding genes upregulated by phosphate starvation), was characterized further. The alkaline response of ENA1 was not affected by mutation of relevant genes involved in osmotic or oxidative signalling, but was decreased in calcineurin and rim101 mutants. Mapping of the ENA1 promoter revealed two pH-responsive regions. The response of the upstream region was fully abolished by the drug FK506 or mutation of CRZ1 (a transcription factor activated by calcium/calcineurin), whereas the response of the downstream region was essentially calcium independent. PHO84 and PHO12 responses were unaffected in crz1 cells, but required the presence of Pho2 and Pho4. In contrast, part of the alkali-induced expression of PHO89 was maintained in pho4 or pho2 cells, but was fully abolished in a crz1 strain or in the presence of FK506. Heterologous promoters carrying the minimal calcineurin-dependent response elements found in ENA1 or FKS2 were able to drive alkaline pH-induced expression. These results demonstrate that the transcriptional response to alkaline pH involves different signalling mechanisms, and that calcium signalling is a relevant component of this response.
[Show abstract][Hide abstract] ABSTRACT: Desiccation has many detrimental effects on the structure and function of biological membranes and proteins and this molecular damage decreases the freshness appearance of dehydrated foods. Phospholipid membranes are destabilised upon water stress by insertion of cellular amphiphiles, phase transition into the gel phase and membrane fusion. Proteins are denatured and electron transport chains are perturbed leading to increased formation of reactive oxygen species which cause irreversible damage of cellular structures. Cells respond to water stress by generating defense proteins and metabolites and eventually develop outstanding desiccation tolerance such as in the case of plant seeds and pollen, fungal spores, crustacean cysts, etc. The molecular bases for this remarkable phenomenon are not completely understood but several important principles have been identified. Three biological systems seem to act in concert to achieve desiccation tolerance: enzymes involved in osmolyte synthesis; proteins specialised in desiccation protection of membranes and proteins (LEA proteins), and antioxidant enzymes and molecules. Both osmolytes and LEA proteins contribute to stabilisation of membrane and protein structures by conferring preferential hydration at moderate desiccation and replacing water at extreme desiccation. Osmolytes also contribute to osmotic adjustment and act as hydroxyl radical scavengers. Genetically modified plants with increased production of these defenses could be useful to improve the quality of dried food.
Food Science and Technology International 01/2003; 9(3):157-161. · 0.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lithium cations exert profound and selective psychopharmacological effects on ameliorate manic-depressive psychosis. Although lithium is an effective drug for both treatment and prophylaxis of bipolar disorder, the precise mechanism of action is not well understood. Lithium acts as both an uncompetitive and non-competitive inhibitor of several lithium- sensitive phosphatases with regard to substrate and magnesium cofactor, respectively. In this work, we report the crystal structure and reaction mechanism of Rattus norvegicus 3'-phosphoadenosine 5'-phosphate and inositol 1,4-bisphosphate phosphatase (RnPIP), a recently identified target of lithium therapy. This Li(+)-sensitive enzyme plays a crucial role in several cellular processes, such as RNA processing, sulphation reactions and probably inositol recycling. RnPIP specifically removes the 3'-phosphate group of 3'-phosphoadenosine 5'-phosphate (PAP) and the 1'-phosphate group of inositol 1,4-bisphosphate (I(1),(4)P(2)) producing AMP and inositol 4'-phosphate, respectively. The crystal structure of RnPIP complexed with AMP, Pi and magnesium ions at 1.69 A resolution provides insight into the reaction mechanism of the hydrolysis of PAP. The core fold of the enzyme is equivalent to that found in other Li(+)-sensitive phosphatases, such as inositol monophosphatase, but molecular modelling of I(1),(4)P(2) in the RnPIP active site reveals important structural determinants that accommodate this additional substrate. RnPIP is potently inhibited by lithium and, as the accumulation of PAP inhibits a variety of proteins, including sulphotransferases and RNA processing enzymes, this dual specificity enzyme represents a potential target of lithium action, in addition to inositol monophosphatases.
Journal of Molecular Biology 02/2002; 315(4):677-85. · 3.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The yeast HAL1 gene facilitates K+/Na+ selectivity and salt tolerance of cells. Ectopic expression of HAL1 in transgenic tomato (Lycopersicon esculentum Mill.) plants minimized the reduction in fruit production caused by salt stress. Maintenance of fruit production by transgenic plants was correlated with enhanced growth under salt stress of calli derived from the plants. The HAL1 transgene enhanced water and K+ contents in both leaf calli and leaves in the presence of salt, which indicates that HAL1 functions in plants using a similar mechanism to that in yeast, namely by facilitating K+/Na+ selectivity under salt stress.
[Show abstract][Hide abstract] ABSTRACT: Salinity is an important limiting factor in plant growth and development. We have cloned a catalytic subunit of the sugar beet protein kinase CK2 (BvCKA2) by functional expression in yeast of a NaCl-induced cDNA library. BvCKA2 was able to increase the yeast tolerance to NaCl and to functionally complement the cka1 cka2 yeast double mutant upon over-expression. Southern blot analysis indicated that, in sugar beet, the BCKA2 gene is a member of a multigene family. The mRNA levels of BvCKA2 were up-regulated in response to NaCl stress which suggests that protein kinase CK2 may be involved in the plant response to salt stress.
[Show abstract][Hide abstract] ABSTRACT: The Sko1p transcriptional repressor regulates a subset of osmoinducible stress defense genes in Saccharomyces cerevisiae by binding to cAMP-responsive elements. We have reported previously that in response to stress Sko1p is phosphorylated by the stress-activated Hog1p mitogen-activated protein kinase, which disrupts its interaction with the Ssn6p x Tup1p corepressor. Here we report that other mechanisms are essential for the regulation of the Sko1p repressor activity upon stress. The nuclear localization of Sko1p depends on the stress-inhibited protein kinase A (PKA). Sko1p is localized in the nucleus of unstressed cells, and it redistributes to the cytosol upon severe salt stress (1 m NaCl). Yeast mutants with low PKA activity localize Sko1p to the cytoplasm in the absence of stress and exhibit deregulated expression of cAMP-responsive element-regulated genes. The central part (315) of Sko1p, containing the PKA phosphorylation sites and the basic domain-leucine zipper domain, is essential for its nuclear localization. Salt-induced export of Sko1p from the nucleus is independent of Hog1p and of the Bcy1p regulatory subunit of PKA. Furthermore, phosphorylation by PKA slightly enhanced DNA binding affinity of Sko1p in vitro, whereas Sko1p dimerization in vivo is not regulated by stress. Sko1p repressor activity is associated to its binding to the Ssn6p x Tup1p complex. Interestingly, the Sko1p NH(2) terminus (1), containing the Hog1p phosphorylation sites, associates in vivo with Tup1p in the absence of Ssn6p, suggesting that Sko1p represses gene transcription by interacting directly with the Tup1p subunit of the Ssn6p x Tup1p complex.
Journal of Biological Chemistry 11/2001; 276(40):37373-8. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF2alpha) is a conserved mechanism regulating protein synthesis in response to various stresses. A screening for negative factors in yeast salt stress tolerance has led to the identification of Gcn2p, the single yeast eIF2alpha kinase that is activated by amino acid starvation in the general amino acid control response. Mutation of other components of this regulatory circuit such as GCN1 and GCN3 also resulted in improved NaCl tolerance. The gcn2 phenotype was not accompanied by changes in sodium or potassium homeostasis. NaCl induced a Gcn2p-dependent phosphorylation of eIF2alpha and translational activation of Gcn4p, the transcription factor that mediates the general amino acid control response. Mutations that activate Gcn4p function, such as gcd7-201, cpc2, and deletion of the translational regulatory region of the GCN4 gene, also cause salt sensitivity. It can be postulated that sodium activation of the Gcn2p pathway has toxic effects on growth under NaCl stress and that this novel mechanism of sodium toxicity may be of general significance in eukaryotes.
Journal of Biological Chemistry 09/2001; 276(33):30753-60. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recent progress has been made in the characterization of cation transporters that maintain ion homeostasis during salt stress in plants. Sodium-proton antiporters at the vacuolar (NHX1) and plasma membrane (SOS1) have been identified in Arabidopsis. SOS1 is regulated by the calcium-activated protein kinase complex SOS2-SOS3. In yeast, a transcription repressor, Sko1, mediates regulation of the sodium-pump ENA1 gene by the Hog1 MAP kinase. The recent visualization at the atomic level of the inhibitory site of sodium in the known target Hal2 has helped identify the interactions determining Na(+) toxicity.
Current Opinion in Cell Biology 09/2001; 13(4):399-404. · 11.41 Impact Factor