Rck1 up-regulates Hog1 activity by down-regulating Slt2 activity in Saccharomyces cerevisiae

We previously reported that the over-expression of KDX1 up-regulates RCK1 gene expression. To further understand the function of Rck1, microarray analysis was performed using a RCK1 over-expressing strain. Based on microarray and Northern blot analyses, we determined that the expression of KDX1 was down-regulated when RCK1 was over-expressed. Furthermore, we determined that phosphorylated forms of Slt2 and Mkk2 were down-regulated by the over-expression of RCK1. Ptp2, a phosphatase that is regulated by the Slt2 MAP kinase pathway, was down-regulated by the over-expression of RCK1. Ptp2 is a negative regulator of Hog1; thus, the phosphorylated form of Hog1 was up-regulated by RCK1 over-expression. A point mutation of lysine 152 to arginine resulted in a failure to up-regulate Hog1 and the subsequent down-regulation of CTT1, which is a Hog1 pathway target gene. Furthermore, using microarray and Northern blot analyses, we determined that genes that are regulated by Msn2/Msn4 were up-regulated by Rck1 and that this was the result of Hog1 activation by RCK1 over-expression. Together, our results suggest that Rck1 inhibits Slt2 MAP kinase pathway activity and then Ptp2, which subsequently activates Hog1.     

CK2 activity is modulated by growth rate in Saccharomyces cerevisiae

CK2 is a highly conserved protein kinase controlling different cellular processes. It shows a higher activity in proliferating mammalian cells, in various types of cancer cell lines and tumors. The findings presented herein provide the first evidence of an in vivo modulation of CK2 activity, dependent on growth rate, in Saccharomyces cerevisiae. In fact, CK2 activity, assayed on nuclear extracts, is shown to increase in exponential growing batch cultures at faster growth rate, while localization of catalytic and regulatory subunits is not nutritionally modulated. Differences in intracellular CK2 activity of glucose- and ethanol-grown cells appear to depend on both increase in molecule number and k_cat. Also in chemostat cultures nuclear CK2 activity is higher in faster growing cells providing the first unequivocal demonstration that growth rate itself can affect CK2 activity in a eukaryotic organism.     

Serine214 of Ras2p plays a role in the feedback regulation of the Ras-cAMP pathway in the yeast Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, Ras proteins are essential for the Ras-cAMP signaling pathway. A serine to alanine substitution at position 214 in the yeast Ras2p resulted in enhanced sensitivity to heat shock, reduced levels of storage glycogen and enhanced both basal cAMP level and glucose-induced cAMP signal. Further work showed that Ras2^Ala214p had a higher GTP-binding capability than wild type Ras2p. These results suggested that serine 214 of Ras2p plays a role in the feedback regulation of the Ras-cAMP pathway.     

Cell ATP level of Saccharomyces cerevisiae sensitively responds to culture growth and drug-inflicted variations in membrane integrity and PDR pump activity

Cellular ATP level in Saccharomyces cerevisiae was measured during culture growth of strain US50-18C overproducing all major PDR pumps and its isogenic mutants variously deleted in these pumps. It was found to be inversely proportional to the intensity of cell metabolism during different growth phases and to the activity of PDR pumps, which are thus among major ATP consumers in the cells. The ATP level was increased when membrane integrity was affected by 0.5% butanol, and further increased by compound 23.1, a semisynthetic phenol lipid derivative that acts as inhibitor of Pdr5p and Snq2p pumps. The magnitude of increase in cell ATP caused by inhibition of Pdr5p pump by compound 23.1 and the Pdr5p pump inhibitor FK506 used for comparison reflects the activity and hence the energy demand of the pump. The rise in cell ATP caused by different PDR pump inhibitors can be thus used as an indicator of pump activity and the potency of the inhibitor.     

Overproduction of geraniol by enhanced precursor supply in Saccharomyces cerevisiae

Monoterpene geraniol, a compound obtained from aromatic plants, has wide applications. In this study, geraniol was synthesized in Saccharomyces cerevisiae through the introduction of geraniol synthase. To increase geraniol production, the mevalonate pathway in S. cerevisiae was genetically manipulated to enhance the supply of geranyl diphosphate, a substrate used for the biosynthesis of geraniol. Identification and optimization of the key regulatory points in the mevalonate pathway in S. cerevisiae increased geraniol production to 36.04 mg L⁻¹. The results obtained revealed that the IDI1-encoded isopentenyl diphosphate isomerase is a rate-limiting enzyme in the biosynthesis of geraniol in S. cerevisiae, and overexpression of MAF1, a negative regulator in tRNA biosynthesis, is another effective method to increase geraniol production in S. cerevisiae.     

Inhibition of p38 MAP kinase activity up-regulates multiple MAP kinase pathways and potentiates 1,25-dihydroxyvitamin D(3)-induced differentiation of human leukemia HL60 cells

Differentiation therapy for neoplastic diseases has potential for supplementing existing treatment modalities but its implementation has been slow. One of the reasons is the lack of full understanding of the complexities of cellular pathways through which signals for differentiation lead to cell maturation. This was addressed in this study using HL60 cells, a well-established model of differentiation of neoplastic cells. SB 203580 and SB 202190, specific inhibitors of a signaling protein p38 MAP kinase, were found to markedly accelerate monocytic differentiation of HL60 cells induced by low concentrations of 1,25-dihydroxyvitamin D(3) (1,25D(3)). Surprisingly, inhibition of p38 activity resulted in sustained enhancement of p38 phosphorylation and of its in vitro activity in the absence of the inhibitor, indicating up-regulation of the upstream components of the p38 pathway. In addition, SB 203580 or SB 202190 treatment of HL60 cells resulted in a prolonged activation of the JNK and, to a lesser extent, the ERK pathways. The data are consistent with the hypothesis that in HL60 cells an interruption of a negative feedback loop from a p38 target activates a common regulator of multiple MAPK pathways. The possibility also exists that JNK and/or ERK pathways amplify a differentiation signal provided by 1,25D(3).     

Effect of low pH on organization of the actin cytoskeleton in Saccharomyces cerevisiae

Cell growth in the yeast Saccharomyces cerevisiae depends on polarization of the actin cytoskeleton. In this study, we investigated how the cell regulates the distribution of actin in response to low pH conditions, focusing on the role of mitogen-activated protein kinases, Hog1 and Slt2. Changing the extracellular pH from 6.0 to 3.0 caused a transient depolarization of the actin cytoskeleton. Actin cables were no longer visible, and actin patches appeared randomly distributed after 30 min at pH 3.0. The deletion strain hog1Δ did not show this low-pH phenotype, suggesting that Hog1 is involved in depolarization of the actin cytoskeleton in response to low-pH stress. Yeast cells incubated at pH 3.0 also showed markedly increased endocytosis compared with the control at neutral pH, as indicated by the uptake of Lucifer Yellow (LY). Both the hog1Δ and slt2Δ mutants took up LY into the vacuole to a similar extent as the wild-type strain. In addition, cells grown at pH 3.0 showed a 2-fold increase in phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2) levels, as did the hog1Δ or slt2Δ cells. Efficient uptake of LY and actin repolarization at pH 3.0 might therefore require activation of PI(4,5)P2 synthesis.     

Screening of Saccharomyces cerevisiae strains with respect to anaerobic growth in non-detoxified lignocellulose hydrolysate

A microplate screening method was used to assess anaerobic growth of 12 Saccharomyces cerevisiae strains in barley straw, spruce and wheat straw hydrolysate. The assay demonstrated significant differences in inhibitor tolerance among the strains. In addition, growth inhibition by the three hydrolysates differed so that wheat hydrolysate supported growth up to 70%, while barley hydrolysate only supported growth up to 50%, with dilute-acid spruce hydrolysate taking an intermediate position.     

The mammalian AMP-activated protein kinase complex mediates glucose regulation of gene expression in the yeast Saccharomyces cerevisiae

The AMP-activated protein kinase (AMPK) controls energy homeostasis in eukaryotic cells. Here we expressed hetero-trimeric mammalian AMPK complexes in a Saccharomycescerevisiae mutant lacking all five genes encoding yeast AMPK/SNF1 components. Certain mammalian complexes complemented the growth defect of the yeast mutant on non-fermentable carbon sources. Phosphorylation of the AMPK α1-subunit was glucose-regulated, albeit not by the Glc7-Reg1/2 phosphatase, which performs this function on yeast AMPK/SNF1. AMPK could take over SNF1 function in glucose derepression. While indirectly acting anti-diabetic drugs had no effect on AMPK in yeast, compound 991 stimulated α1-subunit phosphorylation. Our results demonstrate a remarkable functional conservation of AMPK and that glucose regulation of AMPK may not be mediated by regulatory features of a specific phosphatase.     

Strain engineering of Saccharomyces cerevisiae for enhanced xylose metabolism

Efficient and rapid fermentation of all sugars present in cellulosic hydrolysates is essential for economic conversion of renewable biomass into fuels and chemicals. Xylose is one of the most abundant sugars in cellulosic biomass but it cannot be utilized by wild type Saccharomyces cerevisiae, which has been used for industrial ethanol production. Therefore, numerous technologies for strain development have been employed to engineer S. cerevisiae capable of fermenting xylose rapidly and efficiently. These include i) optimization of xylose-assimilating pathways, ii) perturbation of gene targets for reconfiguring yeast metabolism, and iii) simultaneous co-fermentation of xylose and cellobiose. In addition, the genetic and physiological background of host strains is an important determinant to construct efficient and rapid xylose-fermenting S. cerevisiae. Vibrant and persistent researches in this field for the last two decades not only led to the development of engineered S. cerevisiae strains ready for industrial fermentation of cellulosic hydrolysates, but also deepened our understanding of operational principles underlying yeast metabolism.     

H2O2 induces rapid biophysical and permeability changes in the plasma membrane of Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the diffusion rate of hydrogen peroxide (H₂O₂) through the plasma membrane decreases during adaptation to H₂O₂ by means of a mechanism that is still unknown. Here, evidence is presented that during adaptation to H₂O₂ the anisotropy of the plasma membrane increases. Adaptation to H₂O₂ was studied at several times (15min up to 90min) by applying the steady-state H₂O₂ delivery model. For wild-type cells, the steady-state fluorescence anisotropy increased after 30min, or 60min, when using 2-(9-anthroyloxy) stearic acid (2-AS), or diphenylhexatriene (DPH) membrane probe, respectively. Moreover, a 40% decrease in plasma membrane permeability to H₂O₂ was observed at 15min with a concomitant two-fold increase in catalase activity. Disruption of the ergosterol pathway, by knocking out either ERG3 or ERG6, prevents the changes in anisotropy during H₂O₂ adaptation. H₂O₂ diffusion through the plasma membrane in S. cerevisiae cells is not mediated by aquaporins since the H₂O₂ permeability constant is not altered in the presence of the aquaporin inhibitor mercuric chloride. Altogether, these results indicate that the regulation of the plasma membrane permeability towards H₂O₂ is mediated by modulation of the biophysical properties of the plasma membrane.     

Heterologous expression of mammalian Na/H antiporters in Saccharomyces cerevisiae

Na⁺/H⁺ antiporters, integral membrane proteins that exchange protons for alkali metal cations, play multiple roles in probably all living organisms (preventing cells from excessive amounts of alkali metal cations, regulating intracellular pH and cell volume). In this work, we studied the functionality of rat plasma membrane NHE1–3 exchangers upon their heterologous expression in alkali-metal-cation sensitive Saccharomyces cerevisiae, and searched for conditions that would increase their level in the plasma membrane and improve their functionality. Though three tested exchangers were partially localized to the plasma membrane (and two of them (NHE2 and NHE3) in an active form), the bulk of the synthesized proteins were arrested along the secretory pathway, mainly in the ER. To increase the level of exchangers in the yeast plasma membrane several approaches (truncation of C-terminal regulatory sequences, expression in mutant yeast strains, construction of rat/yeast protein chimeras, various growth conditions and chemical chaperones) were tested. The only increase in the amount of NHE exchangers in the plasma membrane was obtained upon expression in a strain with the npi1 mutation, which significantly lowers the level of Rsp5 ubiquitin ligase in cells. This mutation helped to stabilize proteins in the plasma membrane.     

Characterizing the effect of nitrosative stress in Saccharomyces cerevisiae

Nitrosative stress has various pathophysiological implications. We here present a detailed characterization on the effect of nitrosative stress in Saccharomyces cerevisiae wild-type (Y190) and its isogenic flavohemoglobin mutant (Δyhb1) strain grown in presence of non fermentable carbon source. On addition of sub-toxic dose of nitrosating agent both the strains showed microbiostatic effect. Cellular respiration was found to be significantly affected in both the strains in presence sodium nitroprusside. Although there was no alteration in mitochondrial permeability potential changes and reactive oxygen species production in both the strains but the cellular redox status is differentially regulated in Δyhb1 strain both in cytosol and in mitochondria indicating cellular glutathione is the major player in absence of flavohemoglobin. We also found important role(s) of various redox active enzymes like glutathione reductase and catalase in protection against nitrosative stress. This is the first report of its kind where the effect of nitrosative stress has been evaluated in S. cerevisiae cytosol as well as in mitochondria under respiratory proficient conditions.     

Phase transitions as a function of osmotic pressure in Saccharomyces cerevisiae whole cells, membrane extracts and phospholipid mixtures

Fourier Transform Infrared spectroscopy (FTIR) was used to determine the phase transition temperature of whole Saccharomyces cerevisiae W303-1 A cells as a function of Aw in binary water-glycerol media. A phase transition occurred at 12 °C in water, at 16.5 °C at Aw=0.75, and at 19.5 °C at Aw=0.65. The temperature ranges over which transition occurred increased with decreasing Aw. A total lipid extract of the plasma membranes isolated from S. cerevisiae cells was also studied, with a phase transition temperature determined at 20 °C in pure water and at 27 °C in binary water-glycerol solutions for both Aw levels tested. The pure phospholipids dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) and three binary mixtures of these phospholipids (percentage molar mixtures of DMPC/DMPE of 90.5/9.5, 74.8/25.2, and 39.7/60.3) were studied. For DMPC, there was no influence of Aw on the phase transition temperature (always 23 °C). On the other hand, the phase transition temperature of DMPE increased with decreasing Aw for the three aqueous solutions tested (glycerol, sorbitol and sucrose), from 48 °C in water, to 64 °C for a solution at Aw=0.67. For the DMPC/DMPE mixtures, transitions were found intermediate between those of the two phospholipids, and a cooperative state was observed between species at the gel and at the fluid phases.     

The golden root, Rhodiola rosea, prolongs lifespan but decreases oxidative stress resistance in yeast Saccharomyces cerevisiae

The effect of aqueous extract from R. rosea root on lifespan and the activity of antioxidant enzymes in budding yeast Saccharomyces cerevisiae have been studied. The supplementation of the growth medium with R. rosea extract decreased survival of exponentially growing S. cerevisiae cells under H₂O₂-induced oxidative stress, but increased viability and reproduction success of yeast cells in stationary phase. The extract did not significantly affect catalase activity and decreased SOD activity in chronologically aged yeast population. These results suggest that R. rosea acts as a stressor for S. cerevisiae cells, what sensitizes yeast cells to oxidative stress at exponential phase, but induces adaptation in stationary phase cells demonstrating the positive effect on yeast survival without activation of major antioxidant enzymes.     

Menadione stress in Saccharomyces cerevisiae strains deficient in the glutathione transferases

Using S. cerevisiae as a eukaryotic cell model we have analyzed the involvement of both glutathione transferase isoforms, Gtt1 and Gtt2, in constitutive resistance and adaptive response to menadione, a quinone which can exert its toxicity as redox cycling and/or electrophiles. The detoxification properties, of these enzymes, have also been analyzed by the appearance of S-conjugates in the media. Direct exposure to menadione (20 mM/60 min) showed to be lethal for cells deficient on both Gtt1 and Gtt2 isoforms. However, after pre-treatment with a low menadione concentration, cells deficient in Gtt2 displayed reduced ability to acquire tolerance when compared with the control and the Gtt1 deficient strains. Analyzing the toxic effects of menadione we observed that the gtt2 mutant showed no reduction in lipid peroxidation levels. Moreover, measuring the levels of intracellular oxidation during menadione stress we have shown that the increase of this oxidative stress parameter was due to the capacity menadione possesses in generating reactive oxygen species (ROS) and that both GSH and Gtt2 isoform were required to enhance ROS production. Furthermore, the efflux of the menadione–GSH conjugate, which is related with detoxification of xenobiotic pathways, was not detected in the gtt2 mutant. Taken together, these results suggest that acquisition of tolerance against stress generated by menadione and the process of detoxification through S-conjugates are dependent upon Gtt2 activity. This assessment was corroborated by the increase of GTT2 expression, and not of GTT1, after menadione treatment.     

The induction of trehalose and glycerol in Saccharomyces cerevisiae in response to various stresses

Trehalose and glycerol have been implicated as potential stress protectants that accumulate in yeasts during various stress conditions. We investigated the levels of glycerol and trehalose and the expression profiles of genes involved in their metabolism to determine their involvement in the response of Saccharomyces cerevisiae XQ1 to thermal, sorbitol and ethanol stresses. The results showed that the genes involved in the synthesis and degradation of trehalose and glycerol were stress induced, and that trehalose and glycerol were synthesized simultaneously during the initial stages (a sensitive response period) of diverse stress treatments. Trehalose accumulated markedly under heat treatment, but not under sorbitol or ethanol stress, whereas glycerol accumulated strikingly under sorbitol stress conditions. Interestingly, extracellular trehalose seemed to be involved in protecting cells from damage under unfavorable conditions. Moreover, our results suggest that the stress-activated futile ATP cycles of trehalose and glycerol turnover are of general importance during cellular stress adaptation.