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.

Trehalose protects Saccharomyces cerevisiae from lipid peroxidation during oxidative stress.

Aiming to focus the protective role of the sugar trehalose under oxidative conditions, two sets of Saccharomyces cerevisiae strains, having different profiles of trehalose synthesis, were used. Cells were treated either with a 10% trehalose solution or with a heat treatment (which leads to trehalose accumulation) and then exposed either to menadione (a source of superoxide) or to tert-butylhydroperoxide (TBOOH). According to our results, trehalose markedly increased viability upon exposure to menadione stress, which seems to be correlated with decrease in lipid peroxidation levels. The protective effect of trehalose against oxidative damage produced by menadione was especially efficient under SOD1 deficiency. On the other hand, this sugar does not seem to participate of the mechanism of acquisition of tolerance against TBOOH, since trehalose pretreatment (addition of external trehalose) was not capable of increase cell survival. Therefore, trehalose plays a role in protecting cells, especially membranes, from oxidative injuries. However, this mechanism of defense is dependent on the type of oxidative stress to which cells are submitted.

The interaction of Saccharomyces cerevisiae trehalase with membranes.

Plasma membranes isolated from cells of Saccharomyces cerevisiae previously submitted to a heat-shock showed a 10-fold increase in membrane-bound trehalase activity. Trehalase was purified to a high specific activity and was shown to be inhibited by glucose 6-phosphate and by the addition of a neutral phospholipid-like surfactant. Purified trehalase binds spontaneously to egg phosphatidylcholine small unilamellar vesicles, when in its active, phosphorylated form. When the enzyme was treated with alkaline phosphatase no binding was observed. The significance of this reversible binding for the control of trehalose metabolism in yeast cells is still unknown.

Trehalose metabolism in Saccharomyces cerevisiae during heat-shock.

When different strains of Saccharomyces cerevisiae grown at 23 degrees C were transferred to 36 degrees C, trehalose and glycogen were accumulated. Glycogen accumulation was less extensive and its synthesis started at least 15 min after initiation of trehalose synthesis. The steady-state intracellular concentration of trehalose increased simultaneously with the activities of the enzymes trehalose-6P synthase, UDPG-pyrophosphorylase, phosphoglucomutase and trehalase. A small but significant change was observed in hexokinase activity. Our results directly implicate isoform PII of hexokinase and the minor isoform of phosphoglucomutase in the pathway of trehalose formation during heat-shock. We also showed that the major isoform of phosphoglucomutase increased in activity but was not essential for trehalose accumulation. Studies with the glucose uptake system indicated that trehalose accumulation could be primarily determined by intracellular availability of substrates due to the increase in the rate of glucose uptake. The increased uptake appears to have two components: a kinetic effect of temperature upon glucose transporters and an increase in the numbers of molecules of the transporters, probably mediated by synthesis de novo.

Modulation of trehalase activity in Saccharomyces cerevisiae by an intrinsic protein.

The regulation of cytosolic trehalase activity in yeast has been described as cycles of activation by phosphorylation by cAMP protein kinase. In this paper, evidence is presented for another regulatory mechanism--the binding of an endogenous inhibitory protein. This negative modulator was isolated during the purification procedure of cytosolic cryptic trehalase from repressed wild-type cells of Saccharomyces cerevisiae. However, in derepressed cells the inhibitor was not found nor was it present in ras2 mutant cells submitted to a heat treatment. The trehalase inhibitory activity proved to be a calmodulin ligand protein and, therefore, involved in the modulation of trehalase activity by Ca2+ ions.

Trehalose inhibits ethanol effects on intact yeast cells and liposomes.

The effect of ethanol on stability of intact yeast cells has been investigated. Several strains with differences in trehalose metabolism were examined for their ability to survive in the presence of 10% (v/v) ethanol. A positive correlation was observed between cell viability and trehalose concentration. When leakage of electrolytes from the cells was recorded by observing changes in conductivity of the medium, we found that ethanol increases leakage, but the presence of trehalose reverses that effect. Similar studies were done with liposomes of similar composition to those seen in intact cells in log and stationary phases. In the presence of ethanol, carboxyfluorescein trapped in the liposomes leaked to the medium. When trehalose was added inside, outside or on both sides of the membrane, the ethanol-induced leakage was strongly inhibited. More leakage was observed in liposomes in gel phase state than in liquid-crystalline phase, suggesting that the thermotropic behavior of the lipids in the plasma membrane, together with trehalose, plays a role in enhancing ethanol tolerance.

The role of the trehalose transporter during germination.

Previous studies on the resistance of yeast cells to dehydration pointed towards the protective role of trehalose and the importance of the specific trehalose transporter in guaranteeing survival. The present report demonstrates that the trehalose transporter is essential during the germination process in order to translocate trehalose from the cytosol to the external environment. Diploids that lack the trehalose transporter germinate poorly and do not form 4 spore tetrads although they accumulate trehalose and show trehalase activity. Furthermore, addition of exogenous trehalose to the germination medium enhances germination and normal segregation. The ability to transport trehalose is dominant and seems to be related to a single gene.

Regulation of UDPG-pyrophosphorylase isoforms in Saccharomyces cerevisiae and their roles in trehalose metabolism.

UDPG-pyrophosphorylase (EC 2.7.7.9) from Saccharomyces cerevisiae was studied and the presence of isoforms investigated. Its activity was monitored during growth of cultures in rich media containing glucose, galactose, sucrose, maltose or glycerol as carbon sources. The results suggest that UDPG-pyrophosphorylase is subject to both catabolite repression and catabolite inactivation. The inactivation process seems to be complex: in order to produce maximum inactivation, glucose and ammonium sulfate must be added together. Addition of glucose or ammonium sulfate separately produced little effect upon enzyme activity. Adsorption to and elution from a DEAE-Sephacel column of a crude protein extract prepared from yeast cells collected in stationary phase from a glucose medium showed three activity peaks, which we denominated isoform I, II, and III. Isoform I is constitutive, it was the only form present during exponential growth on glucose medium, and did not suffer any alteration after glucose exhaustion, heat shock or by growing cells on maltose. On the other hand, isoforms II and III were shown to be repressed by glucose, and induced by heat shock. Furthermore, isoform II of UDPG-pyrophosphorylase was present together with isoform I when yeast cells were grown on maltose. The presence of a MAL4C allele rendered isoform II constitutive. Interestingly, a gal3 mutant strain had low UDPG-pyrophosphorylase activity and isoforms I and II were not expressed. These results are discussed in relation to trehalose metabolism.

Role of the trehalose carrier in dehydration resistance of Saccharomyces cerevisiae.

Yeast cells are well known for their ability to survive complete dehydration, a phenomenon that is directly linked to the presence of the sugar trehalose in these cells. This sugar apparently endows the cells with the capacity to survive dehydration. Previous studies on in vitro models showed that trehalose must be present on both sides of the bilayer to stabilize dry membranes. The present report demonstrates that a specific trehalose carrier seems to enable the sugar to protect the yeast cell membrane by translocating trehalose from the cytosol to the extracellular environment. Saccharomyces cerevisiae mutant strains which lack the trehalose carrier did not survive after dehydration although they accumulated endogenous trehalose. Furthermore, when carrier mutants were dehydrated in the presence of exogenous trehalose the cells became more resistant showing increased survival.

Trehalose accumulation in mutants of Saccharomyces cerevisiae deleted in the UDPG-dependent trehalose synthase-phosphatase complex.

In Saccharomyces cerevisiae, trehalose-6-phosphate synthase converts uridine-5'-diphosphoglucose and glucose 6-phosphate to trehalose 6-phosphate which is dephosphorylated by trehalose 6-phosphatase to trehalose. These two steps take place within a complex consisting of three proteins: trehalose-6-phosphate synthase encoded by the GGS1/TPS1 (= FDP1, = BYP1, = CIF1) gene, trehalose 6-phosphatase encoded by the TPS2 gene and by a third protein encoded by both the TSL1 and TPS3 genes. Using three different methods for trehalose determination, we observed trehalose accumulation in ggs1/tps1delta, tps2delta and tsl1delta mutants, and in the double mutants ggs1/tps1delta/tps2delta and also in ggs1/tps1delta deleted mutants suppressed for growth on glucose. All these mutants harbor MAL genes. Trehalose synthesis in these mutants is probably performed by the adenosine-5'-diphosphoglucose-dependent trehalose synthase, (ADPG-dependent trehalose synthase) which was detected in all strains tested. It is noteworthy that trehalose accumulation in these mutants was detected only in cells grown on weakly repressive carbon sources such as maltose and galactose or during the transition phase from fermentable to non-fermentable growth on glucose. alpha-Glucosidase activity was always present in high amounts. We also describe an adenosine-diphosphoglucosepyrophosphorylase (ADPG-pyrophosphorylase) activity in Saccharomyces cerevisiae which increased concomitantly with the accumulation of trehalose during the transition phase from fermentable to non-fermentable growth in MAL-constitutive (MAL2-8c) strains. The same was observed when MAL-induced (MAL1) strains were compared during growth on glucose and maltose. These results led us to conclude that maltose-induced trehalose accumulation is independent of the UDPG-dependent trehalose-6-phosphate synthase/phosphatase complex; that the ADPG-dependent trehalose synthase is responsible for maltose-induced trehalose accumulation probably by forming a complex with a specific trehalose-6-phosphatase activity and that ADPG synthesis is activated during trehalose accumulation under these conditions.

Expression of high-affinity trehalose-H+ symport in Saccharomyces cerevisiae.

The expression of the high-affinity trehalose-H+ symport was investigated in various Saccharomyces cerevisiae strains and culture conditions. Previous kinetic studies of trehalose transport in yeast have revealed the existence of at least two different uptake mechanisms: a high-affinity trehalose-H+ symport activity repressed by glucose, and a constitutive low-affinity transport activity, a putative facilitated diffusion process. Exogenously added trehalose was not an inducer of the high-affinity transport activity, and a correlation between trehalose and maltose uptake by yeast cells was found. Our results indicate that the maltose-H+ symporters encoded by MAL11, MAL21, and MAL41 are not responsible for the trehalose transport activity. The analysis of both trehalose and maltose transport activities in wild-type and in laboratory strains with defined MAL genes showed that the trehalose-H+ symporter was under control of MAL regulatory genes. Our results also suggest that the recently characterized AGT1 gene of S. cerevisiae may encode the high-affinity trehalose-H+ symporter. During diauxic growth on glucose the transport activity was low during the first exponential phase of growth, increased as glucose was exhausted from the medium, and decreased again as the cells reached the late stationary phase. This pattern was coincident with that of the intracellular levels of trehalose. The strong correlation between these two parameters may be of physiological significance during adaptation of yeast cells to stress conditions.

Targets of oxidative stress in yeast sod mutants.

Eukaryotic cells have developed mechanisms to rapidly respond towards the environment by changing the expression of a series of genes. There is increasing evidence that reactive oxygen species (ROS), besides causing damage, may also fulfill an important role as second messengers involved in signal transduction. Recently, we have demonstrated that deletion of SOD1 is beneficial for the acquisition of tolerance towards heat and ethanol stresses. The present report demonstrates that a sod1 mutant was the only one capable of acquiring tolerance against a subsequent stress produced by menadione, although this mutant strain had exhibited high sensitivity to oxidative stress. By measuring the level of intracellular oxidation, lipid peroxidation as well as glutathione metabolism, we have shown that in the SOD1-deleted strain, an unbalance occurs in the cell redox status. These results indicated that the capacity of acquiring tolerance to oxidative stress is related to a signal given by one or all of the above factors.

Regulation of cadmium uptake by Saccharomyces cerevisiae.

In this work, we verified that yeast cells deleted in ZRT1 were not capable of transporting cadmium, suggesting that the transport of this metal into the cell would be carried out through this zinc transporter. On the other hand, cadmium absorption shown by a Deltagsh1 strain (a mutant not able of synthesizing glutathione) was twofold higher than in the control strain. Moreover, the deletion of YCF1 (which encodes a vacuolar glutathione S-conjugate pump) impaired the transport of this metal significantly. Using a mutant strain deficient in YAP1, which codifies a transcription factor that controls the expression of both GSH1 and YCF1, we also observed a twofold increase in cadmium uptake, the same behavior shown by Deltagsh1 cells. Cadmium is compartmentalized in vacuoles through the Ycf1 transporter, in the form of a bis-glutathionato-cadmium complex. We propose that gsh1 cells are unable to form the Cd-GS(2) complex, while ycf1 cells would accumulate high levels of this complex in the cytoplasm. In face of these results we raised the hypothesis that Cd-GS(2) complex controls cadmium uptake through the Zrt1 protein.

Evaluation of the role of Ace1 and Yap1 in cadmium absorption using the eukaryotic cell model Saccharomyces cerevisiae.

In a previous paper, we demonstrated that the cytoplasmic level of glutathione-cadmium complex affects cadmium absorption by Saccharomyces cerevisiae, a usual eukaryotic cell model for studies of stress response. Furthermore, it was also observed that the absorption of this non-essential metal seems to be achieved by Zrt1, a zinc transporter of high affinity. Looking a little further into the control mechanism, we have verified that the deficiency in Ace1 impaired cadmium transport significantly. Ace1 is a transcription factor that activates the expression of CUP1, which encodes the S. cerevisiae metallothionein. On the other hand, the deficiency in the transcription factor Yap1 produced a two-fold increase in cadmium uptake. Cells lacking Yap1 showed low levels of glutathione, which could explain their higher capacity of absorbing cadmium. However, the mutant strain Ace1 deficient exhibited considerable amounts of glutathione. By using RT-PCR analysis, we observed that the lack of Yap1 activates the expression of both CUP1 and ZRT1, while the lack of Ace1 inhibited significantly the expression of these genes. Thus, metallothionein seems also to participate in the regulation of cadmium transport by controlling the expression of ZRT1. We propose that, at low levels of Cup1, the cytoplasmic concentration of essential metals, such as zinc, in free form (not complexated), increases, inhibiting ZRT1 expression. In contrast, at high levels of Cup1, the concentration of these metals falls, inducing ZRT1 expression and favoring cadmium absorption. These results confirm the involvement of zinc transport system with cadmium transport.

Comparative studies between the glucose-induced phosphorylation signal and the heat shock response in mutants of Saccharomyces cerevisiae.

Addition of glucose to derepressed yeast cells, as well as a heat shock treatment, trigger the phosphorylation of trehalase and of trehalose-6-phosphate synthase. In the present paper, evidence is provided for the requirement of the RAS protein in the transduction of the glucose signal. On the other hand, a heat shock at 52 degrees C for 2 min was able to produce a significant phosphorylating effect even in mutant strains deficient in the GTP binding protein. Moreover, it was shown that this treatment does not affect exclusively the cAMP-dependent protein kinase. The use of a series of mutant strains confirmed that low levels of cAMP favor thermotolerance; the role of trehalose in yeast viability is also discussed.

Acquisition of tolerance against oxidative damage in Saccharomyces cerevisiae.

BACKGROUND: Living cells constantly sense and adapt to redox shifts by the induction of genes whose products act to maintain the cellular redox environment. In the eukaryote Saccharomyces cerevisiae, while stationary cells possess a degree of constitutive resistance towards oxidants, treatment of exponential phase cultures with sub-lethal stresses can lead to the transient induction of protection against subsequent lethal oxidant conditions. The sensors of oxidative stress and the corresponding transcription factors that activate gene expression under these conditions have not yet been completely identified. RESULTS: We report the role of SOD1, SOD2 and TPS1 genes (which encode the cytoplasmic Cu/Zn-superoxide dismutase, the mitochondrial Mn-isoform and trehalose-6-phosphate synthase, respectively) in the development of resistance to oxidative stress. In all experimental conditions, the cultures were divided into two parts, one was immediately submitted to severe stress (namely: exposure to H2O2, heat shock or ethanol stress) while the other was initially adapted to 40 degrees C for 60 min. The deficiency in trehalose synthesis did not impair the acquisition of tolerance to H2O2, but this disaccharide played an essential role in tolerance against heat and ethanol stresses. We also verified that the presence of only one Sodp isoform was sufficient to improve cellular resistance to 5 mM H2O2. On the other hand, while the lack of Sod2p caused high cell sensitivity to ethanol and heat shock, the absence of Sod1p seemed to be beneficial to the process of acquisition of tolerance to these adverse conditions. The increase in oxidation-dependent fluorescence of crude extracts of sod1 mutant cells upon incubation at 40 degrees C was approximately 2-fold higher than in sod2 and control strain extracts. Furthermore, in Western blots, we observed that sod mutants showed a different pattern of Hsp104p and Hsp26p expression also different from that in their control strain. CONCLUSIONS: Trehalose seemed not to be essential in the acquisition of tolerance to H2O2 stress, but its absence was strongly felt under water stress conditions such as heat and alcoholic stresses. On the other hand, Sod1p could be involved in the control of ROS production; these reactive molecules could signal the induction of genes implicated within cell tolerance to heat and ethanol. The effects of this deletion needs further investigation.

Identification of an integral membrane 80 kDa protein of Saccharomyces cerevisiae induced in response to dehydration.

Using SDS-PAGE gels we observed the induced synthesis of a protein with a molecular mass of 80 kDa when cells of strains of Saccharomyces cerevisiae were subjected to dehydration. Physiological analysis showed that this protein is not present during growth on glucose but was found in derepressed cells from stationary phase. Furthermore, its synthesis was induced when cells were grown on medium containing alpha-methyl-glucoside as carbon source. However, the 80 kDa protein was not found in cells of mutants unable to transport trehalose. This protein was localized in the cytoplasmic membrane and showed trehalose-binding activity, determined by its partial purification on a trehalose-Sepharose 6B affinity column. The possible involvement of the 80 kDa protein with the trehalose transport system is discussed.

Biotechnological applications of the disaccharide trehalose.

Trehalose is a disaccharide present in a variety of anhydrobiotic organisms which have the ability to promptly resume their metabolism after addition of water. It has been successfully used as a nontoxic cryoprotectant of enzymes, membranes, vaccines, animal and plant cells and organs for surgical transplants. It has been predicted that trehalose can also be used as an ingredient for dried and processed food. Therefore, the recent biotechnological applications of trehalose have imposed the standardization of methods for its production, as well as for its specific quantification.

Effect of hydrostatic pressure of a mutant of Saccharomyces cerevisiae deleted in the trehalose-6-phosphate synthase gene.

Mutants Saccharomyces cerevisiae deleted on the trehalose-6-phosphate synthase gene (tps1) and their parental wild-type cells were submitted to hydrostatic pressure in the range of 0-200 MPa. Experimental evidence showed that viability for both strains decreased with increasing pressure and that tps1 mutants, unable to accumulate trehalose, were more sensitive to hydrostatic pressure than the wild-type cells. Additionally, both tps1 and wild-type cells in the stationary phase, when there is an accumulation of endogenous trehalose, were more resistant to pressure than proliferating cells. Under these conditions, mutant cells were also more sensitive to pressure treatment than the wild type. The present work also showed that mild pressure pretreatment did not induce hydrostatic pressure resistance (barotolerance) in yeast cells.

Active alpha-glucoside transport in Saccharomyces cerevisiae.

The AGT1 permease is a alpha-glucoside-H+ symporter responsible for the active transport of maltose, trehalose, maltotriose, alpha-methylglucoside, melezitose and sucrose. In wild-type as well as in MAL constitutive strains, alpha-methylglucoside seemed to be the best inducer of transport activity, while trehalose had no inducing effect. Based on the initial rates of transport it seems that the sugar preferentially transported by this permease is trehalose, followed by sucrose.