Anita D. Panek

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.

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.

Acquisition of tolerance against oxidative damage in Saccharomyces cerevisiae

BackgroundLiving 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.ResultsWe 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°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°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.ConclusionsTrehalose 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.

Protection against oxidation during dehydration of yeast

Abstract Based on the well-documented notion that oxygen affects the stability of dried cells, the role of the cytosolic and mitochondrial forms of superoxide dismutase (Sod) in the capacity of cells to resist dehydration was examined. Both enzymes are important for improving survival, and the absence of only 1 isoform did not impair tolerance against dehydration. In addition, sod strains showed the same Sod activity as the control strain, indicating that the deficiency in either cytoplasmic Cu/Zn or mitochondrial Mn was overcome by an increase in activity of the remaining Sod. To measure the level of intracellular oxidation produced by dehydration, a fluorescent probe, 2′,7′-dichlorofluorescein, was used. Dry cells exhibited a high increase in fluorescence: both control and sod mutant strains became almost 10-fold more oxidized after dehydration. Furthermore, the disaccharide trehalose was shown to protect dry cells against oxidation.

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.

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.

Cytotoxicity Mechanism of Two Naphthoquinones (Menadione and Plumbagin) in Saccharomyces cerevisiae

BACKGROUND Quinones are compounds extensively used in studies of oxidative stress due to their role in plants as chemicals for defense. These compounds are of great interest for pharmacologists and scientists, in general, because several cancer chemotherapeutic agents contain the quinone nucleus. However, due to differences in structures and diverse pharmacological effects, the exact toxicity mechanisms exerted by quinones are far from elucidatation. METHODOLOGY/PRINCIPAL FINDINGS Using Saccharomyces cerevisiae, we evaluated the main mechanisms of toxicity of two naphthoquinones, menadione and plumbagin, by determining tolerance and oxidative stress biomarkers such as GSH and GSSG, lipid peroxidation levels, as well as aconitase activity. The importance of glutathione transferases (GST) in quinone detoxification was also addressed. The GSSG/GSH ratio showed that menadione seemed to exert its toxicity mainly through the generation of ROS while plumbagin acted as an electrophile reacting with GSH. However, the results showed that, even by different pathways, both drugs were capable of generating oxidative stress through their toxic effects. Our results showed that the control strain, BY4741, and the glutathione transferase deficient strains (gtt1Delta and gtt2Delta) were sensitive to both compounds. With respect to the role of GST isoforms in cellular protection against quinone toxicity, we observed that the Gtt2 deficient strain was unable to overcome lipid peroxidation, even after a plumbagin pre-treatment, indicating that this treatment did not improve tolerance when compared with the wild type strain. Cross-tolerance experiments confirmed distinct cytotoxicity mechanisms for these naphthoquinones since only a pre-treatment with menadione was able to induce acquisition of tolerance against stress with plumbagin. CONCLUSIONS/SIGNIFICANCE These results suggest different responses to menadione and plumbagin which could be due to the fact that these compounds use different mechanisms to exert their toxicity. In addition, the Gtt2 isoform seemed to act as a general protective factor involved in quinone detoxification.

Preservation of frozen yeast cells by trehalose.

Two different methods commonly used to preserve intact yeast cells-freezing and freeze-drying-were compared. Different yeast cells submitted to these treatments were stored for 28 days and cell viability assessed during this period. Intact yeast cells showed to be less tolerant to freeze-drying than to freezing. The rate of survival for both treatments could be enhanced by exogenous trehalose (10%) added during freezing and freeze-drying treatments or by a combination of two procedures: a pre-exposure of cells to 40 degrees C for 60 min and addition of trehalose. A maximum survival level of 71.5 +/- 6.3% after freezing could be achieved at the end of a storage period of 28 days, whereas only 25.0 +/- 1.4% showed the ability to tolerate freeze-drying treatment, if both low-temperature treatments were preceded by a heat exposure and addition of trehalose to yeast cells. Increased survival ability was also obtained when the pre-exposure treatment of yeast cells was performed at 10 degrees C for 3 h and trehalose was added: these treatments enhanced cell survival following freezing from 20.5 +/- 7. 7% to 60.0 +/- 3.5%. Although both mild cold and heat shock treatments could enhance cell tolerance to low temperature, only the heat treatment was able to increase the accumulation of intracellular trehalose whereas, during cold shock exposure, the intracellular amount of trehalose remained unaltered. Intracellular trehalose levels seemed not to be the only factor contributing to cell tolerance against freezing and freeze-drying treatments; however, the protection that this sugar confers to cells can be exerted only if it is to be found on both sides of the plasma membrane.

Trehalose as cryoprotectant for preservation of yeast strains

Abstract Preservation of genetic banks of yeast strains as well as of any kind of eukaryotic cells during dehydration and subsequent rehydration depends upon the maintenance of the integrity of the cell membrane. Trehalose has been successfully used as a non-toxic cryoprotectant for plant cells (Bhandal et al., 1985), as well as for lobster sarcoplasmic vesicles (Rudolph and Crowe, 1985). The hypothesis underlying these observations is that the disaccharide avoids fusion of membranes by replacing water molecules in the bilayer (Crowe et al., 1984). The viability of yeast strains submitted to different drying techniques is reported in this paper. Mutant strains with defects in the regulation of the trehalose-6-phosphate synthase complex were compared. Yeast strains dried in layers at 37°C for 6 h did not lose their viability, however, they died thereafter at 5°C, unless trehalose was used for resuspending the cells before drying. It should be noted that no trehalose accumulation was seen during drying at 37°C under our experimental conditions. In experiments in which cells were frozen at −120°C, addition of 10% trehalose to the suspending buffer had a significant protective effect. On the other hand, a mutant strain with an extremely high trehalose-6-phosphate synthase activity showed an intrinsic capacity for survival which did not depend upon addition of exogenous trehalose. This raises the question of the location of the internal trehalose pool and whether it could replace the externally added cryoprotectant.

Regulation of the trehalose-6-phosphate synthase complex in Saccharomyces

SummaryTrehalose-6-phosphate synthase is another example of an enzyme of carbohydrate metabolism, in Saccharomyces, which could be regulated by interconversion of forms. Deactivation was mediated both in vivo and in vitro by a cyclic AMP-dependent protein kinase. Reversibility of this process was obtained by a phosphatase treatment leading to an increase in activity. The phosphorylated, less active form of the enzyme proved to be more susceptible to activation by ATP.Mg. Mutants with well defined lesions in the cyclic AMP-dependent protein kinase system were used to corroborate our findings of a possible regulatory mechanism of trehalose-6-phosphate synthase activity by interconversion of forms.