Glutathione and gamma-glutamyl transferases are involved in the formation of cadmium-glutathione complex

In a wild-type strain of Saccharomyces cerevisiae, cadmium induces the activities of both gamma-glutamyl transferase (gamma-GT) and glutathione transferase 2 (Gtt2). However, Gtt2 activity did not increase under gamma-GT or Ycf1 deficiencies, suggesting that the accumulation of glutathione-cadmium in the cytosol inhibits Gtt2. On the other hand, the balance between the cytoplasmic and vacuolar level of glutathione seems to regulate gamma-GT activity, since this enzyme was not activated in a gtt2 strain. Taken together, these results suggest that gamma-GT and Gtt2 work together to remove cadmium from the cytoplasm, a crucial mechanism for metal detoxification that is dependent on glutathione.     

The effect of superoxide dismutase deficiency on cadmium stress

Saccharomyces cerevisiae mutant strains deficient in superoxide dismutase (Sod), an antioxidant enzyme, were used to analyze cadmium absorption and the oxidation produced by it. Cells lacking the cytosolic Sod1 removed twice as much cadmium as the control strain, while those deficient in the mitochondrial Sod2 exhibited poor metal absorption. Interestingly, the sod1 mutant did not become more oxidized after exposure to cadmium, as opposed to the control strain. We observed that the deficiency of Sod1 increases the expression of both Cup1 (a metallothionein) and Ycf1 (a vacuolar glutathione S-conjugate pump), proteins involved with protection against cadmium. Furthermore, when sod1 cells were exposed to cadmium, the ratio glutathione oxidized/glutathione reduced did not increase as expected. We propose that a high level of metallothionein expression would relieve glutathione under cadmium stress, while an increased level of Ycf1 expression would favor compartmentalization of this metal into the vacuole. Both conditions would reduce the level of glutathione-cadmium complex in cytosol, contributing to the high capacity of absorbing cadmium by the sod1 strain. Previous results showed that the glutathione-cadmium complex regulates cadmium uptake. These results indicate that, even indirectly, metallothionein also regulates cadmium transport.     

Role of respiration and glutathione in cadmium-induced oxidative stress in Escherichia coli K-12

Cadmium is a widespread pollutant that has been associated with oxidative stress, but the mechanism behind this effect in prokaryotes is still unclear. In this work, we exposed two glutathione deficient mutants (ΔgshA and ΔgshB) and one respiration deficient mutant (ΔubiE) to a sublethal concentration of cadmium. The glutathione mutants show a similar increase in reactive oxygen species as the wild type. Experiments performed using the ΔubiE strain showed that this mutant is more resistant to cadmium ions and that Cd-induced reactive oxygen species levels were not altered. In the light of these facts, we conclude that the interference of cadmium with the respiratory chain is the cause of the oxidative stress induced by this metal and that, contrary to previously proposed models, the reactive oxygen species increase is not due to glutathione depletion, although this peptide is crucial for cadmium detoxification.     

Accumulation of cadmium ions in the methylotrophic yeast Hansenula polymorpha

Intracellular cadmium (Cd²⁺) ion accumulation and the ability to produce specific Cd²⁺ ion chelators was studied in the methylotrophic yeast Hansenula polymorpha. Only one type of Cd²⁺ intracellular chelators, glutathione (GSH), was identified, which suggests that sequestration of this heavy metal in H. polymorpha occurs similarly to that found in Saccharomyces cerevisiae, but different to Schizosaccharomys pombe and Candida glabrata which both synthesize phytochelatins. Cd²⁺ ion uptake in the H. polymorpha wild-type strains appeared to be an energy dependent process. It was found that Δgsh2 mutants, impaired in the first step of GSH biosynthesis, are characterized by increase in net Cd²⁺ ion uptake by the cells, whereas Δgsh1/Δmet1 and Δggt1 mutants impaired in sulfate assimilation and GSH catabolism, respectively, lost the ability to accumulate Cd²⁺ intracellularly. Apparently H. polymorpha, similarly to S. cerevisiae, forms a Cd-GSH complex in the cytoplasm, which in turn regulates Cd²⁺ uptake. Genes GSH1/MET1 and GGT1 are involved in maturation and metabolism of cellular Cd-GSH complex, respectively. Transport of [³H]N-ethylmaleimide-S-glutathione ([³H]NEM-SG) conjugate into crude membrane vesicules, purified from the wild-type cells of H. polymorpha appeared to be MgATP dependent, uncoupler insensitive and vanadate sensitive. We suggest that MgATP dependent transporter involved in Cd-GSH uptake in H. polymorpha, is similar to S. cerevisiae Ycf1-mediated vacuolar transporter responsible for accumulation of organic GS-conjugates and Cd-GSH complex.     

Proteolytic stability of recombinant human serum albumin secreted in the yeast Saccharomyces cerevisiae

 In order to direct the persistent expression of recombinant human serum albumin (HSA) from the GAL10 promoter in the yeast Saccharomyces cerevisiae, we carried out periodic feeding of galactose during shake-flask cultures. Unexpectedly, the recombinant protein secreted was observed to undergo rapid degradation, which was apparently accelerated by carbon-source feeding. The extracellular degradation of HSA occurred even in the strain deficient in the major vacuolar proteases PrA and PrB, and in the strain lacking the acidic protease Yap3p (involved in the generation of HSA-truncated fragments). Interestingly, the degradation correlated closely with the acidification of extracellular pH and thus was significantly overcome either by buffering the culture medium above pH 5.0 or by adding amino acid-rich supplements to the culture medium, which could prevent the acidification of medium pH during cultivation. Addition of arginine or ammonium salt also substantially minimized the degradation of HSA, even without buffering. The extracellular degradation activity was not detected in the cell-free culture supernatant but was found to be associated with intact cells. The results of the present study strongly suggest that the HSA secreted in S. cerevisiae is highly susceptible to the pH-dependent proteolysis mediated by cell-bound protease(s) whose activity and expression are greatly affected by the composition of the medium. Received: 23 August 1999 / Received revision: 8 November 1999 / Accepted: 12 December 1999     

Improved glutathione production by gene expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

To utilize Pichia pastoris to produce glutathione, an intracellular expression vector harboring two genes (gsh1 and gsh2) from Saccharomyces cerevisiae encoding enzymes involved in glutathione synthesis and regulated by the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter was transformed into P. pastoris GS115. Through Zeocin resistance and expression screening, a transformant that had higher glutathione yield (217 mg/L) in flask culture than the host strain was obtained. In fed-batch culture process, this recombinant strain displayed high activity for converting precursor amino acids into glutathione. The glutathione yield and biomass achieved 4.15 g/L and 98.15 g (dry cell weight, DCW)/L, respectively, after 50 h fermentation combined with addition of three amino acids (15 mmol/L glutamic acid, 15 mmol/L cysteine, and 15 mmol/L glycine).     

Apoptosis as a mechanism for removal of mutated cells of Saccharomyces cerevisiae: The role of Grx2 under cadmium exposure

Cadmium is a strong mutagen that acts by inhibiting DNA mismatch repair, while its toxic effect seems to be related to an indirect oxidative stress that involves glutathione (GSH) mobilization. Among the roles of GSH is the protection of proteins against oxidative damage, by forming reversible mixed disulfides with cysteine residues, a process known as protein glutathionylation and catalyzed by glutaredoxins (Grx). In this current study, Saccharomyces cerevisiae cells deficient in GRX2, growing in 80 μM CdSO₄, showed high mitochondrial mutagenic rate, determined by frequency of mutants that had lost mitochondrial function (petite mutants), high tolerance and lower apoptosis induction. The mutant strain also showed decreased levels of glutathionylated-protein after cadmium exposure, which might difficult the signaling to apoptosis, leading to increased mutagenic rates. Taken together, these results suggest that Grx2 is involved with the apoptotic death induced by cadmium, a form of cellular suicide that might lead of removal of mutated cells.     

Response to copper and cadmium stress in wild-type and copper tolerant strains of the lichen alga <Emphasis Type="Italic">Trebouxia erici</Emphasis>: metal accumulation,toxicity and non-protein thiols

Cysteine, glutathione (GSH) and phytochelatins were determined in the cells of both wild and copper tolerant strains of the lichen alga Trebouxia erici following short-term (24 h) exposure to copper and cadmium and long-term (4 weeks) exposure to copper. Both metals caused concentration dependent synthesis of phytochelatins (PC₂–PC₅), but cadmium was a more potent activator of phytochelatin synthesis, even inducing synthesis of PC₅. The copper-tolerant strain did not reveal a higher degree of phytochelatin synthesis than the wild strain, and at 5 μM Cu production of phytochelatins was in fact significantly lower. Lower levels of phytochelatin correlated with significantly decreased intracellular copper content in the copper-tolerant strain. Both strains maintained high GSH levels even at a high copper concentration of 5 μM, and only the highest copper concentration (10 μM) was toxic for both strains, causing a decrease of GSH and PC content in algal cells. Cadmium had less effect on GSH in the cells of both tested strains. In the long term experiments, only relatively small amounts of PC₂ were detected in both strains, but the copper-tolerant strain retained significantly higher levels of reduced glutathione, probably due to the lesser degree of oxidative stress caused by Cu. The significant increase of cysteine synthesis in the copper-tolerant strain found in the present study may be related to copper tolerance in T. erici, while decreased intracellular Cu uptake, detoxification by PCs and increased free proline levels for protection of chloroplast membranes may also be implicated.     

Lack of correlation between glutathione turnover and amino acid absorption by the yeast Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae was grown in the presence of 1.0 mM l-methionine and the half-life of degradation of glutathione determined for the strains Σ1278b (444 min) and the amino-acid-uptake deficient mutant 2512c (368 min). There is no significant difference in these values, yet the rate of uptake of l-methionine is 5–7 times lower in the mutant. In neither strain is the turnover of glutathione sufficient to account for amino acid uptake. We conclude that there is no correlation between the γ-glutamyl cycle and amino acid uptake by this east.     

Vacuolar (lysosomal) trehalase ofSaccharomyces cerevisiae

In the yeastSaccharomyces cerevisiae thePEP4 gene product, protease A, is responsible for activating all soluble vacuolar (lysosomal) enzymes. These vacuolar enzymes remain inactive inpep4 mutants. Vacuolar trehalase activity was diminished in such mutants as well. This suggests that the vacuolar (lysosomal) trehalase is processed in a manner similar to other vacuolar enzymes inS. cerevisiae.     

Kluyveromyces lactis and Saccharomyces cerevisiae, two potent deacidifying and volatile-sulphur-aroma-producing microorganisms of the cheese ecosystem

Cheese flavour is the result of complex biochemical transformations attributed to bacteria and yeasts grown on the curd of smear-ripened cheeses. Volatile sulphur compounds (VSCs) are responsible for the characteristic aromatic notes of several cheeses. In the present study, we have assessed the ability of Kluyveromyces lactis, Kluyveromyces marxianus and Saccharomyces cerevisiae strains, which are frequently isolated from smear-ripened cheeses, to grow and deacidify a cheese medium and generate VSCs resulting from l-methionine degradation. The Kluyveromyces strains produced a wider variety and higher amounts of VSCs than the S. cerevisiae ones. We have shown that the pathway is likely to be proceeding differently in these two yeast genera. The VSCs are mainly generated through the degradation of 4-methylthio-oxobutyric acid in the Kluyveromyces strains, in contrast to the S. cerevisiae ones which have higher l-methionine demethiolating activity, resulting in a direct conversion of l-methionine to methanethiol. The deacidification activity which is of major importance in the early stages of cheese-ripening was also compared in S. cerevisiae and Kluyveromyces strains.     

Optimization of conditions for cadmium selenide quantum dot biosynthesis in Saccharomyces cerevisiae

The biosynthesis of quantum dots has been explored as an alternative to traditional physicochemical methods; however, relatively few studies have determined optimal synthesis parameters. Saccharomyces cerevisiae sequentially treated with sodium selenite and cadmium chloride synthesized CdSe quantum dots in the cytoplasm. These nanoparticles displayed a prominent yellow fluorescence, with an emission maximum of approximately 540 nm. The requirement for glutathione in the biosynthetic mechanism was explored by depleting its intracellular content through cellular treatments with 1-chloro-2,4-dinitrobenzene and buthionine sulfoximine. Synthesis was significantly inhibited by both of these reagents when they were applied after selenite treatment prior to the addition of cadmium, thereby indicating that glutathione contributes to the biosynthetic process. Determining the optimum conditions for biosynthesis revealed that quantum dots were produced most efficiently at entry into stationary phase followed by direct addition of 1 mM selenite for only 6 h and then immediately incubating these cells in fresh growth medium containing 3 mM Cd (II). Synthesis of quantum dots reached a maximum at 84 h of reaction time. Biosynthesis of 800-μg g⁻¹ fresh weight cells was achieved. For the first time, significant efforts have been undertaken to optimize each aspect of the CdSe biosynthetic procedure in S. cerevisiae, resulting in a 70% increased production.

     

Vba4p,a vacuolar membrane protein,is involved in the drug resistance and vacuolar morphology of Saccharomyces cerevisiae

In the vacuolar basic amino acid (VBA) transporter family of Saccharomyces cerevisiae, VBA4 encodes a vacuolar membrane protein with 14 putative transmembrane helices. Transport experiments with isolated vacuolar membrane vesicles and estimation of the amino acid contents in vacuoles showed that Vba4p is not likely involved in the transport of amino acids. We found that the vba4Δ cells, as well as vba1Δ and vba2Δ cells, showed increased susceptibility to several drugs, particularly to azoles. Although disruption of the VBA4 gene did not affect the salt tolerance of the cells, vacuolar fragmentation observed under high salt conditions was less prominent in vba4Δ cells than in wild type, vba1Δ, and vba2Δ cells. Vba4p differs from Vba1p and Vba2p as a vacuolar transporter but is important for the drug resistance and vacuolar morphology of S. cerevisiae.     

The endosomal sorting complex required for transport complex negatively regulates Erg6 degradation under specific glucose restriction conditions

Lipid droplets (LDs) are cytosolic fat storage organelles that play roles in lipid metabolism, trafficking and signaling. Breakdown of LDs in Saccharomyces cerevisiae is mainly achieved by lipolysis and lipophagy. In this study, we found that the endosomal sorting complex required for transport (ESCRT) in S. cerevisiae negatively regulated the turnover of a LD marker, Erg6, under both simplified glucose restriction (GR) and acute glucose restriction (AGR) conditions by monitoring the localization and degradation of Erg6. Loss of Vps27, Snf7 or Vps4, representative subunits of the ESCRT machinery, facilitated the delivery of Erg6‐GFP to vacuoles and its degradation depending on the lipophagy protein Atg15 under simplified GR. Additionally, the lipolysis proteins Tgl3 and Tgl4 were also involved in the enhanced vacuolar localization and degradation of Erg6‐GFP in vps4Δ cells. Furthermore, we found that Atg14, which is required for the formation of putatively liquid‐ordered (Lo) membrane domains on the vacuole that act as preferential internalization sites for LDs, abundantly localized to vacuolar membranes in ESCRT mutants. Most importantly, the depletion or overexpression of Atg14 correspondingly abolished or promoted the observed Erg6 degradation in ESCRT mutant cells. We propose that Atg14 together with other proteins promotes Erg6 degradation in ESCRT mutant cells under specific glucose restriction conditions. These results shed new light on the regulation of ESCRT on LD turnover.     

The two methionine adenosyl transferases in Saccharomyces cerevisiae: Evidence for the existence of dimeric enzymes

Summary In Saccharomyces cerevisiae either of the two genes SAM1 and SAM2 is able to produce a functional methionine adenosyl transferase (MATI and MATII). In a wild-type strain, MATI and MATII are present in dimeric forms: MATI-MATI, MATII-MATII and perhaps MATI-MATII. A hypothesis is presented to explain the possible role of these different forms of methionine adenosyl transferase in S. cerevisiae.     

Lap3 is a selective target of autophagy in yeast, Saccharomyces cerevisiae

Autophagy is a primarily non-selective degradation system of cytoplasmic constituents in lysosomes/vacuoles during starvation. In yeast, autophagy is also involved in the selective transport of Ape1, a vacuolar hydrolase, as a biosynthetic route. Ald6, a soluble cytoplasmic enzyme, is preferentially eliminated from cytoplasm via autophagy. However, little is known about the mechanisms of Ald6 targeting to autophagosomes. Here, we show that Lap3, a soluble cytosolic cysteine protease, is spatially associated with Ape1 and selectively transported to the vacuole during nitrogen starvation. The rate of Lap3 transport is much higher than that of Ald6 and is similar to that of Ape1. Moreover, ATG11 and ATG19, essential factors for Ape1 transport, are important for Lap3 transport. Most Lap3 is degraded within a couple of hours in the vacuole in contrast to Ape1; therefore, we conclude that the machinery required for Ape1 biosynthesis is used for selective degradation of Lap3.     

Functional characterization and expression analysis of a glutathione transporter, BjGT1, from Brassica juncea: evidence for regulation by heavy metal exposure

Glutathione and its derivatives play an important role in the tolerance of plants against heavy metals. A glutathione transporter, BjGT1 (AJ561120), was cloned and functionally characterized from Brassica juncea, a plant which may be used for phytoremediation. The full‐length BjGT1 cDNA showed homology with the high affinity glutathione transporter HGT1 from Saccharomyces cerevisiae and shares 92% identity with a putative glutathione transporter from A. thaliana (At4g16370). When expressed in the S. cerevisiae hgt1Δ strain, BjGT1 complemented the mutant on medium with glutathione as the only sulphur source and mediated the uptake of [³H]GSH. Immunoblot analysis with a peptide‐specific antiserum directed against a C‐terminal sequence revealed high BjGT1 expression in leaf tissue and relatively low expression in stem tissue, whereas BjGT1 protein was not detectable in root tissue. The amounts of BjGT1 mRNA and protein were analysed during a 6 d exposure of B. juncea to 25 µm Cd(NO₃)₂. BjGT1 mRNA was strongly induced by cadmium in stems and leaves. Unexpectedly, the amount of BjGT1 protein in leaves showed a pronounced decrease with a minimum after 96 h of Cd exposure, followed by partial recovery. The strong regulation of BjGT1 by cadmium suggests a role of this glutathione transporter during heavy metal exposure.