SpOPT1, a member of the oligopeptide family (OPT) of the fission yeast Schizosaccharomyces pombe,is involved in the transport of glutathione through the outer membrane of the cell

A protein involved in the transport of glutathione has been identified, cloned and characterized from the fission yeast Schizosaccharomyces pombe. Database searches revealed the Sz. pombe ORF SPAC29B12.10c as a close homologue to several members of the OPT family, including the Saccharomyces cerevisiae high‐affinity glutathione transporter Hgt1p. The gene product of SPAC29B12.10c has been identified as a protein, named SpOPT1, localized within the plasma membrane, transporting the tripeptide glutathione. Disruption of SPAC29B12.10c led to strains inable to grow on media containing glutathione as a sole source of sulphur, due to the inability to internalize the tripeptide. Disruptants contained significantly less glutathione than wild‐type cells. Furthermore, ΔSpopt1 strains were non‐viable in a glutathione biosynthesis‐defective (Δgsh2) background. However, it was possible to complement the disruption of Spopt1 by overexpressing the intact ORF in the disrupted strain. Copyright © 2009 John Wiley & Sons, Ltd.     

Human NKCC2 cation–Cl– co‐transporter complements lack of Vhc1 transporter in yeast vacuolar membranes

Cation–chloride co‐transporters serve to transport Cl^– and alkali metal cations. Whereas a large family of these exists in higher eukaryotes, yeasts only possess one cation–chloride co‐transporter, Vhc1, localized to the vacuolar membrane. In this study, the human cation–chloride co‐transporter NKCC2 complemented the phenotype of VHC1 deletion in Saccharomyces cerevisiae and its activity controlled the growth of salt‐sensitive yeast cells in the presence of high KCl, NaCl and LiCl. A S. cerevisiae mutant lacking plasma‐membrane alkali–metal cation exporters Nha1 and Ena1‐5 and the vacuolar cation–chloride co‐transporter Vhc1 is highly sensitive to increased concentrations of alkali–metal cations, and it proved to be a suitable model for characterizing the substrate specificity and transport activity of human wild‐type and mutated cation–chloride co‐transporters. Copyright © 2013 John Wiley & Sons, Ltd.     

Transport and metabolism of fumaric acid in Saccharomyces cerevisiae in aerobic glucose‐limited chemostat culture

Currently, research is being focused on the industrial‐scale production of fumaric acid and other relevant organic acids from renewable feedstocks via fermentation, preferably at low pH for better product recovery. However, at low pH a large fraction of the extracellular acid is present in the undissociated form, which is lipophilic and can diffuse into the cell. There have been no studies done on the impact of high extracellular concentrations of fumaric acid under aerobic conditions in S. cerevisiae, which is a relevant issue to study for industrial‐scale production. In this work we studied the uptake and metabolism of fumaric acid in S. cerevisiae in glucose‐limited chemostat cultures at a cultivation pH of 3.0 (pH < pK). Steady states were achieved with different extracellular levels of fumaric acid, obtained by adding different amounts of fumaric acid to the feed medium. The experiments were carried out with the wild‐type S. cerevisiae CEN.PK 113‐7D and an engineered S. cerevisiae ADIS 244 expressing a heterologous dicarboxylic acid transporter (DCT‐02) from Aspergillus niger, to examine whether it would be capable of exporting fumaric acid. We observed that fumaric acid entered the cells most likely via passive diffusion of the undissociated form. Approximately two‐thirds of the fumaric acid in the feed was metabolized together with glucose. From metabolic flux analysis, an increased ATP dissipation was observed only at high intracellular concentrations of fumarate, possibly due to the export of fumarate via an ABC transporter. The implications of our results for the industrial‐scale production of fumaric acid are discussed. Copyright © 2015 John Wiley & Sons, Ltd.     

Requirement of glutathione for Sod1 activation during lifespan extension

It has been shown that the activation of cytosolic superoxide dismutase (Sod1) in Saccharomyces cerevisiae is only dependent on Ccs1, which is responsible for insertion of copper into the enzyme catalytic center, and that glutathione (GSH) is not necessary for this process. In this work, we addressed an important role of GSH in Sod1 activation by a Ccs1‐dependent mechanism during oxidative stress and its role in yeast lifespan. Exponential cells of Saccharomyces cerevisiae, treated or not with 0.5 mM menadione for 1 h, were used for evaluation of the effect of a mild oxidative stress pre‐treatment on chronological lifespan. The results showed that menadione induced a lifespan extension in the wild‐type (WT) strain but this adaptive response was repressed in gsh1 and in sod1 strains. Interestingly, menadione treatment increased SOD1 and CCS1 gene expression in both WT and gsh1 strains. However, while these strains showed the same Sod1 activity before treatment, only the WT presented an increase of Sod1 activity after menadione exposure. Glutathionylation seems to be essential for Sod1 activation since no increase in activity was observed after menadione treatment in grx1 and grx2 null mutants. Our results suggest that GSH and glutathionylation are fundamental to protect Sod1 sulfhydryl residues under mild oxidative stress, enabling Sod1 activation and lifespan extension. Copyright © 2010 John Wiley & Sons, Ltd.     

Cloning and characterization of a novel NAD+‐dependent glyceraldehyde‐3‐phosphate dehydrogenase gene from Candida glycerinogenes and use of its promoter

A 3950 bp genomic fragment from Candida glycerinogenes, WL2002‐5, containing the CgGAP gene encoding a glyceraldehyde‐3‐phosphate dehydrogenase homologous to GAP genes in other yeasts using degenerate primers, was cloned and characterized with inverse PCR. Sequence analysis revealed a 1164 bp open reading frame encoding a putative peptide of 387 deduced amino acids, with a molecular mass of 36 kDa. The CgGAP protein consisted of an N‐terminal NAD⁺‐binding domain and a central catalytic domain. Six stress‐response elements were found in the upstream region of the CgGAP gene. The influence of CgGAP on glycolysis was investigated. Functional analysis revealed that Saccharomyces cerevisiae transformed with CgGAP was restored to the wild‐type phenotype when cultured in high‐osmolarity medium, suggesting that it is a functional GAP protein. Promoter studies in S. cerevisiae using the green fluorescent protein (gfp) gene as a reporter showed that the GAP promoter (P_CgGAP) is constitutively expressed in S. cerevisiae cells grown on glucose. Copyright © 2013 John Wiley & Sons, Ltd.     

Mechanisms of Salt Tolerance Conferred by Overexpression of the HAL1 Gene in Saccharomyces cerevisiae

Overexpression of the HAL1 gene improves the tolerance of Saccharomyces cerevisiae to NaCl by increasing intracellular K⁺ and decreasing intracellular Na⁺. The effect of HAL1 on intracellular Na⁺ was mediated by the PMR2/ENA1 gene, corresponding to a major Na⁺ efflux system. The expression level of ENA1 was dependent on the gene dosage of HAL1 and overexpression of HAL1 suppressed the salt sensitivity of null mutants in calcineurin and Hal3p, other known regulators of ENA1 expression. The effect of HAL1 on intracellular K⁺ was independent of the TRK1 and TOK1 genes, corresponding to a major K⁺ uptake system and to a K⁺ efflux system activated by depolarization, respectively. Overexpression of HAL1 reduces K⁺ loss from the cells upon salt stress, a phenomenon mediated by an unidentified K⁺ efflux system. Overexpression of HAL1 did not increase NaCl tolerance in galactose medium. NaCl poses two types of stress, osmotic and ionic, counteracted by glycerol synthesis and sodium extrusion, respectively. As compared to glucose, with galactose as carbon source glycerol synthesis is reduced and the expression of ENA1 is increased. As a consequence, osmotic adjustment through glycerolsynthesis, a process not affected by HAL1, is the limiting factor for growth on galactose under NaCl stress. © 1997 John Wiley & Sons, Ltd.     

The yeast Saccharomyces cerevisiae Pdr16p restricts changes in ergosterol biosynthesis caused by the presence of azole antifungals

Pdr16p belongs to the family of phosphatidylinositol transfer proteins in yeast. The absence of Pdr16p results in enhanced susceptibility to azole antifungals in Saccharomyces cerevisiae. In the major fungal human pathogen Candida albicans, CaPDR16 is a contributing factor to clinical azole resistance. The current study was aimed at better understanding the function of Pdr16p, especially in relation to azole resistance in S. cerevisiae. We show that deletion of the PDR16 gene increased susceptibility of S. cerevisiae to azole antifungals that are used in clinical medicine and agriculture. Significant differences in the inhibition of the sterol biosynthetic pathway were observed between the pdr16Δ strain and its corresponding wild‐type (wt) strain when yeast cells were challenged by sub‐inhibitory concentrations of the azoles miconazole or fluconazole. The increased susceptibility to azoles, and enhanced changes in sterol biosynthesis upon exposure to azoles of the pdr16Δ strain compared to wt strain, are not the results of increased intracellular concentration of azoles in the pdr16Δ cells. We also show that overexpression of PDR17 complemented the azole susceptible phenotype of the pdr16Δ strain and corrected the enhanced sterol alterations in pdr16Δ cells in the presence of azoles. Pdr17p was found previously to be an essential part of a complex required for intermembrane transport of phosphatidylserine at regions of membrane apposition. Based on these observations, we propose a hypothesis that Pdr16p assists in shuttling sterols or their intermediates between membranes or, alternatively, between sterol biosynthetic enzymes or complexes. Copyright © 2013 John Wiley & Sons, Ltd.     

Chromosome VIII disomy influences the nonsense suppression efficiency and transition metal tolerance of the yeast Saccharomyces cerevisiae

The SUP35 gene of the yeast Saccharomyces cerevisiae encodes the translation termination factor eRF3. Mutations in this gene lead to the suppression of nonsense mutations and a number of other pleiotropic phenotypes, one of which is impaired chromosome segregation during cell division. Similar effects result from replacing the S. cerevisiae SUP35 gene with its orthologues. A number of genetic and epigenetic changes that occur in the sup35 background result in partial compensation for this suppressor effect. In this study we showed that in S. cerevisiae strains in which the SUP35 orthologue from the yeast Pichia methanolica replaces the S. cerevisiae SUP35 gene, chromosome VIII disomy results in decreased efficiency of nonsense suppression. This antisuppressor effect is not associated with decreased stop codon read‐through. We identified SBP1, a gene that localizes to chromosome VIII, as a dosage‐dependent antisuppressor that strongly contributes to the overall antisuppressor effect of chromosome VIII disomy. Disomy of chromosome VIII also leads to a change in the yeast strains’ tolerance of a number of transition metal salts. Copyright © 2015 John Wiley & Sons, Ltd.     

Vectors for Cu2+-inducible production of glutathione S-transferase-fusion proteins for single-step purification from yeast

We describe six new yeast episomal vectors which encode glutathione S-transferase (GST) affinity tags. These allow for the production of GST-fusion proteins in Saccharomyces cerevisiae under the control of the CUP1 promoter. Affinity chromatography with glutathione-Sepharose permits convenient purification of the fusion protein from a yeast lysate. The presence of a protease cleavage site facilitates subsequent removal of the GST tag. The expression and single-step purification of both GST and a functional GST-metallothionein fusion from yeast are shown as an example of the application of these vectors.     

Inhibition of the decrease of volatile esters and terpenes during storage of a white wine and a model wine medium by glutathione and N-acetylcysteine

Glutathione and N‐acetylcysteine, each at 20 mg L^?1, were tested as inhibitors of the decrease of volatile esters and terpenes during storage of Debina white wine. Moreover, the inhibition of the decrease of isoamyl acetate, ethyl hexanoate and linalool in a model wine medium by glutathione and N‐acetylcysteine, each at 0–20 mg L^?1, was also tested. Several volatiles, such as isoamyl acetate, ethyl hexanoate, ethyl octanoate, ethyl decanoate and linalool, decreased during wine storage. Glutathione or N‐acetylcysteine significantly restricted the decrease of these volatiles. In the model medium, each thiol inhibited the decrease of the three volatiles in a dose‐dependent manner. N‐acetylcysteine inhibited the decrease of all three volatiles at 2.5 mg L^?1 while glutathione at 2.5 or 5.0 mg L^?1. The present results indicate that glutathione and N‐acetylcysteine may be taken into account as potent inhibitors of the disappearance of aromatic esters and terpenes in wines.     

Hygromycin‐resistance vectors for gene expression in Pichia pastoris

Pichia pastoris is a common host organism for heterologous protein expression and metabolic engineering. Zeocin‐, G418‐, nourseothricin‐ and blasticidin‐resistance genes are the only dominant selectable markers currently available for selecting P. pastoris transformants. We describe here new P. pastoris expression vectors that confer a hygromycin resistance base on the Klebsiella pneumoniae hph gene. To demonstrate the application of the vectors for intracellular and secreted protein expression, green fluorescent protein (GFP) and human serum albumin (HSA) were cloned into the vectors and transformed into P. pastoris cells. The resulting strains expressed GFP and HSA constitutively or inducibly. The hygromycin resistance marker was also suitable for post‐transformational vector amplication (PTVA) for obtaining strains with high plasmid copy numbers. A strain with multiple copies of the HSA expression cassette after PTVA had increased HSA expression compared with a strain with a single copy of the plasmid. To demonstrate compatibility of the new vectors with other vectors bearing antibiotic‐resistance genes, P. pastoris was transformed with the Saccharomyces cerevisiae genes GSH1, GSH2 or SAM2 on plasmids containing genes for resistance to Zeocin, G418 or hygromycin. The resulting strain produced glutathione and S‐adenosyl‐l ‐methionine at levels approximately twice those of the parent strain. The new hygromycin‐resistance vectors allow greater flexibility and potential applications in recombinant protein production and other research using P. pastoris. Copyright © 2014 John Wiley & Sons, Ltd.     

Antioxidant and melanogenesis‐inhibitory activities of collagen peptide from jellyfish (Rhopilema esculentum)

BACKGROUND: Jellyfish collagen was hydrolysed with trypsin and properase E, and jellyfish collagen peptide (JCP) was purified from the enzymatic hydrolysate using ion exchange chromatography and gel filtration. The antioxidant activity of JCP in a linoleic acid emulsion system, its superoxide anion‐ and hydroxyl radical‐scavenging activities and its copper‐chelating ability were evaluated in vitro. Initial investigations of JCP's ability to inhibit melanogenesis were carried out using cultured B16 melanoma cells. RESULTS: The molecular weight distribution of JCP was from 400 to 1200 Da. Amino acid analysis showed that JCP was rich in Gly, Pro, Ser, Ala, Glu and Asp and had a total hydrophobic amino acid content of 384.2 g kg^?1. JCP showed high antioxidant activity (IC₅₀147.8 µg mL^?1), superoxide anion‐scavenging activity (IC₅₀21.9 µg mL^?1), hydroxyl radical‐scavenging activity (IC₅₀16.7 µg mL^?1) and copper‐chelating ability (IC₅₀88.7 µg mL^?1) in vitro. It also significantly inhibited intracellular tyrosinase activity, decreased melanin content and enhanced glutathione synthesis (P < 0.05). Furthermore, JCP decreased intracellular cAMP levels and suppressed tyrosinase mRNA expression. CONCLUSION: Based on the results of this study, JCP exerts anti‐melanogenic actions via its antioxidant properties and copper‐chelating ability. JCP could be used as a natural skin‐lightening agent in the medicine and food industries. Copyright © 2009 Society of Chemical Industry     

Decapping of stabilized,polyadenylated mRNA in yeast pab1 mutants

Interaction of the poly(A) binding protein, Pab1p, with mRNA plays an important role in gene expression. This work describes an analysis of pab1 mutants in Saccharomyces cerevisiae. Yeast pab1 mutants were found to be sensitive to elevated concentrations of copper (Cu) and 3‐aminotriazole (3‐AT) in the growth medium. This phenotype arises because these pab1 mutants underaccumulate mRNA, including the CUP1 and HIS3 mRNAs, the products of which are required for Cu and 3‐AT resistance, respectively. To determine the cause of the mRNA underaccumulation, mRNA turnover and production were examined in the pab1‐53 mutant. It was found that although the pattern of mRNA decay was altered, and substantial decapping of polyadenylated mRNA could be detected, mRNA was not destabilized in this strain. It was also found that the pab1 mutant was impaired in the production of mRNA. These data show that the decreased level of mRNA in the pab1‐53 mutant arises from poor production, and they suggest that yeast Pab1p is involved in an aspect of nuclear mRNA metabolism. They also indicate that deadenylation can be uncoupled from decapping without significant changes in an mRNA's stability, and that this uncoupling can be tolerated by yeast. Copyright © 1999 John Wiley & Sons, Ltd.