The term ‘sake yeast’ is generally used to indicate the Saccharomyces cerevisiae strains that possess characteristics distinct from others including the laboratory strain S288C and are well suited for sake brewery. Here, we report the draft whole-genome shotgun sequence of a commonly used diploid sake yeast strain, Kyokai no. 7 (K7). The assembled sequence of K7 was nearly identical to that of the S288C, except for several subtelomeric polymorphisms and two large inversions in K7. A survey of heterozygous bases between the homologous chromosomes revealed the presence of mosaic-like uneven distribution of heterozygosity in K7. The distribution patterns appeared to have resulted from repeated losses of heterozygosity in the ancestral lineage of K7. Analysis of genes revealed the presence of both K7-acquired and K7-lost genes, in addition to numerous others with segmentations and terminal discrepancies in comparison with those of S288C. The distribution of Ty element also largely differed in the two strains. Interestingly, two regions in chromosomes I and VII of S288C have apparently been replaced by Ty elements in K7. Sequence comparisons suggest that these gene conversions were caused by cDNA-mediated recombination of Ty elements. The present study advances our understanding of the functional and evolutionary genomics of the sake yeast.

Epigallocatechin gallate (EGCG) is the most abundant polyphenolic flavonoid in green tea. Catechin and its derivatives, including EGCG, are widely believed to function as antioxidants. Here we demonstrate that both EGCG and green tea extract (GTE) cause oxidative stress-related responses in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe under weak alkaline conditions in terms of the activation of oxidative-stress-responsive transcription factors. GTE as well as EGCG induced the nuclear localization of Yap1 in S. cerevisiae, which was repressed by the addition of catalase but not by the addition of superoxide dismutase. The same phenomena were observed for the nucleocytoplasmic localization of Msn2 in S. cerevisiae and Pap1, a Yap1 homologue, in S. pombe. The formation of intramolecular disulfide bonds has been proposed to be crucial for the H 2 O 2 -induced nuclear localization of Yap1, and we verified the importance of cysteine residues of Yap1 in response to EGCG and GTE. Additionally, we show that EGCG and GTE produce H 2 O 2 in a weak alkaline medium. Finally, we conclude that tea polyphenols are able to act as prooxidants to cause a response to oxidative stress in yeasts under certain conditions.Green tea is one of the beverages consumed in the highest quantity in the world. Epidemiologic research has revealed that individuals who drink a lot of green are less likely to develop cancer (28,30,51,64). Very recently, a relationship between the consumption of green tea and a reduced risk of type 2 diabetes was reported (23). Green tea contains many ingredients considered to promote health such as polyphenolic flavonoids, of which epigallocatechin gallate (EGCG) is the major constituent. Evidence is mounting that EGCG has anticarcinogenic activity in vitro (3,8,27,61), which may support the results of the epidemiologic research on the correlation between drinking green tea and the risk of morbidity from cancer.Many studies have been done on the biological activity of green tea extract (GTE) and individual catechins in vitro. EGCG is widely accepted as an antioxidant. For example, EGCG scavenges superoxide anion radicals (O 2 ·Ϫ ), hydrogen peroxide (H 2 O 2 ), hydroxy radicals (HO · ), peroxyl radicals, singlet oxygen, and peroxynitrite (5,16,17,45,47,49,56). The one-electron reduction potential of EGCG under standard conditions is 550 mV, a value lower than that of glutathione (920 mV) and comparable to that of ␣-tocopherol (480 mV) (13,24,25). Besides directly scavenging reactive oxygen/nitrogen species, EGCG chelates redox-active metal ions, such as iron and copper, leading to a reduction in the production of reactive oxygen species. Accordingly, many food supplements or beverages containing a high concentration of EGCG (Ͼ1 mM) have been developed, and therefore, the physiological function of EGCG in vivo with a high-dose ingestion remains to be elucidated.In contrast to its antioxidative activity, recent experiments in vitro indicate that EGCG produces reactive oxy...

Methylglyoxal, a toxic metabolite synthesized in vivo during glycolysis, inhibits cell growth. One of the mechanisms protecting eukaryotic cells against its toxicity is the glyoxalase system, composed of glyoxalase I and II (glo1 and glo2), which converts methylglyoxal into D-lactic acid in the presence of glutathione. Here we have shown that the two principal oxidative stress response pathways of Schizosaccharomyces pombe, Sty1 and Pap1, are involved in the response to methylglyoxal toxicity. The mitogen-activated protein kinase Sty1 is phosphorylated and accumulates in the nucleus following methylglyoxal treatment. Moreover, glo2 expression is induced by methylglyoxal and environmental stresses in a Sty1-dependent manner. The transcription factor Pap1 also accumulates in the nucleus, activating the expression of its target genes following methylglyoxal treatment. Our studies showed that the C-terminal cysteine-rich domain of Pap1 is sufficient for methylglyoxal sensing. Furthermore, the redox status of Pap1 is not changed by methylglyoxal. We propose that methylglyoxal treatment triggers Pap1 and Sty1 nuclear accumulation, and we describe the molecular basis of such activation mechanisms. In addition, we discuss the potential physiological significance of these responses to a natural toxic metabolite.

Methylglyoxal (MG) is Methylglyoxal (MG,2 CH 3 COCHO) is a typical 2-oxoaldehyde derived from glycolysis, a ubiquitous energy-generating system present in all types of organism; nevertheless, MG inhibits the growth of cells (1). For example, MG induces apoptosis in some mammalian cells by provoking production of reactive oxygen species (2). Actually, cells exhibiting MG-induced apoptosis, such as Jurkat cells and HL60 cells, are sensitive to reactive oxygen species (3, 4). On the other hand, MG induces necrosis in the budding yeast Saccharomyces cerevisiae (5). In this case, MG does not enhance production of reactive oxygen species, and therefore, mutants lacking antioxidant enzymes (for example sod1⌬, sod2⌬, and gpx3⌬) do not show increased sensitivity to MG (5).Because the major source of MG is glycolysis, and an overaccumulation of MG adversely affects cellular function, cells of all types possess a ubiquitous enzymatic pathway, the glyoxalase system, for detoxification of MG. The glyoxalase system consists of glyoxalase I and glyoxalase II. The former catalyzes the conversion of MG to S-D-lactoylglutathione in the presence of glutathione. The latter hydrolyzes the resultant glutathione thiolester to D-lactic acid and glutathione (see Fig. 1). Previously, we have reported that the disruption of GLO1, the structural gene for glyoxalase I, enhances susceptibility to MG in S. cerevisiae (6). A mutant defective in glyoxalase II was also sensitive to MG (7). On the other hand, S. cerevisiae has another enzymatic route by which to convert MG to lactic acid, i.e. MG is reduced to L-lactaldehyde by the action of an NADPH-dependent methylglyoxal reductase, and L-lactaldehyde is further oxidized to L-lactic acid by an NAD ϩ -dependent lactaldehyde dehydrogenase (8, 9). Chen et al. (10) have reported that GRE2 encodes methylglyoxal reductase, although, as far as we could determine, gre2⌬ cells were not sensitive to MG. 3 Aguilera and Prieto (11) have reported that GRE3 encodes aldose reductase that is involved in the metabolism of MG; however, a gre3⌬ mutant did not exhibit an MG-sensitive phenotype. Consequently, the glyoxalase system is the major pathway to detoxify MG in S. cerevisiae (12). We have confirmed that a glyoxalase I-deficient mutant of the fission yeast Schizosaccharomyces pombe was also sensitive to MG (13).Aberrant metabolism of MG has been implicated in diseases such as colon cancer, diabetes, Alzheimer disease, and autism (14 -17). For example, in type 2 diabetes, the basal activity of the mitogen-activated protein (MAP) kinase family is increased, and the up-regulation of p38, one of the MAP kinases, is associated with a loss of expression of the GLUT4 glucose transporter that results in a lowering of glucose uptake, which may exacerbate hyperglycemia (18). Therefore, it is implied that there is a correlation between high MG levels and increased basal activation of p38 in type 2 diabetes. However, it remains to be clarified whether a high MG level is a consequence of diabetes, or diabetes induces abe...

Methylglyoxal (MG) is a ubiquitous metabolite derived from glycolysis; however, this aldehyde kills all types of cell. We analyzed the properties of MG-induced cell death of the budding yeast Saccharomyces cerevisiae. The MCA1 gene encodes a caspase homologue that is involved in H2O2-induced apoptosis in yeast, although the disruption of MCA1 did not repress sensitivity to MG. In addition, the intracellular oxidation level did not increase under conditions in which MG kills the cell. Furthermore, the disruption of genes encoding antioxidant enzymes did not affect the susceptibility to MG. Here, we demonstrate that yeast cells killed by MG do not exhibit the characteristics of apoptosis in a TUNEL assay or an annexin V staining, but show those of necrosis upon propidium iodide staining. We demonstrate that MG at high concentrations provokes necrotic cell death without the generation of reactive oxygen species in S. cerevisiae.

The GPX2 gene encodes a homologue of mammalian phospholipid hydroperoxide glutathione peroxidase in Saccharomyces cerevisiae. Previously, we have reported that the oxidative stress-induced expression of GPX2 is strictly regulated by Yap1 and Skn7 transcription factors. Here, we found that the expression of GPX2 is induced by CaCl 2 in a calcineurin (CN)/ Crz1-dependent manner, and the CN-dependent response element was specified in the GPX2 promoter. Neither Yap1 nor Skn7 was required for Ca 2þ -dependent induction of GPX2, therefore, distinct regulation for the oxidative stress response and Ca 2þ signal response for GPX2 exists in yeast cells.

Thioredoxin (TRX) is an important antioxidant present in all types of organisms. Besides its role as an antioxidant, TRX protects the gastric mucosa due to its antiinflammatory effect. In addition, TRX decreases allergenicity; therefore, the oral administration of TRX is of considerable interest with respect to its clinical use as well as the development of functional foods containing TRX. We have attempted to enrich the cellular TRX content in Saccharomyces cerevisiae, and found that green tea extract (Sunphenon), which is rich in catechins (polyphenols), activates the Yap1 transcription factor, leading to the induction of TRX2, a target of Yap1. Production of yeast TRX was monitored by both a TRX2-lacZ reporter expression assay and Western blotting using an anti-yeast TRX antibody. Maximal production of TRX was achieved in a medium containing 0.1% green tea extract at pH 7.6. We discuss the underlying mechanism by which green tea extract activates Yap1.

The GPX2 gene encodes a homologue of phospholipid hydroperoxide glutathione peroxidase in Saccharomyces cerevisiae. The GPX2 promoter contains three elements the sequence of which is completely consistent with the optimal sequence for the Yap1 response element (YRE). Here, we identify the intrinsic YRE that functions in the oxidative stress response of GPX2. In addition, we discovered a cis-acting element (5'-GGCCGGC-3') within the GPX2 promoter proximal to the functional YRE that is necessary for H(2)O(2)-induced expression of GPX2. We present evidence showing that Skn7 is necessary for the oxidative stress response of GPX2 and is able to bind to this sequence. We determine the optimal sequence for Skn7 to regulate GPX2 under conditions of oxidative stress to be 5'-GGC(C/T)GGC-3', and we designate this sequence the oxidative stress-responsive Skn7 response element.