Yap1p, a crucial transcription factor in the oxidative stress response of Saccharomyces cerevisiae, is transported in and out of the nucleus under nonstress conditions. The nuclear export step is specifically inhibited by H 2 O 2 or the thiol oxidant diamide, resulting in Yap1p nuclear accumulation and induction of transcription of its target genes. Here we provide evidence for sensing of H 2 O 2 and diamide mediated by disulfide bond formation in the C-terminal cysteine-rich region (c-CRD), which contains 3 conserved cysteines and the nuclear export signal (NES). The H 2 O 2 or diamide-induced oxidation of the c-CRD in vivo correlates with induced Yap1p nuclear localization. Both were initiated within 1 min of application of oxidative stress, before the intracellular redox status of thioredoxin and glutathione was affected. The cysteine residues in the middle region of Yap1p (n-CRD) are required for prolonged nuclear localization of Yap1p in response to H 2 O 2 and are thus also required for maximum transcriptional activity. Using mass spectrometry analysis, the H 2 O 2 -induced oxidation of the c-CRD in vitro was detected as an intramolecular disulfide linkage between the first (Cys 598 ) and second (Cys 620 ) cysteine residues; this linkage could be reduced by thioredoxin. In contrast, diamide induced each pair of disulfide linkage in the c-CRD, but in this case the cysteine residues in the n-CRD appeared to be dispensable for the response. Our data provide evidence for molecular mechanisms of redox signal sensing through the thiol-disulfide redox cycle coupled with the thioredoxin system in the Yap1p NES.

Methylglyoxal (MG) is a typical 2-oxoaldehyde derived from glycolysis, although it inhibits the growth of cells in all types of organism. Hence, it has been questioned why such a toxic metabolite is synthesized via the ubiquitous energy-generating pathway. We have previously reported that expression of GLO1, coding for the major enzyme detoxifying MG, was induced by osmotic stress in a high osmolarity glycerol ( Glycolysis is a ubiquitous energy-generating pathway in organisms. Triosephosphate isomerase is one of the enzymes involved in this anaerobic oxidation pathway for glucose. During the triosephosphate isomerase reaction, methylglyoxal (MG, 1 CH 3 COCHO) is synthesized by the ␤-elimination reaction from an enediolate phosphate intermediate (1, 2). Although MG is synthesized during normal metabolism of glucose, this aldehyde inhibits the growth of cells from microorganisms to higher eukaryotes, eventually killing them, and therefore, the raison d'etre of MG has been of considerable interest.By the systematic biochemical analysis of the metabolic fate of MG using the budding yeast Saccharomyces cerevisiae as a model organism, we revealed that MG is metabolized to lactic acid by two different routes (for reviews, see Refs. 3 and 4). One route is a glyoxalase system in which glyoxalase I and glyoxalase II are involved. In this route, MG is condensed with glutathione to give S-D-lactoylglutathione by the action of glyoxalase I, and the glutathione thiolester is then hydrolyzed to lactic acid and glutathione by glyoxalase II. The other route is a reduction/oxidation system consisting of methylglyoxal reductase and lactaldehyde dehydrogenase. MG is reduced to lactaldehyde by NADPH-dependent methylglyoxal reductase, and lactaldehyde is further oxidized to lactic acid by NAD ϩ -dependent lactaldehyde dehydrogenase. To evaluate the physiological importance of these enzymatic routes in vivo, we cloned the structural gene for glyoxalase I (GLO1) and demonstrated that a glo1⌬ mutant showed hypersensitivity to MG (5). Similarly, Bito et al. (6) reported that a mutant defective in glyoxalase II (glo2⌬) was also hypersensitive to MG. On the other hand, Chen et al. (7) recently reported that GRE2 is one of the genes encoding NADPH-dependent methylglyoxal reductase activity, although a gre2⌬ mutant did not exhibit sensitivity to MG as far as we discerned.2 Alternatively, the GRE3 gene encodes an aldose reductase, and the corresponding gene product is supposed to be involved in MG metabolism, although the disruption of GRE3 did not enhance susceptibility to MG (8). These results indicate that the glyoxalase system is the most important pathway for MG detoxification in S. cerevisiae. This was also the case in the fission yeast (9).To obtain a clue as to the cellular function of MG and its metabolic pathway, we have attempted to find the conditions that alter the intracellular MG level and/or MG-metabolizing enzyme activity in S. cerevisiae. We found that the expression of GLO1 is specifically induced by osmotic stress in a h...

Methylglyoxal (MG) is synthesized during glycolysis, although it inhibits cell growth in all types of organisms. Hence, it has long been asked why such a toxic metabolite is synthesized in vivo. Glyoxalase I is a major enzyme detoxifying MG. Here we show that the Yap1 transcription factor, which is critical for the oxidativestress response in Saccharomyces cerevisiae, is constitutively concentrated in the nucleus and activates the expression of its target genes in a glyoxalase I-deficient mutant. Yap1 contains six cysteine residues in two cysteine-rich domains (CRDs), i.e., three cysteine residues clustering near the N terminus (n-CRD) and the remaining three cysteine residues near the C terminus (c-CRD). We reveal that any of the three cysteine residues in the c-CRD is sufficient for MG to allow Yap1 to translocate into the nucleus and to activate the expression of its target gene. A Yap1 mutant possessing only one cysteine residue in the c-CRD but no cysteine in the n-CRD and deletion of the basic leucine zipper domain can concentrate in the nucleus with MG treatment. However, substitution of all the cysteine residues in Yap1 abolishes the ability of this transcription factor to concentrate in the nucleus following MG treatment. The redox status of Yap1 is substantially unchanged, and protein(s) interaction with Yap1 through disulfide bond is hardly detected in cells treated with MG. Collectively, neither intermolecular nor intramolecular disulfide bond formation seems to be involved in Yap1 activation by MG. Moreover, we show that nucleocytoplasmic localization of Yap1 closely correlates with growth phase and intracellular MG level. We propose a novel regulatory pathway underlying Yap1 activation by a natural metabolite in the cell.

a b s t r a c tEukaryotic translation initiation factor 5A (eIF5A) is a protein subject to hypusination, which is essential for its function. eIF5A is also acetylated, but the role of that modification is unknown. Here, we report that acetylation regulates the subcellular localization of eIF5A. We identified PCAF as the major cellular acetyltransferase of eIF5A, and HDAC6 and SIRT2 as its major deacetylases. Inhibition of the deacetylases or impaired hypusination increased acetylation of eIF5A, leading to nuclear accumulation. As eIF5A is constitutively hypusinated under physiological conditions, we suggest that reversible acetylation plays a major role in controlling the subcellular localization of eIF5A.

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...

The glyoxalase system consists of glyoxalase I and glyoxalase II. Glyoxalase I catalyzes the conversion of methylglyoxal (CH(3)COCHO), a metabolite derived from glycolysis, with glutathione to S-D-lactoylglutathione, while glyoxalase II hydrolyses this glutathione thiolester to D-lactic acid and glutathione. Since methylglyoxal is toxic due to its high reactivity, the glyoxalase system is crucial to warrant the efficient metabolic flux of this reactive aldehyde. The budding yeast Saccharomyces cerevisiae has the sole gene (GLO1) encoding the structural gene for glyoxalase I. Meanwhile, this yeast has two isoforms of glyoxalase II encoded by GLO2 and GLO4. The expression of GLO1 is regulated by Hog1 mitogen-activated protein kinase and Msn2/Msn4 transcription factors under highly osmotic stress conditions. The physiological significance of GLO1 expression in response to osmotic stress is to combat the increase in the levels of methylglyoxal in cells during the production of glycerol as a compatible osmolyte. Deficiency in GLO1 in S. cerevisiae causes pleiotropic phenotypes in terms of stress response, because the steady state level of methylglyoxal increases in glo1Δ cells thereby constitutively activating Yap1 transcription factor. Yap1 is crucial for oxidative stress response, although methylglyoxal per se does not enhance the intracellular oxidation level in yeast, but it directly modifies cysteine residues of Yap1 that are critical for the nucleocytoplasmic localization of this b-ZIP transcription factor. Consequently, glyoxalase I can be defined as a negative regulator of Yap1 through modulating the intracellular methylglyoxal level.

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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.