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Shingo Izawa

Publications and source records attributed to Shingo Izawa.

At least 19 recordsLinked to original sources
PubMed2026

Acetic acid-induced translational repression involves eIF2B body formation and Ded1 sequestration into stress granules in yeast.

Elucidating the physiological impact of acetic acid stress and the corresponding yeast responses is essential for advancing fundamental biology and improving industrial alcoholic fermentation. Despite numerous genome-wide studies, information on the effects of acetic acid stress on yeast translational regulation remains limited. We found that a sublethal concentration of acetic acid (35 mM, 0.2% v/v) causes translational repression, accompanied by the formation of eIF2B bodies and the phosphorylation of eIF2α, both of which are involved in the regulation of translation initiation. Acetic acid also caused the sequestration of Ded1, a DEAD-box RNA helicase crucial for translation initiation, into stress granules. Removal of acetic acid restored translational activity and the proper localization of eIF2B and Ded1, indicating the reversibility of acetic acid-induced translational repression. Furthermore, when yeast cells were pretreated with 0.05% acetic acid, translational repression under subsequent 0.2% acetic acid stress was attenuated in wild-type cells but not in hrk1Δ cells. This indicates that Hrk1, a Pma1 activator, is required to sufficiently enhance tolerance to acetic acid-induced translational repression. These findings provide novel insights into the physiological effects of acetic acid stress on translational activity and translation-related factors in yeast cells.

Saccharomyces cerevisiae↗
PubMed2007

Efficient extraction of thioreodoxin from Saccharomyces cerevisiae by ethanol.

Thioredoxin, an antioxidant protein, is a promising molecule for development of functional foods because it protects the gastric mucosa and reduces the allergenicity of allergens. To establish a method for obtaining an ample amount of yeast thioredoxin, we found here that thioredoxin is released from Saccharomyces cerevisiae by treatment with 20% ethanol. We also found that Japanese sake contains a considerable amount of thioredoxin.

Ethanol↗
PubMed2006

Msn2p/Msn4p-activation is essential for the recovery from freezing stress in yeast.

Since it seems quite difficult for frozen cells to repair the damage caused by freezing, the adequate responses appear to be induced during and/or after the thawing process to recover from the damage due to freezing. In this study, the cellular events happening upon the return from freezing at -30 degrees C to a growth temperature (28 degrees C) were investigated. Yap1p, an oxidative stress-responsive transcription factor, was not activated in the thawed cells, indicating that no serious oxidative stress was generated in the frozen-thawed cells. On the other hand, Msn2p and Msn4p, general stress-responsive transcription factors, were activated in the thawed cells and caused the increased expression of a number of Msn2p/Msn4p-target genes including SOD1, SOD2, and several HSP genes. Since almost no expression of Msn2p/Msn4p-target genes was induced before thawing, these results indicate that Msn2p and Msn4p play a role during the recovery process from freezing.

Adaptation, Physiological↗
PubMed2006

Green tea polyphenols function as prooxidants to activate oxidative-stress-responsive transcription factors in yeasts.

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.

Camellia sinensis↗
PubMed2006

Methylglyoxal as a signal initiator for activation of the stress-activated protein kinase cascade in the fission yeast Schizosaccharomyces pombe.

Methylglyoxal (MG) is a typical 2-oxoaldehyde derived from glycolysis. We have recently found that MG activates transcription factors such as Yap1 and Msn2, and triggers a Hog1 mitogen-activated protein kinase cascade in Saccharomyces cerevisiae. Regarding the activation of Hog1 by MG, we found that Sln1, an osmosensor possessing histidine kinase activity, functions as a sensor of MG (Maeta, K., Izawa, S., and Inoue, Y. (2005) J. Biol. Chem. 280, 253-260). To gain further insight into the role of MG as a signal initiator, here we analyze the response of Schizosaccharomyces pombe to extracellular MG. Spc1, a stress-activated protein kinase (SAPK), was phosphorylated following the treatment with MG. No phosphorylation was observed in a wis1Delta mutant. The His-to-Asp phosphorelay system consisting of three histidine kinases (Phk1, Phk2, and Phk3), a phosphorelay protein (Spy1), and a response regulator (Mcs4) exists upstream of the Spc1-SAPK pathway. The phosphorylation of Spc1 following MG treatment was observed in phk1Deltaphk2Deltaphk3Delta and spy1Delta cells, but not in mcs4Delta cells. These results suggest that S. pombe has an alternative module(s) that directs the MG signal to the SAPK pathway via Mcs4. Additionally, we found that the transcription factor Pap1 is concentrated in the nucleus in response to MG, independent of the Spc1-SAPK pathway.

Basic-Leucine Zipper Transcription Factors↗
PubMed2006

Asr1, an alcohol-responsive factor of Saccharomyces cerevisiae, is dispensable for alcoholic fermentation.

Yeast Asr1 is the first reported protein whose intracellular distribution changes specifically in response to alcohol (Betz et al. (2004) J Biol Chem 279:28174-28181). It was reported that Asr1 is required for tolerance to alcohol and plays an important role in the alcohol stress response. Therefore, Asr1 is of interest to brewers and winegrowers attempting to improve the techniques of alcoholic fermentation. We verified the importance of Asr1 in the alcohol stress response during alcoholic fermentation. Although we reconfirmed the alcohol-responsive changes in the intracellular localization of Asr1, we could not detect the effects of Asr1-deficiency on Japanese sake brewing or winemaking. In addition, we could not reconfirm the hypersensitivity of Asr1-deficient mutants to alcohol and sodium dodecyl sulfate. Instead, we conclude that Asr1 is not required and nor important for tolerance to alcohol stress.

Active Transport, Cell Nucleus↗
PubMed2005

GPX2, encoding a phospholipid hydroperoxide glutathione peroxidase homologue, codes for an atypical 2-Cys peroxiredoxin in Saccharomyces cerevisiae.

We have previously reported that Saccharomyces cerevisiae has three glutathione peroxidase homologues (GPX1, GPX2, and GPX3) (Inoue, Y., Matsuda, T., Sugiyama, K., Izawa, S., and Kimura, A. (1999) J. Biol. Chem. 274, 27002-27009). Of these, the GPX2 gene product (Gpx2) shows the greatest similarity to phospholipid hydroperoxide glutathione peroxidase. Here we show that GPX2 encodes an atypical 2-Cys peroxiredoxin which uses thioredoxin as an electron donor. Gpx2 was essentially in a reduced form even in mutants defective in glutathione reductase or glutaredoxin under oxidative stressed conditions. On the other hand, Gpx2 was partially oxidized in a mutant defective in cytosolic thioredoxin (trx1Deltatrx2Delta) under non-stressed conditions and completely oxidized in tert-butyl hydroperoxide-treated cells of trx1Deltatrx2Delta and thioredoxin reductase-deficient mutant cells. Alanine scanning of cysteine residues of Gpx2 revealed that an intramolecular disulfide bond was formed between Cys37 and Cys83 in vivo. Gpx2 was purified to determine whether it functions as a peroxidase that uses thioredoxin as an electron donor in vitro. Gpx2 reduced H2O2 and tert-butyl hydroperoxide in the presence of thioredoxin, thioredoxin reductase, and NADPH (for H2O2, Km= 20 microm, kcat = 9.57 x 10(2) s(-1); for tert-butyl hydroperoxide, Km= 62.5 microm, kcat = 3.68 x 10(2) s(-1)); however, it showed remarkably less activity toward these peroxides in the presence of glutathione, glutathione reductase, and NADPH. The sensitivity of yeast cells to tert-butyl hydroperoxide was found to be exacerbated by the co-existence of Ca2+, a tendency that was most obvious in gpx2Delta cells. Although the redox state of Gpx2 was not affected by Ca2+, the Gpx2 level was markedly increased in the presence of both tert-butyl hydroperoxide and Ca2+. Gpx2 is likely to play an important role in the protection of cells from oxidative stress in the presence of Ca2+.

Base Sequence↗
PubMed2005

Characterization of Rat8 localization and mRNA export in Saccharomyces cerevisiae during the brewing of Japanese sake.

Ethanol affects the nuclear export of mRNA in a similar way to heat shock in Saccharomyces cerevisiae. We recently reported that the nuclear accumulation of Rat8 caused by ethanol stress correlates well with blocking of the export of bulk poly(A)(+) mRNA. Here, we characterize the localization of Rat8 and bulk poly(A)(+) mRNA in sake (Japanese rice wine) yeast during the brewing of sake. In wine must and synthetic dextrose medium, sake yeast showed the same responses to ethanol regarding changes in the localization of Rat8 as wine yeast and a laboratory strain: i.e., cells began the nuclear accumulation of Rat8 at an ethanol concentration of 6% and completed it at 9%. In contrast, during the sake-brewing process, sake yeast showed unique phenomena: i.e., cells did not start the nuclear accumulation of Rat8 until the ethanol concentration of the sake mash reached around 12% and they showed a normal localization of Rat8 around the nuclear envelope at the late stage of fermentation. These results provide new information about the transport of mRNA in yeast cells during actual alcoholic fermentation.

Alcoholic Beverages↗
PubMed2005

Unique regulation of glyoxalase I activity during osmotic stress response in the fission yeast Schizosaccharomyces pombe: neither the mRNA nor the protein level of glyoxalase I increase under conditions that enhance its activity.

Glyoxalase I is a ubiquitous enzyme that catalyzes the conversion of methylglyoxal, a toxic 2-oxoaldehyde derived from glycolysis, to S-D-lactoylglutathione. The activity of glyoxalase I in the fission yeast Schizosaccharomyces pombe was increased by osmotic stress induced by sorbitol. However, neither the mRNA levels of its structural gene nor its protein levels increased under the same conditions. Cycloheximide blocked the induction of glyoxalase I activity in cells exposed to osmotic stress. In addition, glyoxalase I activity was increased in stress-activated protein kinase-deficient mutants (wis1 and spc1). We present evidence for the post-translational regulation of glyoxalase I by osmotic stress in the fission yeast.

Blotting, Northern↗
PubMed2005

Diagnosis of cell death induced by methylglyoxal, a metabolite derived from glycolysis, in Saccharomyces cerevisiae.

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.

Cell Death↗
PubMed2005

Enrichment of yeast thioredoxin by green tea extract through activation of Yap1 transcription factor in Saccharomyces cerevisiae.

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.

Membrane Proteins↗
PubMed2004

Methylglyoxal, a metabolite derived from glycolysis, functions as a signal initiator of the high osmolarity glycerol-mitogen-activated protein kinase cascade and calcineurin/Crz1-mediated pathway in Saccharomyces cerevisiae.

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 (HOG)-mitogen-activated protein (MAP) kinase-dependent manner in Saccharomyces cerevisiae. Here we show that MG activates the HOG-MAP kinase cascade. Two osmosensors, Sln1 and Sho1, have been identified to function upstream of the HOG-MAP kinase cascade, and we reveal that MG initiates the signal transduction to this MAP kinase cascade through the Sln1 branch. We also demonstrate that MG activates the Msn2 transcription factor. Moreover, MG activated the uptake of Ca(2+) in yeast cells, thereby stimulating the calcineurin/Crz1-mediated Ca(2+) signaling pathway. We propose that MG functions as a signal initiator in yeast.

Glycolysis↗
PubMed2004

Stress response in yeast mRNA export factor: reversible changes in Rat8p localization are caused by ethanol stress but not heat shock.

Ethanol stress (10% v/v) causes selective mRNA export in Saccharomyces cerevisiae in a similar manner to heat shock (42 degrees C). Bulk poly(A)(+) mRNA accumulates in the nucleus, whereas heat shock protein mRNA is exported under such conditions. Here we investigated the effects of stress on mRNA export factors. In cells treated with ethanol stress, the DEAD box protein Rat8p showed a rapid and reversible change in its localization, accumulating in the nucleus. This change correlated closely with the blocking of bulk poly(A)(+) mRNA export caused by ethanol stress. We also found that the nuclear accumulation of Rat8p is caused by a defect in the Xpo1p/Crm1p exportin. Intriguingly, the localization of Rat8p did not change in heat shocked cells, suggesting that the mechanisms blocking bulk poly(A)(+) mRNA export differ for heat shock and ethanol stress. These results suggest that changes in the localization of Rat8p contribute to the selective export of mRNA in ethanol stressed cells, and also indicate differences in mRNA export between the heat shock response and ethanol stress response.

Active Transport, Cell Nucleus↗
PubMed2004

Distinct regulatory mechanism of yeast GPX2 encoding phospholipid hydroperoxide glutathione peroxidase by oxidative stress and a calcineurin/Crz1-mediated Ca2+ signaling pathway.

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(2+)-dependent induction of GPX2, therefore, distinct regulation for the oxidative stress response and Ca(2+) signal response for GPX2 exists in yeast cells.

Calcineurin↗
PubMed2004

Gle2p is essential to induce adaptation of the export of bulk poly(A)+ mRNA to heat shock in Saccharomyces cerevisiae.

The export of bulk poly(A)(+) mRNA is blocked under heat-shocked (42 degrees C) conditions in Saccharomyces cerevisiae. We found that an mRNA export factor Gle2p rapidly dissociated from the nuclear envelope and diffused into the cytoplasm at 42 degrees C. However, in exponential phase cells pretreated with mild heat stress (37 degrees C for 1 h), Gle2p did not dissociate at 42 degrees C, and the export of bulk poly(A)(+) mRNA continued. Cells in stationary phase also continued with the export of bulk poly(A)(+) mRNA at 42 degrees C without the dissociation of Gle2p from the nuclear envelope. The dissociation of Gle2p was caused by increased membrane fluidity and correlated closely with blocking of the export of bulk poly(A)(+) mRNA. Furthermore, the mutants gle2Delta and rip1Delta could not induce such an adaptation of the export of bulk poly(A)(+) mRNA to heat shock. Our findings indicate that Gle2p plays a crucial role in mRNA export especially under heat-shocked conditions. Our findings also indicate that the nuclear pore complexes that Gle2p constitutes need to be stabilized for the adaptation and that the increased membrane integrity caused by treatment with mild heat stress or by survival in stationary phase is likely to contribute to the stabilization of the association between Gle2p and the nuclear pore complexes.

Adaptation, Biological↗
PubMed2004

Regulation of the yeast phospholipid hydroperoxide glutathione peroxidase GPX2 by oxidative stress is mediated by Yap1 and Skn7.

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.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Intracellular glycerol influences resistance to freeze stress in Saccharomyces cerevisiae: analysis of a quadruple mutant in glycerol dehydrogenase genes and glycerol-enriched cells.

Glycerol is well known as a cryoprotectant similar to trehalose. However, there is little information about the effects of intracellular glycerol on the freeze-thaw stress tolerance of yeast. Through analysis of a quadruple-knockout mutant of glycerol dehydrogenase genes (ara1 Delta gcy1 Delta gre3 Delta ypr1 Delta) in Saccharomyces cerevisiae, we revealed that the decrease in glycerol dehydrogenase activity led to increased levels of intracellular glycerol. We also found that this mutant showed higher tolerance to freeze stress than wild type strain W303-1A. Furthermore, we demonstrated that intracellular-glycerol-enriched cells cultured in glycerol medium acquire tolerance to freeze stress and retain high leavening ability in dough even after frozen storage for 7 days. These results suggest the possibility of using intracellular-glycerol-enriched cells to develop better frozen dough.

Fermentation↗