Yoshifumi Takatsume

Whole-Genome Sequencing of Sake Yeast Saccharomyces cerevisiae Kyokai no. 7

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 Interestingly, two regions in chromosomes I and VII of S288C have apparently been replaced by Ty elements in K7.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.

Green Tea Polyphenols Function as Prooxidants To Activate Oxidative-Stress-Responsive Transcription Factors in Yeasts

ABSTRACT 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 H2O2-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 H2O2 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.

The Glycolytic Metabolite Methylglyoxal Activates Pap1 and Sty1 Stress Responses in Schizosaccharomyces pombe

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.

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 H2O2‐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.

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 wis1Δ 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 phk1Δphk2Δphk3Δ and spy1Δ cells, but not in mcs4Δ 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.

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 CaCl2 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 Ca2+‐dependent induction of GPX2, therefore, distinct regulation for the oxidative stress response and Ca2+ signal response for GPX2 exists in yeast cells.

Calcineurin/Crz1 destabilizes Msn2 and Msn4 in the nucleus in response to Ca(2+) in Saccharomyces cerevisiae.

Although methylglyoxal is derived from glycolysis, it has adverse effects on cellular function. Hence, the intrinsic role of methylglyoxal in vivo remains to be determined. Glyoxalase 1 is a pivotal enzyme in the metabolism of methylglyoxal in all types of organisms. To learn about the physiological roles of methylglyoxal, we have screened conditions that alter the expression of the gene encoding glyoxalase 1, GLO1, in Saccharomyces cerevisiae. We show that the expression of GLO1 is induced following treatment with Ca2+ and is dependent on the MAPK (mitogen-activated protein kinase) Hog1 protein and the Msn2/Msn4 transcription factors. Intriguingly, the Ca2+-induced expression of GLO1 was enhanced in the presence of FK506, a potent inhibitor of calcineurin. Consequently, the Ca2+-induced expression of GLO1 in a mutant that is defective in calcineurin or Crz1, the sole transcription factor downstream of calcineurin, was much greater than that in the wild-type strain even without FK506. This phenomenon was dependent upon a cis-element, the STRE (stress-response element), in the promoter that is able to mediate the response to Ca2+ signalling together with Hog1 and Msn2/Msn4. The level of Ca2+-induced expression of GLO1 reached a maximum in cells overexpressing MSN2 even when FK506 was not present, whereas in cells overexpressing CRZ1 the level was greatly reduced and increased markedly when FK506 was present. We also found that the levels of Msn2 and Msn4 proteins in Ca2+-treated cells decreased gradually and that FK506 blocked the degradation of Msn2/Msn4. We propose that Crz1 destabilizes Msn2/Msn4 in the nuclei of cells in response to Ca2+ signalling.

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.