Dai Hirata

Protein phosphatase type 2B (calcineurin)-mediated, FK506-sensitive regulation of intracellular ions in yeast is an important determinant for adaptation to high salt stress conditions

To assess the physiological function of Ca(2+)‐dependent protein phosphatase (PP2B) in the yeast Saccharomyces cerevisiae, the phenotypes of PP2B‐deficient mutants were investigated. Although PP2B was dispensable for growth under normal conditions, the mutations did, however, cause growth inhibition under certain stress circumstances. The growth of the mutants was inhibited by NaCl and LiCl, but not by KCl, CaCl2, MgCl2 or nonspecific osmotic stresses. Upon shift to high NaCl medium, intracellular Na+ levels of both wild type yeast and the mutants initially increased at a comparable rate. However, internal Na+ in wild type cells started to decline more rapidly than the mutant cells during cultivation in high NaCl medium, indicating that PP2B is important in maintaining a gradient across the membrane. The protection against salt stress was achieved, at least in part, by the stimulation of Na+ export. The maintenance of a high level of internal K+ in high NaCl medium was also PP2B‐dependent. In the presence of the immunosuppressant FK506, the growth behaviour and intracellular Na+ and K+ of wild type cells in high NaCl medium became very similar to those of the PP2B‐deficient mutant in a manner dependent on the presence of the FK506 binding protein.

Saccharomyces cerevisiae YDR1, which encodes a member of the ATP-binding cassette (ABC) superfamily, is required for multidrug resistance

A multidrug resistance gene, YDR1, of Saccharomyces cerevisiae, which encodes a 170-kDa protein of a member of the ABC superfamily, was identified. Disruption of YDR1 resulted in hypersensitivity to cycloheximide, cerulenin, compactin, staurosporine and fluphenazine, indicating that YDR1 is an important determinant of cross resistance to apparently-unrelated drugs. The Ydr1 protein bears the highest similarity to the S. cerevisiae Snq2 protein required for resistance to the mutagen 4-NQO. The drug-specificity analysis of YDR1 and SNQ2 by gene disruption, and its phenotypic suppression by the overexpressed genes, revealed overlapping, yet distinct, specificities. YDR1 was responsible for cycloheximide, cerulenin and compactin resistance, whereas, SNQ2 was responsible for 4-NQO resistance. The two genes had overlapping specificities toward staurosporine and fluphenazine. The transcription of YDR1 and SNQ2 was induced by various drugs, both relevant and irrelevant to the resistance caused by the gene, suggesting that drug specificity can be mainly attributed to the functional difference of the putative transporters. The transcription of these genes was also increased by heat shock. The yeast drug-resistance system provides a novel model for mammalian multidrug resistance.

Role of calcineurin and Mpk1 in regulating the onset of mitosis in budding yeast.

Signalling via calcium is probably involved in regulating eukaryotic cell proliferation, but details of its mechanism of action are unknown,. In Schizosaccharomyces pombe, the onset of mitosis is determined by activation of a complex of the p34cdc2 protein kinase and a cyclin protein that is specific to the G2 phase of the cell cycle. This activation requires dephosphorylation of p34cdc2 (ref. 3). Wee1, a tyrosine kinase that inhibits p34cdc2 by phosphorylating it, is needed to determine the length of G2 phase. Here we show that calcium-activated pathways in Saccharomyces cerevisiae control the onset of mitosis by regulating Swe1, a Wee1 homologue. Zds1 (also known as Oss1 and Hst1) (refs 4–7) is important in repressing the transcription of SWE1 in G2 phase. In the presence of high calcium levels, cells lacking Zds1 are delayed in entering mitosis. Calcineurin and Mpk1 (refs 12, 13) regulate Swe1 activation at the transcriptional and post-translational levels, respectively, and both are required for the calcium-induced delay in G2 phase. These cellular pathways also induce a G2-phase delay in response to hypotonic shock.

Effect of Ethanol on Cell Growth of Budding Yeast: Genes That Are Important for Cell Growth in the Presence of Ethanol

The budding yeast Saccharomyces cerevisiae has been used in the fermentation of various kinds of alcoholic beverages. But the effect of ethanol on the cell growth of this yeast is poorly understood. This study shows that the addition of ethanol causes a cell-cycle delay associated with a transient dispersion of F-actin cytoskeleton, resulting in an increase in cell size. We found that the tyrosine kinase Swe1, the negative regulator of Cdc28-Clb kinase, is related to the regulation of cell growth in the presence of ethanol. Indeed, the increase in cell size due to ethanol was partially abolished in the SWE1-deleted cells, and the amount of Swe1 protein increased transiently in the presence of ethanol. These results indicated that Swe1 is involved in cell size control in the presence of ethanol, and that a signal produced by ethanol causes a transient up-regulation of Swe1. Further we investigated comprehensively the ethanol-sensitive strains in the complete set of 4847 non-essential gene deletions and identified at least 256 genes that are important for cell growth in the presence of ethanol.

Stress-induced transcriptional activation mediated by YAP1 and YAP2 genes that encode the Jun family of transcriptional activators in Saccharomyces cerevisiae

The Saccharomyces cerevisiae YAP2 gene encoding an AP-1-like transcriptional activator protein was cloned by selection for genes that confer pleiotropic drug resistance when present in high copy number. The novel YAP2 gene encodes a protein of 45827 daltons and is homologous in part to a known transcriptional activator protein encoded by YAP1/PDR4/SNQ3/PAR1. Homology was found only in both terminal regions. The N-terminal portion contains a region rich in basic amino acids, followed by a “leucine zipper” motif. Overexpression of YAP2 led to the induction of expression of an AP-1 recognition element (ARE)-dependent promoter. The yap1 disruptant has been shown to be sensitive to H2O2. In this study, we demonstrated that the yap1 disruptant is also unable to grow in medium containing 150 μM cadmium, whereas the yap2 disruptant exhibited no significant phenotypes. However, YAP2 in high copy number did suppress cadmium sensitivity, but not H2O2 sensitivity of the yap1 disruptant. YAP1 was able to mediate both cadmium- and H2O2-induced transcriptional activation of an ARE-dependent promoter. A high-copy-number plasmid bearing YAP2 mediated cadmium-induced transcriptional activation of this promoter. The inductions were prevented by the antioxidant N-acetyl-l-cysteine.

Adaptation to high-salt stress in Saccharomyces cerevisiae is regulated by Ca2+/calmodulin-dependent phosphoprotein phosphatase (calcineurin) and cAMP-dependent protein kinase.

Ca2−/calmodulin-dependent phosphoprotein phosphatase (calcineurin, PP2B) of Saccharomyces cerevisiae is implicated in adaptation to high-salt conditions. Calcineurin mediates high salt-induced expression of the ENA1/PMR2 gene encoding the P-type ATPase, which is suggested to be involved in Na+ efflux. We identified the PDE1 gene encoding the low-affinity cAMP phosphodiesterase as a multicopy suppressor of the Li+- and Na+-sensitive calcineurin null mutant, suggesting that cAMP is a negative regulator of adaptation to high-salt stress. Genetic analysis indicated that calcineurin and cAMP act antagonistically in a common pathway for adaptation. The bcy1 disruption, which leads to constitutive cAMP-dependent protein kinase (PKA) activity, inhibited high NaCl-induced expression of the ENA1/PMR2 gene, caused an elevation of the intracellular Na+ level and a growth defect in high-NaCl medium, all of which were analogous to the defects of a calcineurin mutant. A reduced cAMP level resulting from multiple copies of the PDE1 gene caused increased expression of the ENA1/PMR2 gene in response to high NaCl. We propose a model for the regulation of cation homeostasis, in which calcineurin antagonizes PKA to activate transcription of the ENA1/PMR2 gene in response to high-salt conditions.

GSK‐3 kinase Mck1 and calcineurin coordinately mediate Hsl1 down‐regulation by Ca2+ in budding yeast

The Ca2+‐activated pathways of Saccharomyces cerevisiae induce a delay in the onset of mitosis through the activation of Swe1, a negative regulatory kinase that inhibits the Cdc28–Clb complex. Calcineurin and Mpk1 activate Swe1 at the transcriptional and post‐translational level, respectively, and both pathways are essential for the cell cycle delay. Our genetic screening identified the MCK1 gene, which encodes a glycogen synthetase kinase‐3 family protein kinase, as a component of the Ca2+ signaling pathway. Genetic analyses indicated that Mck1 functions downstream of the Mpk1 pathway and down‐regulates Hsl1, an inhibitory kinase of Swe1. In medium with a high concentration of Ca2+, Hsl1 was delocalized from the bud neck and destabilized in a manner dependent on both calcineurin and Mck1. Calcineurin was required for the dephosphorylation of autophosphorylated Hsl1. The E3 ubiquitin ligase complex SCFCdc4, but not the anaphase‐promoting complex (APC), was essential for Hsl1 destabilization. The Ca2+‐activated pathway may play a role in the rapid inactivation of Hsl1 at the cell cycle stage(s) when APC activity is low.

Identification of Saccharomyces cerevisiae Isoleucyl-tRNA Synthetase as a Target of the G1-specific Inhibitor Reveromycin A

To dissect the action mechanism of reveromycin A (RM-A), a G1-specific inhibitor, aSaccharomyces cerevisiae dominant mutant specifically resistant to RM-A, was isolated from a strain in which the genes implicated in nonspecific multidrug resistance had been deleted. The mutant gene (YRR2–1) responsible for the resistance was identified as an allele of the ILS1 gene encoding tRNAIle synthetase (IleRS). The activity of IleRS, but not several other aminoacyl-tRNA synthetases examined in wild type cell extract, was highly sensitive to RM-A (IC50 = 8 ng/ml). The IleRS activity of the YRR2–1 mutant was 4-fold more resistant to the inhibitor compared with that of wild type. The mutation IleRSN660D, near the KMSKS consensus sequence commonly found in the class I aminoacyl transferases, was found to be responsible for RM-A resistance. Moreover, overexpression of theILS1 gene from a high-copy plasmid conferred RM-A resistance. These results indicated that IleRS is a target of RM-Ain vivo. A defect of the GCN2 gene led to decreased RM-A resistance. IleRS inhibition by RM-A led to transcriptional activation of the ILS1 gene viathe Gcn2-Gcn4 general amino acid control pathway, and this autoregulation seemed to contribute to RM-A resistance.

Fission yeast MO25 protein is localized at SPB and septum and is essential for cell morphogenesis

Cell morphogenesis is of fundamental significance in all eukaryotes for development, differentiation, and cell proliferation. In fission yeast, Drosophila Furry‐like Mor2 plays an essential role in cell morphogenesis in concert with the NDR/Tricornered kinase Orb6. Mutations of these genes result in the loss of cell polarity. Here we show that the conserved proteins, MO25‐like Pmo25, GC kinase Nak1, Mor2, and Orb6, constitute a morphogenesis network that is important for polarity control and cell separation. Intriguingly, Pmo25 was localized at the mitotic spindle pole bodies (SPBs) and then underwent translocation to the dividing medial region upon cytokinesis. Pmo25 formed a complex with Nak1 and was required for both the localization and kinase activity of Nak1. Pmo25 and Nak1 in turn were essential for Orb6 kinase activity. Further, the Pmo25 localization at the SPBs and the Nak1‐Orb6 kinase activities during interphase were under the control of the Cdc7 and Sid1 kinases in the septation initiation network (SIN), suggesting a functional linkage between SIN and the network for cell morphogenesis/separation following cytokinesis.

Yeast gene YRR1, which is required for resistance to 4‐nitroquinoline N‐oxide, mediates transcriptional activation of the multidrug resistance transporter gene SNQ2

We have cloned and characterized a Saccharomyces cerevisiae gene YRR1 that is important for resistance to the mutagen 4‐nitroquinoline N‐oxide (4‐NQO). The wild‐type YRR1 gene encodes a protein that contains a Zn(II)2Cys6‐type zinc‐finger motif. Disruption of the YRR1 gene leads to hypersensitivity to 4‐NQO. A dominant mutation (YRR1‐1 ) that confers strong resistance to 4‐NQO has been identified. Epistasis analysis demonstrated that 4‐NQO resistances mediated by the YRR1 and YRR1‐1 alleles require the presence of the SNQ2 gene that encodes a multidrug resistance ATP binding cassette superfamily protein responsible for 4‐NQO export. Northern blot analysis of SNQ2 mRNA levels indicated that Yrr1p is involved in basal and drug‐induced transcriptional activation of SNQ2, whereas Pdr1p/Pdr3p transcription factors are mainly involved in basal SNQ2 expression. In the YRR1‐1 mutant, the level of SNQ2 mRNA is constitutively elevated. These results establish that Yrr1p is important for 4‐NQO resistance by mediating transcriptional activation of the SNQ2 gene in response to the stress imposed by 4‐NQO. The gain‐of‐function mutation of Yrr1‐1p was attributable to the duplication of a 12‐amino‐acid sequence generated near the carboxy terminus.