Activation of the DNA damage checkpoint perturbs asymmetric localization of Kar9 to spindle pole bodies in Saccharomyces cerevisiae.

During cell cycle progression in Saccharomyces cerevisiae, spindle pole bodies (SPBs) are duplicated during the G1/S-phase transition. SPBs are crucial for the organization of both the spindle and astral microtubules, and their orientation defines the direction of nuclear division. In this process, an old SPB, which serves as the template SPB during the duplication process, is oriented toward the bud side. The patterning microtubule plus-end tracking protein, Kar9, plays an important role in the orientation of SPBs by asymmetrically localizing to the old SPB. Here, methylglyoxal (MG), a metabolite derived from glycolysis, was found to perturb asymmetric Kar9 localization and influence proper positioning of the old SPB. MG activated the DNA damage checkpoint pathway, and MG-induced perturbation of asymmetric Kar9 localization was abolished by the deletion of MEC1, a sensor for the DNA damage checkpoint pathway. Methyl methanesulfonate, a DNA-alkylating agent, also perturbed asymmetric Kar9 localization. Our results suggest that activation of the DNA damage checkpoint pathway perturbs the asymmetric Kar9 localization required for proper positioning of SPBs.

Methylglyoxal induces multiple serine phosphorylation in insulin receptor substrate 1 via the TAK1-p38-mTORC1 signaling axis in adipocytes.

Certain metabolic intermediates produced during metabolism are known to regulate a wide range of cellular processes. Methylglyoxal (MG), a natural metabolite derived from glycolysis, has been shown to negatively influence systemic metabolism by inducing glucose intolerance, insulin resistance, and diabetic complications. MG plays a functional role as a signaling molecule that initiates signal transduction. However, the specific relationship between MG-induced activation of signal transduction and its negative effects on metabolism remains unclear. Here, we found that MG activated mammalian target of rapamycin complex 1 (mTORC1) signaling via p38 mitogen-activated protein kinase in adipocytes, and that the transforming growth factor-ß-activated kinase 1 (TAK1) is needed to activate p38-mTORC1 signaling following treatment with MG. We also found that MG increased the phosphorylation levels of serine residues in insulin receptor substrate (IRS)-1, which is involved in its negative regulation, thereby attenuating insulin-stimulated tyrosine phosphorylation in IRS-1. The negative effect of MG on insulin-stimulated IRS-1 tyrosine phosphorylation was exerted due to the MG-induced activation of the TAK1-p38-mTORC1 signaling axis. The involvement of the TAK1-p38-mTORC1 signaling axis in the induction of IRS-1 multiple serine phosphorylation was not unique to MG, as the proinflammatory cytokine, tumor necrosis factor-α, also activated the same signaling axis. Therefore, our findings suggest that MG-induced activation of the TAK1-p38-mTORC1 signaling axis caused multiple serine phosphorylation on IRS-1, potentially contributing to insulin resistance.

Roles of phosphatidylserine and phospholipase C in the activation of TOR complex 2 signaling in Saccharomyces cerevisiae.

Target of rapamycin (TOR) forms two distinct complexes, TORC1 and TORC2, to exert its essential functions in cellular growth and homeostasis. TORC1 signaling is regulated in response to nutrients such as amino acids and glucose; however, the mechanisms underlying the activation of TORC2 signaling are still poorly understood compared to those for TORC1 signaling. In the budding yeast Saccharomyces cerevisiae, TORC2 targets the protein kinases Ypk1 and Ypk2 (hereafter Ypk1/2), and Pkc1 for phosphorylation. Plasma membrane stress is known to activate TORC2-Ypk1/2 signaling. We have previously reported that methylglyoxal (MG), a metabolite derived from glycolysis, activates TORC2-Pkc1 signaling. In this study, we found that MG activates the TORC2-Ypk1/2 and TORC2-Pkc1 signaling, and that phosphatidylserine is involved in the activation of both signaling pathways. We also demonstrated that the Rho family GTPase Cdc42 contributes to the plasma membrane stress-induced activation of TORC2-Ypk1/2 signaling. Furthermore, we revealed that phosphatidylinositol-specific phospholipase C, Plc1, contributes to the activation of both TORC2-Ypk1/2 and TORC2-Pkc1 signaling.

Role of RhoGAP Rgd1 in Pkc1 signaling-related actin repolarization under heat shock stress in Saccharomyces cerevisiae.

BACKGROUND: A serine/threonine kinase Pkc1 is the sole protein kinase C in the budding yeast Saccharomyces cerevisiae, and plays an important role in the regulation of polarized growth and stress responses such as those due to heat shock. Exposure of cells to high temperature transiently arrests polarized growth and leads to depolarization of the actin cytoskeleton, followed by actin repolarization during adaptation to heat shock stress. Actin repolarization is ensured by the activation of Pkc1 signaling; however, the molecular mechanisms underlying this phenomenon remain poorly understood. METHODS: Using an overexpression construct of a constitutively active mutant of Pkc1 (Pkc1R398P), we explored the Pkc1 target molecules involved in actin repolarization. RESULTS: PKC1R398P overexpression as well as heat shock stress increased the phosphorylation levels of Rho GTPase-activating protein (RhoGAP) Rgd1. Rgd1 was found to contribute to Pkc1-signaling-related actin repolarization during adaptation to heat shock stress in a GAP activity-independent manner, with Ser148 in Rgd1 playing a crucial role. Furthermore, Rgd1 was involved in the maintenance of phosphorylation status of the mitogen-activated protein (MAP) kinase Mpk1, a downstream effector of Pkc1, under heat shock stress. CONCLUSIONS: Rgd1 is a target of Pkc1 signaling under conditions of heat shock stress, and required for the normal process of actin repolarization during adaptation to heat shock stress. GENERAL SIGNIFICANCE: Our results provide insights into the molecular mechanism underlying Pkc1-mediated modulation of actin repolarization under heat shock stress.

Methylglyoxal inhibits nuclear division through alterations in vacuolar morphology and accumulation of Atg18 on the vacuolar membrane in Saccharomyces cerevisiae.

Methylglyoxal (MG) is a natural metabolite derived from glycolysis, and it inhibits the growth of cells in all kinds of organisms. We recently reported that MG inhibits nuclear division in Saccharomyces cerevisiae. However, the mechanism by which MG blocks nuclear division remains unclear. Here, we show that increase in the levels of phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) is crucial for the inhibitory effects of MG on nuclear division, and the deletion of PtdIns(3,5)P2-effector Atg18 alleviated the MG-mediated inhibitory effects. Previously, we reported that MG altered morphology of the vacuole to a single swelling form, where PtdIns(3,5)P2 accumulates. The changes in the vacuolar morphology were also needed by MG to exert its inhibitory effects on nuclear division. The known checkpoint machinery, including the spindle assembly checkpoint and morphological checkpoint, are not involved in the blockade of nuclear division by MG. Our results suggest that both the accumulation of Atg18 on the vacuolar membrane and alterations in vacuolar morphology are necessary for the MG-induced inhibition of nuclear division.

Contribution of phosphatidylserine to Rho1- and Pkc1-related repolarization of the actin cytoskeleton under stressed conditions in Saccharomyces cerevisiae.

The budding yeast Saccharomyces cerevisiae undergoes polarized cell growth, which is established in association with actin polarization. Rho1, one of the Rho-type GTPases in S. cerevisiae, is crucial for maintaining polarized cell growth and actin polarization and controlling the downstream signaling pathway, the Pkc1-Mpk1 MAP kinase cascade, through a physical interaction with Pkc1, the sole protein kinase C in this yeast. The Pkc1-Mpk1 MAP kinase cascade is important for the repolarization of actin under heat shock-stressed conditions. We recently reported that phosphatidylserine (PS), a membrane phospholipid component, played a pivotal role in the physical interaction between Rho1 and Pkc1 as well as the activation of the Pkc1-Mpk1 MAP kinase cascade. However, it currently remains unclear whether PS is involved in actin polarization by regulating the physical interaction between Rho1 and Pkc1. We herein demonstrated that the C1 domain of Pkc1, which is responsible for the interaction with Rho1, was crucial for Rho1-regulated actin polarization. We also found that actin repolarization under heat shock-stressed conditions was impaired in a mutant defective in CHO1 encoding PS synthase. These results suggest that PS contributes to actin polarization in which Rho1 and Pkc1 play a crucial role.

Toxicity of dihydroxyacetone is exerted through the formation of methylglyoxal in Saccharomyces cerevisiae: effects on actin polarity and nuclear division.

Dihydroxyacetone (DHA) is the smallest ketotriose, and it is utilized by many organisms as an energy source. However, at higher concentrations, DHA becomes toxic towards several organisms including the budding yeast Saccharomyces cerevisiae In the present study, we show that DHA toxicity is due to its spontaneous conversion to methylglyoxal (MG) within yeast cells. A mutant defective in MG-metabolizing enzymes (glo1Δgre2Δgre3Δ) exhibited higher susceptibility to DHA. Intracellular MG levels increased following the treatment of glo1Δgre2Δgre3Δ cells with DHA. We previously reported that MG depolarized the actin cytoskeleton and changed vacuolar morphology. We herein demonstrated the depolarization of actin and morphological changes in vacuoles following a treatment with DHA. Furthermore, we found that both MG and DHA caused the morphological change in nucleus, and inhibited the nuclear division. Our results suggest that the conversion of DHA to MG is a dominant contributor to its cytotoxicity.

Phosphatidylinositol 3,5-bisphosphate is involved in methylglyoxal-induced activation of the Mpk1 mitogen-activated protein kinase cascade in Saccharomyces cerevisiae.

Methylglyoxal (MG) is a natural metabolite derived from glycolysis, and this 2-oxoaldehyde has been implicated in some diseases including diabetes. However, the physiological significance of MG for cellular functions is yet to be fully elucidated. We previously reported that MG activates the Mpk1 (MAPK) cascade in the yeast Saccharomyces cerevisiae To gain further insights into the cellular functions and responses to MG, we herein screened yeast-deletion mutant collections for susceptibility to MG. We found that mutants defective in the synthesis of phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) are more susceptible to MG. PtdIns(3,5)P2 levels increased following MG treatment, and vacuolar morphology concomitantly changed to a single swollen shape. MG activated the Pkc1-Mpk1 MAPK cascade in which a small GTPase Rho1 plays a crucial role, and the MG-induced phosphorylation of Mpk1 was impaired in mutants defective in the PtdIns(3,5)P2 biosynthetic pathway. Of note, heat shock-induced stress also provoked Mpk1 phosphorylation in a Rho1-dependent manner; however, PtdIns(3,5)P2 was dispensable for the heat shock-stimulated activation of this signaling pathway. Our results suggest that PtdIns(3,5)P2 is specifically involved in the MG-induced activation of the Mpk1 MAPK cascade and in the cellular adaptation to MG-induced stress.

Role of phosphatidylserine in the activation of Rho1-related Pkc1 signaling in Saccharomyces cerevisiae.

Protein kinase C (PKC) belongs to a family of serine/threonine kinases and is evolutionary conserved among eukaryotes. It contains several functional domains, with the C1 domain being identified as a membrane-targeting module. Diacylglycerol (DAG) and phorbol esters bind to the C1 domain to enhance its kinase activity. The C1 domain is conserved in PKC (Pkc1) in the budding yeast Saccharomyces cerevisiae; however, its kinase activity does not respond to DAG. Although the C1 domain of Pkc1 physically interacts with the small GTPase Rho1, the interaction between C1 domain and lipids has not yet been characterized. We herein provide evidence to show the physical interaction between the C1 domain of Pkc1 and phosphatidylserine (PS), but not DAG. The stress-induced activation of Pkc1 signaling was abolished in a cho1 mutant, which was defective in PS synthase. The deletion of CHO1 perturbed the appropriate localization of Pkc1 at the bud tip, and impaired the physical interaction between Pkc1 and GTP-bound Rho1 in vivo. Our results suggest that PS is necessary for Pkc1 signaling due to its role in regulating the localization of Pkc1 as well as the physical interaction between Rho1 and Pkc1.

Methylglyoxal activates the target of rapamycin complex 2-protein kinase C signaling pathway in Saccharomyces cerevisiae.

Methylglyoxal is a typical 2-oxoaldehyde derived from glycolysis. We show here that methylglyoxal activates the Pkc1-Mpk1 mitogen-activated protein (MAP) kinase cascade in a target of rapamycin complex 2 (TORC2)-dependent manner in the budding yeast Saccharomyces cerevisiae. We demonstrate that TORC2 phosphorylates Pkc1 at Thr(1125) and Ser(1143). Methylglyoxal enhanced the phosphorylation of Pkc1 at Ser(1143), which transmitted the signal to the downstream Mpk1 MAP kinase cascade. We found that the phosphorylation status of Pkc1(T1125) affected the phosphorylation of Pkc1 at Ser(1143), in addition to its protein levels. Methylglyoxal activated mammalian TORC2 signaling, which, in turn, phosphorylated Akt at Ser(473). Our results suggest that methylglyoxal is a conserved initiator of TORC2 signaling among eukaryotes.

Involvement of glutathione peroxidase 1 in growth and peroxisome formation in Saccharomyces cerevisiae in oleic acid medium.

Saccharomyces cerevisiae is able to use some fatty acids, such as oleic acid, as a sole source of carbon. ß-oxidation, which occurs in a single membrane-enveloped organelle or peroxisome, is responsible for the assimilation of fatty acids. In S. cerevisiae, ß-oxidation occurs only in peroxisomes, and H(2)O(2) is generated during this fatty acid-metabolizing pathway. S. cerevisiae has three GPX genes (GPX1, GPX2, and GPX3) encoding atypical 2-Cys peroxiredoxins. Here we show that expression of GPX1 was induced in medium containing oleic acid as a carbon source in an Msn2/Msn4-dependent manner. We found that Gpx1 was located in the peroxisomal matrix. The peroxisomal Gpx1 showed peroxidase activity using thioredoxin or glutathione as a reducing power. Peroxisome biogenesis was induced when cells were cultured with oleic acid. Peroxisome biogenesis was impaired in gpx1∆ cells, and subsequently, the growth of gpx1∆ cells was lowered in oleic acid-containing medium. Gpx1 contains six cysteine residues. Of the cysteine-substituted mutants of Gpx1, Gpx1(C36S) was not able to restore growth and peroxisome formation in oleic acid-containing medium, therefore, redox regulation of Gpx1 seems to be involved in the mechanism of peroxisome formation.

Temperature dependent N-glycosylation of plasma membrane heat shock protein Hsp30p in Saccharomyces cerevisiae.

The HSP30 gene of the budding yeast Saccharomyces cerevisiae encodes a seven-transmembrane heat shock protein expressed in response to various types of stress including heat shock. Although Hsp30p contains a potential N-glycosylation consensus sequence (Asn(2)-Asp(3)-Thr(4)), whether it is actually N-glycosylated has not been verified. Here we demonstrate that N-glycosylation is induced at Asn(2) of Hsp30p by severe heat shock, ethanol stress, and acetic acid stress. Mild heat shock and glucose depletion induced the expression but not N-glycosylation of Hsp30p, indicating the N-glycosylation to be dependent on temperature and environmental conditions. N-glycosylation did not affect on the intracellular localization of Hsp30p but its physiological role under severe heat shock conditions. Since limited information is available on stress-responsive or condition-induced N-glycosylation, our findings provide new insight into the regulation of cellular stress response in yeast.

Reduction of glucose uptake through inhibition of hexose transporters and enhancement of their endocytosis by methylglyoxal in Saccharomyces cerevisiae.

Diabetes mellitus is characterized by an impairment of glucose uptake even though blood glucose levels are increased. Methylglyoxal is derived from glycolysis and has been implicated in the development of diabetes mellitus, because methylglyoxal levels in blood and tissues are higher in diabetic patients than in healthy individuals. However, it remains to be elucidated whether such factors are a cause, or consequence, of diabetes. Here, we show that methylglyoxal inhibits the activity of mammalian glucose transporters using recombinant Saccharomyces cerevisiae cells genetically lacking all hexose transporters but carrying cDNA for human GLUT1 or rat GLUT4. We found that methylglyoxal inhibits yeast hexose transporters also. Glucose uptake was reduced in a stepwise manner following treatment with methylglyoxal, i.e. a rapid reduction within 5 min, followed by a slow and gradual reduction. The rapid reduction was due to the inhibitory effect of methylglyoxal on hexose transporters, whereas the slow and gradual reduction seemed due to endocytosis, which leads to a decrease in the amount of hexose transporters on the plasma membrane. We found that Rsp5, a HECT-type ubiquitin ligase, is responsible for the ubiquitination of hexose transporters. Intriguingly, Plc1 (phospholipase C) negatively regulated the endocytosis of hexose transporters in an Rsp5-dependent manner, although the methylglyoxal-induced endocytosis of hexose transporters occurred irrespective of Plc1. Meanwhile, the internalization of hexose transporters following treatment with methylglyoxal was delayed in a mutant defective in protein kinase C.

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.

Glutathione peroxidase 2 in Saccharomyces cerevisiae is distributed in mitochondria and involved in sporulation.

Gpx2, one of three glutathione peroxidase homologs (Gpx1, Gpx2, and Gpx3) in Saccharomyces cerevisiae, is an atypical 2-Cys peroxiredoxin that prefers to use thioredoxin as a reducing agent in vitro. Despite Gpx2 being an antioxidant, no obvious phenotype of gpx2Δ mutant cells in terms of oxidative stress has yet been found. To gain a clue as to Gpx2's physiological function in vivo, here we identify its intracellular distribution. Gpx2 was found to exist in the cytoplasm and mitochondria. In mitochondria, Gpx2 was associated with the outer membrane of the cytoplasmic-side, as well as the inner membrane of the matrix-side. The redox state of the mitochondrial Gpx2 was regulated by Trx1 and Trx2 (cytoplasmic thioredoxin), and by Trx3 (mitochondrial matrix thioredoxin). In addition, we found that the disruption of GPX2 reduced the sporulation efficiency of diploid cells.

Acetate but not propionate induces oxidative stress in bakers' yeast Saccharomyces cerevisiae.

The influence of acetic and propionic acids on baker's yeast was investigated in order to expand our understanding of the effect of weak organic acid food preservatives on eukaryotic cells. Both acids decreased yeast survival in a concentration-dependent manner, but with different efficiencies. The acids inhibited the fluorescein efflux from yeast cells. The inhibition constant of fluorescein extrusion from cells treated with acetate was significantly lower in parental strain than in either PDR12 (ABC-transporter Pdr12p) or WAR1 (transcriptional factor of Pdr12p) defective mutants. The constants of inhibition by propionate were virtually the same in all strains used. Yeast exposure to acetate increased the level of oxidized proteins and the activity of antioxidant enzymes, while propionate did not change these parameters. This suggests that various mechanisms underlie the yeast toxicity by acetic and propionic acids. Our studies with mutant cells clearly indicated the involvement of Yap1p transcriptional regulator and de novo protein synthesis in superoxide dismutase up-regulation by acetate. The up-regulation of catalase was Yap1p independent. Yeast pre-incubation with low concentrations of H2O2 caused cellular cross-protection against high concentrations of acetate. The results are discussed from the point of view that acetate induces a prooxidant effect in vivo, whereas propionate does not.

Glyoxalase system in yeasts: structure, function, and physiology.

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.

Modification of yeast characteristics by soy peptides: cultivation with soy peptides represses the formation of lipid bodies.

We have previously reported that the cultivation of yeast cells with soy peptides can improve the tolerance of yeast to freeze-thaw stress (Izawa et al. Appl Microbiol Biotechnol 75:533-538, 2007), indicating that soy peptides can modify the characteristics of yeast cells. To gain a greater understanding of the potencies of soy peptides, we further investigated the effects of cultivation with soy peptides on yeast physiology and found that soy peptides repress the formation of lipid bodies (also called lipid droplets or lipid particles), in which neutral lipids are accumulated. Compared with casein peptone, bacto peptone, yeast nitrogen base, and free amino acid mixtures having the same amino acid composition as soy peptides, cultivation with soy peptides caused decreased levels of mRNAs of neutral lipid synthesis-related genes, such as DGA1, and repressed the formation of lipid bodies and accumulation of triacylglycerol. These results indicate that soy peptides affect the lipid metabolism in yeast cells, and also demonstrate a potentiality of edible natural ingredients as modifiers of the characteristics of food microorganisms.

Vacuolar morphology of Saccharomyces cerevisiae during the process of wine making and Japanese sake brewing.

Although ethanol and osmotic stress affect the vacuolar morphology of Saccharomyces cerevisiae, little information is available about changes in vacuolar morphology during the processes of wine making and Japanese sake (rice wine) brewing. Here, we elucidated changes in the morphology of yeast vacuoles using Zrc1p-GFP, a vacuolar membrane protein, so as to better understand yeast physiology during the brewing process. Wine yeast cells (OC-2 and EC1118) contained highly fragmented vacuoles in the sake mash (moromi) as well as in the grape must. Although sake yeast cells (Kyokai no. 9 and no. 10) also contained highly fragmented vacuoles during the wine-making process, they showed quite a distinct vacuolar morphology during sake brewing. Since the environment surrounding sake yeast cells in the sake mash did not differ much from that surrounding wine yeast cells, the difference in vacuolar morphology during sake brewing between wine yeast and sake yeast was likely caused by innate characters.

Kinetics and redox regulation of Gpx1, an atypical 2-Cys peroxiredoxin, in Saccharomyces cerevisiae.

The budding yeast Saccharomyces cerevisiae has three homologues of glutathione peroxidase (GPX1, GPX2, and GPX3). Two structural homologues of the mammalian glutathione peroxidase, Gpx2 and Gpx3, have been proven to be atypical 2-Cys peroxiredoxins, which prefer to use thioredoxin as an electron donor. Here, we show that Gpx1 is also an atypical 2-Cys peroxiredoxin, but uses glutathione and thioredoxin almost equally. We determined the redox state of Gpx1 in vivo.