11. Cell signalling - Wiley Online Library

11. Cell signalling

YEAST 2007

Yeast 2007; 24: S1-S175

XXIIIrd International Conference on Yeast Genetics and Molecular Biology 11 - Cell signalling

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11-01 Evidence for NO-mediated apoptosis in yeast. Bruno Almeida (1), Sabrina Buttner (2), Steffen Ohlmeier (3), Alexandra Silva (1), Ana Mesquita (1), Belém Sampaio-Marques (1), Nuno Osório (1), Alexander Kollau (4), Bernhard Mayer (4), Cecília Leão (1), Fernando Rodrigues (1), Frank Madeo (2), Paula Ludovico (1) (1) Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Campus de Gualtar, Braga, 4710-057, Portugal; (2) Institute for Molecular Biosciences, Universitätsplatz 2, A-8010 Graz, Austria; (3) Proteomics Core Facility, Biocenter Oulu, Department of Biochemistry, University of Oulu, Oulu, Finland; (4) Department of Pharmacology and Toxicology, KFUG, Universitätsplatz 2, A-8010 Graz, Austria Nitric oxide (NO) is a small molecule with edge roles in diverse physiological functions in biological systems, among them the control of the apoptotic signalling cascade. By combining proteomic, genetic, and biochemical approaches we demonstrate that NO and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are crucial mediators of yeast apoptosis. Using classical methods, we present results showing that H2O2 treatment induces NO production responsible for GAPDH S-nitrosation and modulation of reactive oxygen species production. Blockage of NO synthesis with Nomega-nitro-L-arginine methyl ester leads to a decrease of GAPDH S-nitrosation and cell death while this process is enhanced by increasing the extracellular arginine, supporting the link between NO signalling and intracellular L-arginine content. Evidence is presented showing that NO and GAPDH S-nitrosation also mediate cell death during chronological life span pointing to a physiological role of NO in yeast apoptosis.

11-02 Systems Biology of the yeast AMP-activated Protein Kinase Pathway. Gemma Beltran, Daniel Bosch, Tian Ye, Karin Elbing, Stefan Hohmann Cell and Molecular Biology / Microbiology, Göteborg University, Lundberg Laboratory Box 462, Göteborg, 40530, Sweden AMP-activated protein kinase (AMPK) is the central component of a protein kinase cascade involved in regulating energy levels in mammalian cells. The Saccharomyces cerevisiae homologue, Snf1, is an essential component of signal transduction pathways, monitoring glucose and fructose levels by counteracting the Mig1-dependent repression of a large set of genes. Snf1 is a trimeric complex which consists of the catalytic α subunit (Snf1p), scaffolding β subunits (Gal83p, Sip1p, Sip2p) and a regulatory subunit (Snf4p). Snf1p is activated by the phosphorylation of a single threonine, in response to the opposing activities of three redundant upstream kinases (Sak1p, Elm1p, Tos3p) and the Protein Phosphatase 1 (Glc7p-Reg1p complex). A detailed molecular mechanism on the control of AMPK signalling pathways is still missing. AMPKIN, a European Commission funded initiative, aims to generate, optimise and verify as many different steps as possible in the AMPK pathway of yeast, by applying a systems biology approach. Quantitative dynamic datasets for activation and deactivation of the Snf1 pathway have been generated, including protein levels of Snf1 pathway components, subcellular localisation, mRNA levels, protein activities and metabolite levels. We present preliminary data on Snf1 phosphorylation levels in relation to the amount of glucose on the media, as well as indirect measurements of Snf1p activity by analysing expression of the SUC2 gene as a reporter.

11-03 Starvation of yeast auxotrophs results in PPM1-dependent exponential death and uncontrolled glucose fermentation. Viktor Boer, Sasan Amini, David Botstein Molecular Biology, Lewis-Sigler Institute, Washington road, Princteon, New Jersey, 08540, USA Yeast has evolved robust responses to deal with starvation for nutrients wild type yeast requires in its natural diet. We investigated the response of yeast to starvation for an auxotrophic requirement, i.e. a condition for which no evolved response is to be expected. Several auxotrophic mutants were tested, in a number of S. cerevisiae and S. bayanus strains. Unlike the starvation for phosphate or sulfate, starvation for auxotrophic requirements (except methionine) resulted in exponential loss of viability. When growth is limited by an auxotrophic requirement, we observed elevated glucose consumption rates, particularly when net cell growth slows and then stops. This phenotype is reminiscent of the uncontrolled fermentation characteristic of most tumor cells (Warburg effect). We devised an enrichment procedure for mutants of a leucine auxotroph that maintain viability after repeated cycles of leucine starvation. Mutants were recovered with 100-fold increased survival. Furthermore, these mutants displayed reduced glucose consumption rates. We were able to identify the causal mutations by using both bulk segregant analysis and single feature polymorphism detection on DNA tiling arrays. In two independent selection experiments, we found mutations in the gene PPM1, encoding a carboxy methyl transferase responsible for methylation of the conserved terminal leucine of the global regulator PP2Ac. Our current focus is to identify how and which downstream pathways are affected.

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11-04 Calcium Metabolism and Sugar-Induced Activation of Plasma Membrane H+-ATPase in the Yeast Saccharomyces cerevisiae. Rogelio Brandão (1), Anamaria Cardoso (1), Maria José Trópia (1), Luciano Fietto (2), Ieso Castro (1) (1) Nucleo de Pesquisas em Ciências Biológicas, Universidade Federal de Ouro Preto, Campus do Morro do Cruzeiro, Ouro Preto, Minas Gerais, 35.400-000, Brazil; (2) Departamento de Bioquímica, Universidade Federal de Viçosa, Viçosa, MG - Brazil The plasma membrane H+-ATPase of yeast cells is a predominant membrane protein that is essential for nutrient uptake by secondary active transport systems. Glucose, the external signal more studied in yeast, triggers post-translational modifications that increase the H+-ATPase activity. We have demonstrated that this activation is strongly dependent on calcium metabolism and that several proteins are directly involved in this activation (Trópia et al. Biochem. Biophys. Res. Comm. 343:1234-124, 2006). In this work, we show that activation of the enzyme is dependent of the calcium availability in the cytosol. We have measured the levels of total cellular calcium in a strain lacking the phospholipase C and our results indicated that the vacuolar Ca2+-ATPase, Pmc1p, is involved with the reduction of the activity ATPase in the mutant plc1Δ, as already observed in the mutants snf3Δ and pgm2Δ. Moreover, we found evidence indicating that the vacuolar channel, Yvc1p, is somehow related to the sugar-induced activation of this enzyme. Thus, and by using different mutants, our results indicate a relationship between the activation of the H+-ATPase and calcium signaling in Saccharomyces cerevisiae cells. Together with our previous data, these new results permit us to propose an entire pathway involved in the sugar-induced activation of this enzyme.

11-05 The G2/M Checkpoint Response Detects UV-induced Double-Strand Breaks. A. John Callegari (1), Thomas Kelly (2) (1) 430 East 67th Street, Rockefeller Research Labs Room 609, New York, New York 10021, USA; (2) Molecular Biology, SloanKettering Institute, 430 East 67th Street, New York, New York, 10021, USA Eukaryotic cells irradiated with physiologic UV doses carry lesions into S-phase and activate a post-replication checkpoint response that delays mitosis. At high UV doses, the cell cycle response is different. Cells irradiated in G1 and G2 phase delay the cell cycle in the stage at which they are irradiated, then exhibit an additional post-replication delay. The reason for this biphasic response is unknown. One possibility is that the G1/S and G2/M checkpoints do not occur unless the number of lesions crosses a threshold level. Alternatively, different forms of DNA damage may result from the proximity of lesions at high UV doses. For instance, excision repair gaps containing lesions or double-strand breaks might be formed. We used time-lapse microscopy to measure the UV response of a fission yeast strain defective in DNA polymerase eta, a polymerase which repairs gapped DNA with pyrimidine dimers in the template. Our results indicate that the substrate for this polymerase is not an important signal in generating the G2/M checkpoint. In contrast, a similar analysis of the homologous recombination mutant, ∆rhp51, suggests that homologous recombination intermediates are generated after high doses of UV but not after a physiologic dose. These observations are consistent with a model in which the excision of closely-spaced UV lesions creates double-strand breaks that trigger a G2/M checkpoint response.

11-06 Efficient Tor signaling requires a functional Class C Vps protein complex. Sara A. Zurita-Martinez (1), Rekha Puria (1), Xuewen Pan (2), Jef D. Boeke (3), Maria E. Cardenas (4) (1) Molecular Genetics and Microbiology, Duke University Medical Center, 321 CARL Bldg. Research Dr., Durham, North Carolina, 27710, USA; (2) Department of Biochemistry & Molecular Biology, Baylor College of Medicine, Houston, Texas 77030 USA; (3) Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 USA; (4) Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina 27710 USA The Tor kinases regulate responses to nutrients and control cell growth. Unlike most organisms that only contain one Tor protein, Saccharomyces cerevisiae expresses two, Tor1 and Tor2, which are thought to share all of the rapamycin-sensitive functions attributable to Tor signaling. We conducted a genetic screen that defined the global TOR1 synthetic fitness or lethal interaction gene network. This screen identified mutations in distinctive functional categories that impaired vacuolar function, including components of the EGO/GSE and PAS complexes that reduce fitness. In addition, tor1 is lethal in combination with mutations in Class C Vps complex components. This finding is intriguing giving that Tor1 has been localized to internal membranes that resemble those associated with the endocytic pathway. However, we find that Tor1 does not regulate any known functions of the Class C Vps complex in protein sorting. Instead, Class C vps mutants fail to recover from rapamycin-induced cell cycle arrest or to survive nitrogen starvation and have low levels of amino acids. Remarkably, addition of glutamate or glutamine restores viability to a tor1 pep3 mutant strain. We conclude that Tor1 is more efficient than Tor2 at providing rapamycin-sensitive Tor signaling under conditions of amino acid limitation, and that an intact Class C Vps complex is required to mediate intracellular amino acid homeostasis necessary for Tor1 signaling.

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11-07 Effect of Radicicol on Ca2+-Dependent Cell-Cycle Regulation in Budding Yeast. Ruthada Chanklan, Eiji Aihara, Saori Koga, Masaki Mizunuma, Tokichi Miyakawa Molecular Biotechnology, Graduate School of Advanced Sciences of Matter (AdSM), Hiroshima University, Kagamiyama, Higashi-Hiroshima, 739-8530, Japan The activation of Ca2+-signaling by exposure of zds1-deletion strain Saccharomyces cerevisiae cells to external CaCl2 causes a severe defect of growth which is accompanied by the characteristic physiological changes. A positive screening from microbial metabolites for the activities that alleviated the deleterious physiological effects of external CaCl2 identified radicicol, an inhibitor of Hsp90, as an inhibitor of the Ca2+-dependent regulatory pathway. Radicicol alleviated the analogous physiological effects caused by the genetic activations of calcineurin or Swe1. Western blot analysis of Swe1 level indicated that the effect of radicicol was attributable to the inhibition of the Ca2+-dependent accumulation of Swe1.

11-08 Investigation of the oxidative stress sensing mechanism of Swi6 for cell cycle regulation in Saccharomyces cerevisiae. Joyce Chiu (1), Merridee Wouters (2), Ian Dawes (1) (1) School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW, 2052, Australia; (2) Victor Chang Cardiac Research Institute, Sydney, NSW, 2010, Australia Yeast cells bud when growth conditions are favourable during G1 phase. When subjected to oxidative stress, cells arrest at G1 delaying entry into S phase thus allowing cellular repair to occur. Hence, there exist mechanisms that coordinate oxidative stress sensing and regulation of cell cycle progression. Screening of a subset of the yeast deletion mutant collection was performed to identify deletants that were unable to arrest in G1 phase when treated with linoleic acid hydroperoxide (LoaOOH). This screen identified SWI6, encoding a G1/S phase cell-cycle transcription factor, as a possible sensor of oxidative stress for cell-cycle arrest. Analysis of the Swi6 structure revealed a potentially reactive cysteine residue at position 404 that could participate in dimerisation through disulfide bond formation. To study the role of Swi6 in response to oxidative stress in yeast, the Cys404 residue was mutated to an alanine. Bud count and FACS analysis of swi6 deletant strains carrying wild type or C404A mutated SWI6 showed that loss of the cysteine resulted in an inability of cells to arrest at G1 when treated with LoaOOH. Western blot analysis did not identify any dimerisation of wild type Swi6 when cells were treated with LoaOOH indicating that Cys404 may be modified through an alternative mechanism. We are currently using immunoprecipitation to identify the interacting partners of Swi6 that are lost in its alanine mutant form.

11-09 Pkc1p interacts with PKA and Pde2p in order to increase intracellular levels of glycogen, cAMP, programmed cell death and to depolarize the actin cytoskeleton. Emma Collinson, Brian Chan, Ian Dawes School of Biotechnology and Biomolecular Sciences, University of New South Wales, Cnr Botany/High Sts Randwick, Sydney, NSW, 2052, Australia The ability of cells to respond appropriately to alterations in the environment is essential for survival of stress situations, and processes for stress sensing and subsequent modulation of cell physiology require involvement of signal transduction pathways. The Ras-cyclic AMP (Ras-cAMP) pathway is an essential nutrient sensing pathway in yeast, and also important for stress tolerance of Saccharomyces cerevisiae. We have previously shown that Rom2p (a positive regulator of the cell integrity pathway) negatively regulates the Ras cAMP pathway. Here we show that Rom2p does not directly negatively regulate the Ras cAMP pathway, instead, it signals through Pkc1p and it is Pkc1p that interacts with the Ras cAMP pathway. Our data indicate that Pkc1p specifically interacts with the subunits of PKA and Pde2p in order to increase glycogen levels and cAMP levels. This Pkc1pdependent increase in cAMP levels is accompanied by an increase in yeast programmed cell death and actin depolarisation, which is dependent on Pde2p.

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11-10 Rgd1p, the RhoGAP of Rho3p and Rho4p, is a phosphorylated protein that interacts with various phospholipids. François Doignon, Helder Fernandes, Valérie Prouzet-Mauleon, Olivier Roumanie, Xavier Gatti, Sandra Claret, Didier Thoraval, Marc Crouzet RDPR - UMR CNRS 5095, Université V., Segalen Bordeaux2, 146 rue Léo Saignat, 33076 Bordeaux CEDEX, France Rho GTPases belong to the Ras superfamily of small G proteins. They are involved in signal transduction, cytoskeleton organization, exocytosis and cytokinesis by cycling between a GTP and a GDP bound form. Misregulation of Rho GTPase activity is implicated in the development of various cancers. In previous studies, we have characterized Rgd1 as a RhoGAP protein (negative regulator) dedicated to the down-regulation of Rho3p and Rho4p in the yeast S. cerevisiae. These two Rho GTPases are more specifically involved in exocytosis and actin cytoskeleton organization. The RhoGAP domain of Rgd1p presents strong sequence similarities with the RhoGAP domain of MgcRacGAP, a negative regulator for RhoA in multicellular organisms. To decipher more precisely how Rho3p and Rho4p are down-regulated, we are studying the Rgd1p regulation. We already found that Rgd1p interacts with a specific set of phospholipids: the phosphatidylserine and the members of PtdIns family. In addition, we showed that the interaction between Rgd1p and the phospholipids was dependent on the phosphorylation of the RhoGAP. Using mass spectrometry and protein-phosphatase approaches, we have identified several phosphorylation sites along Rgd1p. Through an in vitro assay, we are now studying the effect of Rgd1p phosphorylation and phospholipid binding on its RhoGAP activity toward Rho3p and Rho4p.

11-11 Novel type of pheromone-induced invasion of Saccharomyces cerevisiae. Ivana Frydlova, Marek Basler, Ivana Malcova, Pavla Vasicova, Jiri Hasek Laboratory of Cell Reproduction, Institute of Microbiology AS CR, Videnska 1083, Prague 4, 142 20, Czech Republic The ability to invade a solid substrate is a prerequisite for pathogenic activity of fungi. Even though invasion in Saccharomyces cerevisiae is less prominent than in other fungi, this organism is a useful model system for these studies. We report here on invasion displayed by MATα cells of S. cerevisiae lacking the Isw2 protein, a subunit of ISW2 chromatin remodelling complex. The absence of other ISW2 complex subunits, Dls1 and Dpb4, has not the same effect as isw2 deletion but it enhances its invasive phenotype. Many features of the isw2∆ MATα invasion resemble pheromone-induced invasive growth, including its dependence on the Fig2 surface protein. We show here that another pheromone-induced surface protein, Aga1, is necessary for the isw2∆ MATα invasive behaviour. It suggests that mating agglutinin Aga1 commonly ensuring the cell-cell contact during mating can also play a role in the agar adhesion necessary for cells to be invasive. We also found that the Flo11p-independent invasive behaviour of isw2∆ cells specifically requires Fus3 kinase. Its function in the invasion of isw2∆ MATα cells cannot be substituted by Kss1 kinase, which plays a basic role in invasive growth signalling. Fus3p- and Aga1p-dependence thus indicates that invasion of isw2∆ MATα cells differs from the pheromone induced invasive growth.

11-12 Involvement of Ca2+-signaling in the regulation of life span in Saccharomyces cerevisiae. Anri Gengyo, Masaki Mizunuma, Tokichi Miyakawa Molecular Biology, Hiroshima University, Kagamiyama1-3-1, Higashi-Hiroshima, 739-8530, Japan The zds1 Saccharomyces cerevisiae strain exhibits characteristic Ca2+-dependent phenotypes, such as growth inhibition, G2 cellcycle arrest and polarized bud growth in the presence of external CaCl2. These phenotypes are due to the activation of Swe1 and Cln2 by a Ca2+-dependent regulatory mechanism, which is mediated by the coordinated action of calcineurin and Mpk1 pathway. From analyses of scz mutations that suppressed the Ca2+ phenotypes of zds1 strain, the scz18 mutation was identified as a mutant allele of SIR3 gene. The Ca2+-dependent phenotypes were all suppressed by disruption of the SIR3 gene, suggesting that the effect of the scz18 mutation was due to the loss of function of the SIR3 gene. It had been previously reported that Sir3 is involved in regulation of the expression of the genes in MAT loci and telomere end. More recently, Sir3 was also implicated in aging. We investigated if Ca2+-signalling is involved in aging. The life spans of various strains were compared in the absence and presence of external CaCl2 by determination of replicative senescence of nascent daughter cells. In wild-type strain, average number of division decreased from 25.5 to 19.3 times by CaCl2. The lifespan of sir3 strain in YPD was shorter (20.1 times) than in wild-type strain and it was similar to that of wild-type in the presence of exogenous CaCl2 (19.6 times). Taken together, it was suggested that Ca2+-signal negatively regulate lifespan and SIR3 may be involved in this process.

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11-13 New insights into the regulation of GATA-family transcriptional activators Gln3 and Gat1 in response to rapamycin. Isabelle Georis (1), Jennifer J. Tate (2), Terrance G. Cooper (2), Evelyne Dubois (1) (1) Laboratoire de Microbiologie - ULB, Institut de Recherches Microbiologiques J.-M. Wiame, Av. E. Gryzon 1, Brussels, 1070, Belgium; (2) Department of Molecular Sciences, University of Tennessee, Memphis, Tennessee, USA The TOR-inhibitor rapamycin (Rap) induces dephosphorylation of Gln3 and relocation of Gln3-Myc13 to the nucleus, both Sit4dependent, and Nitrogen Catabolite Repression (NCR) sensitive transcription in glutamine-grown cells. However, these events do not always consistently correlate to give a clear picture of NCR gene activation. Therefore, we investigated the Sit4 requirement for Rap-induced transcription in TB50 (TB) and Σ1278b (Σ) genetic backgrounds. Rap induced high level TB DAL5 (a typical NCR gene) transcription, even though Gln3-Myc13 is cytoplasmic in a TB sit4. This pointed to Gat1 as the major DAL5 activator, which correlated with transcription decreasing more in a gat1 than in a gln3. In a Σ sit4, Gln3-Myc13 was also cytoplasmic, but here Rap failed to induce DAL5 transcription, despite Gat1-Myc13 being nuclear. ChIP experiments demonstrated Rap induced Gln3-Myc13 and Gat1-Myc13 binding to the DAL5 promoter in both TB and Σ. In contrast to Gat1-Myc13, however, Gln3-Myc13 bound only when Sit4 was functional, which correlated with its intracellular localization. Finally, Gln3-Myc13 did not bind in a gat1, whereas Gat1-Myc13 binding was largely Gln3-independent. In sum, Gat1 – unlike Gln3 – does not require Sit4 for its Rapinduced nuclear localization. Gln3 nuclear targeting alone is not sufficient for binding to the DAL5 promoter. The ability of bound Gat1 to activate NCR sensitive transcription is strain-dependent. Support: COCOF for ED, IG ; GM35642 for TC.

11-14 Functional characterization of a novel mitotic regulator. Tanja Herrmann, Fouzia Ahmad, Gislene Pereira German Cancer Research Center, Molekular Biology of Centrosomes and Cilia, Im Neuenheimer Feld 280, Heidelberg, 69120, Germany In anaphase, after the full elongation of the mitotic spindle and separation of the sister chromatids, cyclin-dependent kinase (CDK) activity must be downregulated to allow the transition out of mitosis (mitotic exit) and cytokinesis to occur. In budding yeast, the conserved phosphatase Cdc14 plays a major role in CDK inactivation. Until anaphase, Cdc14 is sequestered in an inhibitory complex located in the nucleolus. The release of Cdc14 from the nucleolus activates Cdc14 and is regulated by two networks. The FEAR (Cdc14 Early Anaphase Release) network functions early in anaphase and is responsible for the activation of only a certain pool of Cdc14. Cdc14 released via this pathway regulates several processes, including chromosome segregation and microtubule stability, however it is not sufficient to trigger CDK inactivation and subsequent mitotic exit. For the later processes to occur, activation of the Mitotic Exit Network (MEN) is essential. The MEN is a GTPase driven signaling transduction cascade associated with the yeast centrosome, named the spindle pole body (SPB). In order to understand how the FEAR and MEN pathways are regulated on a molecular level, we performed a genetic screen to identify new MEN and FEAR components. One putative candidate, named BIT4, was found as a positive regulator of mitotic exit. We found that Bit4 interacts with MEN components and is localized at the SPB. Here we will present the initial functional characterization of Bit4.

11-15 The MAP kinase encoded by SMK1 negatively regulates 1,3-β-glucan production during spore wall morphogenesis. Linda S. Huang, Hugh K. Doherty Department of Biology, University of Massachusetts Boston, 100 Morrissey Boulevard, Boston, Massachusetts, 02125, USA Sporulation in S. cerevisiae is a developmental program of meiosis followed by spore formation. The S. cerevisiae spore wall contains two inner layers made of mannan and glucan (like the vegetative cell wall) surrounded by two outer spore-specific layers made of chitosan and dityrosine. We found that the MAP kinase Smk1p physically interacts with Gsc2p/Fks2p, a sporulationexpressed subunit of 1,3-β-glucan synthase important for the glucan layer of the spore wall. Loss of GSC2 suppresses the smk1 chitosan-deposition defect, suggesting that SMK1 is a negative regulator of GSC2. Consistent with this model, we see that 1,3-βglucan synthase activity is elevated in smk1 mutants. Our working model is that negative regulation of 1-3-β-glucan production by SMK1 facilitates the transition between early phases of spore wall deposition, when inner layers are preferentially deposited, and late phases, when outer layers are preferentially deposited. To begin to dissect the molecular mechanism by which SMK1 regulates GSC2, we have identified putative MAP kinase docking sites on Gsc2p. We have found that one of these sites is necessary for the physical association of Gsc2p and Smk1p as well as for proper sporulation. We are currently examining the consequences of this disruption on Gsc2p activity and spore morphogenesis.

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11-16 Msa2, an RNA binding protein negatively regulates meiosis in fission yeast. Yasuo Oowatari, Jeong Hee Tae, Satoshi Katayama, Makoto Kawamukai Life and Environmental Science, Shimane University, Nishikawatsu, Matsue, Shimane, 690-8504, Japan Fission yeast proliferates in a haploidic state throughout the cell cycle under nutrient rich conditions, but initiates sexual differentiation when the nutrient conditions become poor. Nine sam mutants have been isolated as to confer a hyper-sporulated phenotype to cells. In those sam mutants, they skip a requirement of starvation to initiate meiosis. We isolated multi-copy suppressors of sam1 called msa1 and msa2, both encoding RNA-binding proteins. Msa2p negatively regulates the onset of sexual differentiation by repressing the Ste11p-regulated genes. When we conducted a two-hybrid screening with Msa2p as bait, one of identified proteins was turned out to be Cpc2p, a positive regulator of meiosis. Cpc2p is a fission yeast ortholog of the mammalian RACK1 protein, which is a highly conserved member of WD-repeat proteins and is capable of interacting with protein kinase C. We confirmed that Msa2p and Cpc2p physically associated in the fission yeast cells by co-immunoprecipitation. Epistatic analysis of msa2 and cpc2 suggested that Msa2p is an upstream regulator for Cpc2p. Msa2p was phosphorylated under nutrient starvation and existence of mating pheromones. Msa2p possesses two potential MAPK phosphorylation sites at Thr40 and Thr126. Msa2p reduced its inhibitory ability on mating by replacement of Thr126 by Asp, but not by Ala, suggesting phosphorylation negatively controlled the activity of Msa2. A novel regulatory mechanism to control the onset of meiosis will be discussed.

11-17 Structural and functional characterization of FHA-SCD Binding Involving Rad53 and Dun1 in Saccharomyces cerevisiae. Hyun Lee (1), Chunhua Yuan (2), Anjali Mahajan (2), Eric Chen (1), Ming-Daw Tsai (2) (1) Genomics Research Center, Academia Sinica, 128, Academia Road, Sec.2, Nankang Dist., Taipei, 115, Taiwan; (2) Department of Chemistry,The Ohio State University, 100 W. 18th Avenue, Columbus, Ohio 43210, USA Rad53 and Dun1 proteins of Saccharomyces cerevisiae play a crucial role within cell signaling pathways responsible for coping with DNA damage. DNA damage activates Mec1 kinase which, in association with Rad9 kinase, phosphorylates Rad53. Activated Rad53 then activates Dun1 by phosphorylation. This Mec1/Rad53/Dun1 pathway regulates ribonucleotide reductase (RNR) activity by modulationg the concentration of the RNR inhibitor Sml1. Rad53 and Dun1 are known to autophosphorylate in order to amplify the cell signal, and autophosphorylation is regulated by interaction between the fork-head associated (FHA) domain and SQ/TQ-rich cluster (SCD). Here we report the molecular basis, specificity, and biological significance of SCD interactions with three FHA domains: (1) Binding studies with NMR and SPR indicate that Dun1 FHA prefers multiply phosphorylated SCD, Rad53 FHA1 prefers singly phosphorylated SCD, and Rad53 FHA2 does not bind to SCD. (2) NMR was then used to map out the binding site and uncover the structural basis of different ligand specificity of the three FHA domains. (3) Autophosphorylation levels of both Rad53 and Dun1 were significantly decreased when Rad53 FHA1 and Dun1 FHA were mutated. (4) Autophosphorylation of Dun1 in vitro was found to be concentration-dependent, and nine autophosphorylation sites were identified by mass spectrometry.

11-18 Activation of the S. cerevisiae Heat Shock Transcription Factor by Yak1 Protein Kinase in Response to Nutrient Signals. Peter Lee, Bo-Ram Cho, Ji-Sook Hahn School of Chemical & Biological Engineering, Seoul National University, SAN 56-1, Seoul, 151-744, South Korea Heat Shock Transcription Factor (HSF) plays a central role in cellular homeostasis by activating gene expression in response to multiple stresses including heat shock, oxidative stress, and glucose starvation. Although the mechanisms for the stress-specific activation of HSF are largely unknown, differential phosphorylation has been suggested as one of the mechanisms by which a single HSF protein can be activated by a variety of stresses. Here we demonstrate that Yak1 kinase, which is under the negative control of PKA and TOR, activates Hsf1. Yak1 phosphorylates Hsf1 in vitro and in vivo, and activates expression of a subset of HSF targets by glucose starvation and heat shock. Inhibition of PKA activity by overexpression of the regulatory subunit Bcy1 induces phosphorylation of Hsf1 even in the yak1 deletion mutant, suggesting that Yak1 is not the only kinase acting downstream of PKA to phosphorylate Hsf1.These results suggest a regulatory mechanism linking Hsf1 activation to two major nutrient signaling pathways mediated by PKA and TOR.

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11-19 The sensitivity of Saccharomyces mutants to palmitoleic acid may provide a means to study the control of membrane fluidity in eukaryotes. Daniel Lockshon (1), Emily O. Kerr (1), Robert Learmonth (2), Brian K. Kennedy (1) (1) Department of Biochemistry, University of Washington, Box 357350, Seattle, Washington, 98195, USA; (2) Centre for Systems Biology, University of Southern Queensland, Toowoomba, 4350, Australia The mechanisms which control the fluidity of eukaryotic membranes are unknown. We have identified S. cerevisiae deletion strains whose growth is impaired by palmitoleic (PO; C16:1) but not oleic (C18:1) acid. PO-sensitivity is suppressed by oleate thus perhaps identifying a signaling pathway that controls the ratio of these fatty acids in membrane phospholipid. Growth of these mutants is also inhibited by a known fluidizer, benzyl alcohol, thus indicating that PO has a fluidizing effect. Removal of Pkc1, known to play a key role in cell wall integrity control, leads to acute PO-sensitivity. Removal of Bck1, Mkk1, Mkk2, Slt2, or Swi6 downstream components of the cell wall integrity pathway, cause modest PO-sensitivity. Suppression by 1M sorbitol of the PO-sensitivity of these four mutants implies that PO/oleate ratio influences the cell wall. Acute PO-sensitivity of the pkc1Δ strain, even in the presence of 1M sorbitol, suggests the cell wall to be more severely compromised by PO addition to this strain. Alternatively, the failure to control the PO/oleate ratio could have an additional effect on the pkc1 strain, perhaps by disabling a 2nd pathway downstream of Pkc1 thus allowing PO addition to cause excess membrane fluidity. We are attempting to distinguish these two models by a variety of genetic, biochemical, and physical methods. Most notably, the effect of PO on the fluidity of the plasma membrane is being examined by measuring the depolarization of laurdan fluorescence.

11-20 Functional Characterization of the S. cerevisiae ClC channel (Gef1p). Ataúlfo Martínez-Torres (1), Alfonso Carabez Trejo (1), Leanne Coynne (2), Robert Halliwell (2), Ricardo Miledi (1), Angélica López Rodríguez (1) (1) Instituto de Neurobiología, Universidad Nacional Autónoma de México, Carr. México-SLP Km. 15, Querétaro, Querétaro, 76230, México; (2) Department of Physiology and Pharmacology, Thomas J. Long School of Pharmacy & Health Sciences, University of the Pacific, Stockton, CA 95211 USA In the yeast S. cerevisiae the gene GEF1 encodes for a protein regarded as a member of the voltage activated Cl- channel family (ClC), a gene family associated with multiple physiological roles including maintenance of osmotic equilibrium, intracellular pH and membrane potential of plasma membranes. Microinjection of plasmids carrying the gene GEF1 into X. laevis oocytes induced the expression of a protein (Gef1p) conducting Cl-, the ion channel remained permanently open and was effectively and reversibly blocked by the Cl- channel blocker, NPPB. This channel was also found when GEF1 was transfected into HEK-293 cells in culture and was again inhibited by NPPB as well as by niflumic acid, another Cl- channel blocker. Furthermore, the flow of Cl- in the yeast was monitored by the emission of fluorescence by the halide indicator SPQ; which was rapidly quenched in a wild type strain, in contrast to both a GEF1 knock-out strain and yeast grown in the presence of NPPB that quenched the indicator at slower rates. We conclude that GEF1 is at least partially responsible for the transport of Cl- in the yeast and the channel remains permanently open. Project supported by CONACYT 44943Q, UNAM.PAPIIT IN208-003-2, 228205 y 204806. Thanks to E. Ruiz and IA. Martínez by technical support.

11-21 Two redundant inhibitors of the MAPK-responsive transcription factor Ste12 differentially modulate noise during pheromone signaling. Emma McCullagh, Anupama Seshan, Hana El-Samad, Hiten Madhani Biochemistry and Biophysics, University of California, San Francisco, 600 16th St., San Francisco, California, 94102, USA Variability, or noise, in the transcriptional responses of individual isogenic cells is a ubiquitous, yet incompletely understood, phenomenon. Studies in bacteria and yeast have demonstrated that variability within cells (intrinsic) can be explained by stochastic events in gene expression whereas the sources of variability between cells (extrinsic) is poorly understood. The pheromone response MAPK pathway provides a tractable system for studying noise during eukaryotic signal transduction and gene activation. The target activator, Ste12, is regulated by redundant inhibitors Dig1 and Dig2, which bind to the activation and DNA-binding domains of Ste12, respectively. To test the hypothesis that there exist pathway-specific factors that modulate noise in the pathway output, we measured the noise from three pheromone-inducible promoters and a pheromone independent reporter using promoter-fluorescent protein gene fusions. While Dig1 and Dig2 redundantly inhibit Ste12, we found that Dig1, but not Dig2, has a role in suppressing noise from Ste12-dependent promoters: cells lacking Dig1 have significant increases in extrinsic noise. Remarkably, the increase in noise in dig1Δ strains is abolished when the STE12 promoter is replaced by different promoters. Thus the effect of Dig1 on noise may be due to a role in limiting variability in activator levels through feedback regulation on the STE12 promoter. Fluctuations in levels of gene activators may be a general source of extrinsic noise.

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11-22 Implication of Stm1 in down-regulating Cln2 level and hyper-polarized bud growth in response to calcium signaling in Saccharomyces cerevisiae. Masaki Mizunuma, Tomohiro Machida, Tokichi Miyakawa Molecular Biotechnology, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, 739-8530, Japan Cell polarity control is essential for morphogenesis and development. The calcium-activated pathways in yeast induces the formation of hyper-polarized bud and delayed the cell-cycle progression in G2. The effect of calcium on cell-cycle regulation and polarized bud growth are pronounced on a zds1 background lacking the negative regulator for SWE1 and CLN2 transcription. We have previously shown that the zds1 strain in medium containing 300 mM CaCl2 is severely inhibited, showing G2 arrest and hyper-polarized bud growth. To identify negative regulators for the calcium-induced phenotypes, we screened for genes whose overexpression could suppress the calcium-sensitivity of the zds1 strain. By this screening, STM1 gene was obtained as a suppressor. STM1 encodes a guanine quadruplex (G4) and purine-motif triplex nucleic-acid-binding protein. Stm1 has been implicated in several biological processes, ranging from apoptosis to telomere biosynthesis. The hyper-polarized bud growth induced by calcium was suppressed by overexpression of the STM1 gene, whereas the G2 delay was not suppressed by it. It has been known that hyper-polarized bud growth is dependent on the elevation of Cln2 level. By overexpression of the STM1 gene, the abundance of Cln2 in the zds1 strain decreased rapidly, suggesting that Stm1 down-regulated the Cln2 level. Moreover, genetic analyses indicated that Stm1 is downregulated by the Sir complexes in the calcium-induced hyper-polarized bud growth.

11-23 Hog1 MAP kinase phosphorylation of the plasma membrane Fps1 targets this aquaglyceroporin for endocytic degradation, rendering yeast resistant to acetic acid. Mehdi Mollapour, Peter W. Piper Molecular Biology and Biotechnology, The University of Sheffield, Western Bank, Sheffield, S10 2TN, UK Aquaporins and aquaglyceroporins form the membrane channels that mediate fluxes of water and small solute molecule into, and out of, cells. Eukaryotes often use Mitogen Activated Protein Kinase (MAPK) cascades for the intracellular signaling of stress. This work reveals an aquaglyceroporin becoming destabilized by direct MAPK phosphorylation. Hog1 stress-activated MAPK undergoes a transient activation in yeast exposed to high, toxic levels of acetic acid. This Hog1 then phosphorylates the plasma membrane aquaglyceroporin Fps1. This phosphorylation is the signal for Fps1 to be ubiquitinated and endocytosed, then degraded in the vacuole. Fps1 membrane channel facilitates passive diffusional influx of undissociated acetic acid (pKa 4.75) to the cell at low pH. Consistent with this generating resistance, sensitivity to acetic acid is seen with diverse mutations that prevent this endocytic removal of Fps1 from the plasma membrane (loss of Hog1; loss of the soluble domains on, or T231A S537A double mutation of, Fps1 preventing its in vivo phosphorylation; or mutations generating a general loss of endocytosis of cell surface proteins (doa4Δ and end3Δ)). Remarkably, such targetting of Fps1 for degradation may be the major requirement for an active Hog1 in acetic acid resistance, since Hog1 is dispensible for such resistance when the cells lack Fps1. Evidence is presented that Hog1 MAPK is in physical association with the N-terminal domain of Fps1, even in unstressed cells.

11-24 Stp1, a transcription factor for amino acid permeases, regulates TOR signaling pathway in Saccharomyces cerevisiae. Chun-Shik Shin, Won-Ki Huh School of Biological Sciences, and Research Center for Functional Cellulomics, Seoul National University, Seoul 151-747, South Korea The TOR (target of rapamycin) signal transduction pathway is an important mechanism for controlling cell growth in all eukaryotic cells. In Saccharomyces cerevisiae, extracellular amino acids lead to activation of TOR pathway and subsequent cell growth. When extracelluar amino acids are sensed, the transcription factor Stp1 is activated by proteolytic truncation and is translocated to nucleus, where it activates transcription of its target amino acid permease genes. Through genome-wide translocalization study using GFP-tagged strains, we found that Stp1-GFP signal disappeared from the nucleus under inactivation of TOR by rapamycin treatment. Interestingly, disappearance of Stp1 turned out to result from degradation of Stp1, not from translocalization to cytoplasm. Furthermore, stp1∆ cells were more sensitive to rapamycin than wild-type cells. Based on these findings, we propose that extracellular amino acid signal is transferred for activation of TOR pathway through Stp1.

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11-25 Understanding the Fidelity of MAP Kinase Signaling. Teresa Shock, Hiten Madhani Biochemistry, University of California, San Francisco, 600 16th Street, San Francisco, California, 94143-2200, USA Mitogen-activated protein kinase (MAPK) signaling cascades play a fundamental role in eukaryotic cells, acting as signal transducers involved in many biological processes including growth, differentiation and response to stress. In yeast, the mating, filamentation, and high osmolarity glycerol (HOG) pathways elicit specific responses to distinct stimuli, yet share multiple components. Unexpectedly, we observed that both the MEK and the MAPKs of the mating and filamentation pathways are phosphorylated on their activating residues during signaling through the HOG pathway. Therefore, mechanisms must exist downstream of the MAPKs to ensure that mating and filamentation genes are not transcribed in response to osmotic stress. In addition, we found that this is a novel mechanism, distinct from the degradation mechanism used to prevent cross-talk between the filamentation and mating pathways. We tested whether specificity is achieved before or after the mating and filamentation specific transcription factors, Ste12 and Tec1, are recruited to their gene targets, and found that in the absence of Hog1 there is a large increase in the levels of Tec1 binding to filamentation specific promoters. This indicates that Hog1 plays an essential role in inhibiting erroneous signaling by preventing Tec1 from binding to its target promoters. These results point to a new mode of regulation used by eukaryotic cells to perform the indispensable function of maintaining signaling specificity.

11-26 Cross-Talks between MAPK activity and Cell Cycle progression during Genotoxic Stress in S. pombe. Geetanjali Sundaram (1), Santanu Pal Chaudhuri (2), Dhrubajyoti Chattopadhyay (1) (1) Biochemistry, University of Calcutta, c/o Prof. Dhrubajyoti Chattopadhyay35 Ballygunje Circular Road, Kolkata-700019, 700019, India; (2) Northwestern University, Feinberg School of Medicine, 333 East Superior Street, Suite 490, Chicago, Illinois 60611 Response to Genotoxic Stress (Cigarette Smoke Extract; CSE) in S. pombe involves the activation of both the Sty1 MAPK pathway and the Rad3-Cds1 dependent S phase checkpoint in S. pombe. Mutants in the two pathways are highly sensitive to CSE and the rad3 sty1 double mutant exhibits a sensitivity much higher that the additive phenotype of the respective single mutants, indicating potential overlaps between the activities of the key molecules of both the signaling pathways. We have investigated the cross-talks between these two pathways and our observations prove that the activation of the Sty1 dependent transcription factor Atf1 in response to CSE treatment is also affected by Rad3 activity. At the same time the MAPK sty1 can also independently regulate the cell cycle by physically associating with the cell cycle regulatory phosphatase Cdc25. Coupling these with our previous observations we propose a model for the synergistic action between them which ultimately leads to minimization of the damage and cell survival.

11-27 Stress-responsive Gln3 localization in S. cerevisiae is separable from and can overwhelm nitrogen source regulation. Jennifer J. Tate, Terrance G. Cooper Molecular Sciences, University of Tennessee, 858 Madison Ave., Memphis, Tennessee, 38163, USA Intracellular distribution of GATA-family transcription activator, Gln3, depends heavily on a cell's nutritional environment. Gln3Myc13 is cytoplasmic in cells provided with repressive nitrogen sources (glutamine), and nuclear with derepressive nitrogen sources, (proline), or after rapamycin (Rap), or methionine sulfoximine (Msx) treatment. Gln3-Myc13 phosphorylation also responds to growth conditions and Tor1,2 pathway inhibitors, i.e., it decreases in Rap-treated cells and increases in Msx-treated, nitrogen- or carbon-starved cells and in a sit4 mutant. Here, we demonstrate that a broad spectrum of environmental stresses (temperature, osmotic, oxidative) also increases Gln3-Myc13 phosphorylation. In parallel, these stresses elicit rapid (