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Aim The goal of the study was to investigate the cellular tolerance mechanism in response to honokiol exposure. Methods and results The broth microdilution method was employed to test the sensitivity of different Saccharomyces cerevisiae strains to honokiol. Intracellular levels of reactive oxygen species (ROSs) were determined by DCFH-DA staining. The phosphorylation of Hog1 was evaluated by Western blot analysis. The mRNA expressions of genes involved in the Ras-cyclic AMP (cAMP) pathway were analysed by real-time reverse transcription polymerase chain reaction. We found that the sod1▵ mutant was hypersensitive to honokiol and produced more ROS compared with wild-type and sod2▵ cells. Hog1 was phosphorylated in response to honokiol exposure and deletion of HOG1 increased the sensitivity to honokiol. The expressions of genes involved in the Ras-cAMP pathway were down-regulated after honokiol exposure; exogenous cAMP significantly reduced the phosphorylation of Hog1, although the level was higher than the control level. Conclusions In addition to SOD1, the Ras-cAMP cascade and Hog1 MAP kinase pathway is essential for protecting against honokiol-induced oxidative stress. Significance and impact of the study Our results provide insight into the understanding of the action mechanism of honokiol.

By performing an integrated comparative analysis on the physiology and transcriptome of four different S. cerevisiae strains growing on galactose and glucose, it was inferred that the transcription factors Bas1p, Pho2p, and Gcn4p play a central role in the regulatory events causing the Crabtree effect in S. cerevisiae. The analysis also revealed that a point mutation in the RAS2 observed in a galactose-adapted strain causes a lower Crabtree effect and growth rate on glucose by decreasing the activity of Gcn4p while at the same time is at the origin of higher growth rate on galactose due to a lower activity of the transcriptional repressor Sok2p. The role of Gcn4p on the trade-off effect observed on glucose was confirmed experimentally. This was done by showing that the point mutation in RAS2 does not result in a lower growth rate on glucose if it is introduced in a GCN4-negative background.

Previously, we reported that Rck1 regulates Hog1 and Slt2 activities and affects MAP kinase activity in Saccharomyces cerevisiae. Recently, we found that Rck1 up-regulates phospho-Kss1 and phospho-Fus3. Kss1 has been known as a component in the pseudohyphal growth pathway, and we attempted to identify the function of Rck1 in pseudohyphal growth. Rck1 up-regulated Ras2 at the protein level, not the transcriptional level. Additionally, FLO11 transcription was up-regulated by RCK1 over-expression. RCK1 expression was up-regulated during growth on SLAD + 1% butanol medium. On nitrogen starvation agar plates, RCK1 over-expression induced pseudohyphal growth of colonies, and cells over-expressing RCK1 showed a filamentous morphology when grown in SLAD medium. Furthermore, 1-butanol greatly induced filamentous growth when RCK1 was over-expressed. Moreover, invasive growth was activated in haploid cells when RCK1 was over-expressed. The growth defect of cells observed on 1-butanol medium was recovered when RCK1 was over-expressed. Interestingly, Ras2 and phospho-Kss1 were up-regulated by Rck1 independently. Together, these results suggest that Rck1 promotes pseudohyphal growth by activating Ras2 and Kss1 via independent pathways in S. cerevisiae.

The acidification of the medium is observed during yeast cell growth. This process contributes to the emission of organic acids, mainly acetic acid. Acetic acid is known as the inducer of apoptosis in the yeast Saccharomyces cerevisiae. In this study, we showed that hydrochloric acid can also induce apoptosis in yeast cells, and the apoptotic phenotype triggered by treating yeast cells with hydrochloric acid is modulated by the Ras/PKA pathway. The Ras/PKA pathway is highly conserved between all eukaryotic organisms, as well as cell processes that are related to apoptosis and aging. In this research, we demonstrated that the activation of the Ras/PKA pathway by insertion of Ras2(Val19) allele or deletion of PDE2 gene increases cell death, displaying the markers of apoptosis in an acidic environment. Downregulation of the pathway by deletion of RAS2, RAS1, PDE1, and insertion of the Ha-ras gene increases the cell viability and diminishes cell death with the apoptotic phenotypes. The deletion of PDE1 gene and double deletion of both phosphodiesterase genes prevent the induction of apoptosis in the cells. Modulations in the Ras/PKA pathway affect cell viability and apoptosis during natural gradual acidification of the medium as well as in acid stress conditions.

Fungal infections are an increasing clinical problem. Decreased treatment effectiveness is associated with biofilm formation and drug recalcitrance is thought to be biofilm specific. However, no systematic investigations have tested whether resistance mechanisms are shared between biofilm and planktonic populations. We performed multiplexed barcode sequencing (Bar-seq) screening of a pooled collection of gene-deletion mutants cultivated as biofilm and planktonic cells. Screening for resistance to the ergosterol-targeting fungicide amphotericin B (AmB) revealed that the two growth modes had significant overlap in AmB-persistent mutants. Mutants defective in sterol metabolism, ribosome biosynthesis and the TORC1 and Ras pathways showed increased persistence when treated with AmB. The ras1, ras2 and tor1 mutants had a high-persister phenotype similar to wild-type biofilm and planktonic cells exposed to the TORC1 pathway inhibitor rapamycin. Inhibition of TORC1 with rapamycin also increased the proportion of persisters in Candida albicans and Candida glabrata. We propose that decreased TORC1-mediated induction of ribosome biosynthesis via Ras can lead to formation of AmB-persister cells regardless of whether the cells are in planktonic or biofilm growth mode. Identification of common pathways leading to growth mode-independent persister formation is important for developing novel strategies for treating fungal infections.

In a previous study, system level analysis of adaptively evolved yeast mutants showing improved galactose utilization revealed relevant mutations. The governing mutations were suggested to be in the Ras/PKA signaling pathway and ergosterol metabolism. Here, site-directed mutants having one of the mutations RAS2 Lys77 , RAS2 Tyr112 , and ERG5 Pro370 were constructed and evaluated. The mutants were also combined with overexpression of PGM2 , earlier proved as a beneficial target for galactose utilization. The constructed strains were analyzed for their gross phenotype, transcriptome and targeted metabolites, and the results were compared to those obtained from reference strains and the evolved strains. The RAS2 Lys77 mutation resulted in the highest specific galactose uptake rate among all of the strains with an increased maximum specific growth rate on galactose. The RAS2 Tyr112 mutation also improved the specific galactose uptake rate and also resulted in many transcriptional changes, including ergosterol metabolism. The ERG5 Pro370 mutation only showed a small improvement, but when it was combined with PGM2 overexpression, the phenotype was almost the same as that of the evolved mutants. Combination of the RAS2 mutations with PGM2 overexpression also led to a complete recovery of the adaptive phenotype in galactose utilization. Recovery of the gross phenotype by the reconstructed mutants was achieved with much fewer changes in the genome and transcriptome than for the evolved mutants. Our study demonstrates how the identification of specific mutations by systems biology can direct new metabolic engineering strategies for improving galactose utilization by yeast.

Air–liquid biofilm formation appears to be an adaptive mechanism that promotes foraging of Saccharomyces cerevisiae flor strains in response to nutrient starvation. The FLO11geneplaysa centralroleinthisphenotypeasitsexpressionallowsyeastcellstorise to the liquid surface. Here, we investigated the role of ammonium depletion in air–liquid biofilm formation and FLO11 expression in a S. cerevisiae flor strain. The data obtained show that increasing ammonium concentrations from 0 to 450mM reduce air–liquid biofilm in terms of biomass and velum formation and correlate with a reduction of FLO11 expression. Rapamycin inhibition of the TOR pathway and deletion of RAS2 gene significantlyreducedbiofilmformationandFLO11expression.Takentogether,thesedata suggest that ammonium depletion is a key factor in the induction of air–liquid biofilm formation and FLO11 expression in S. cerevisiae flor strains. Copyright © 2011 John Wiley & Sons, Ltd.

Background TC21 plays an important role in highly aggressive tumor formation, and it was overexpressed in several human cancers, including breast cancer, oral squamous cell carcinoma (SCC), and esophageal SCC. In light of this, we explored the expression of TC21 in overall skin cancers in order to evaluate the relationship between TC21 and malignant skin tumors. Methods We examined six normal skin tissues and 18 malignant skin tumor tissues, including six malignant melanomas (MM), six SCCs, and six basal cell carcinomas (BCCs) using western blotting for the expression of TC21. In another set, 16 specimens of MM, 16 SCC, and 16 BCC were analyzed for the expression of TC21 using immunohistochemical staining. To evaluate the amount of expression of TC21, the Raytest TINA software was used for western blotting and a histochemical score (HSCORE) was used for immunohistochemical evaluation. Results The western blotting and immunohistochemistry showed that TC21 was expressed in all malignant skin tumors and not in normal skin tissues. The relative protein expression was an average of 0.004 in normal skin, 1.042 in MM, 0.621 in SCC, and 0.485 in BCC. In immunohistochemistry, HSCORE for normal skin was an average of 0.05, MM was 2.42, SCC was 2.11, and BCC was 1.22. Conclusions This article is the first study demonstrating expression of TC21 in human skin malignant tumors and suggests that TC21 is more expressed in highly aggressive skin tumors.

The GTPase Ras1 activates the yeast-to-hypha transition in Candida albicans by activating cAMP synthesis. Here, we have characterized Ras2. Ras2 belongs to a group of atypical Ras proteins in some fungal species that share poor identity with other Ras GTPases with many variations in conserved motifs thought to be crucial for Ras-associated activities. We find that recombinant Ras2 is enzymatically as active as Ras1. However, only RAS1 can rescue the lethality of the Saccharomyces cerevisiae ras1 ras2 mutant, suggesting functional divergence of the two genes. ras2Delta is normal in hyphal growth, but deleting RAS2 in the ras1Delta background greatly aggravates the hyphal defect, indicating that Ras2 also has a role in hyphal development. Strikingly, while RAS1 deletion causes a approximately 20-fold decrease in cellular cAMP, further deletion of RAS2 restores it to approximately 30% of the wild-type level. Consistently, while the ras1Delta mutant enters the stationary phase prematurely, the double mutant does so normally. Moreover, ras1Delta cells exhibit increased resistance to H(2)O(2) and higher sensitivity to the heavy metal Co(2+), whereas ras2Delta cells show the opposite phenotypes. Together, our data reveal a novel regulatory mechanism by which two antagonizing Ras GTPases balance each other in regulating multiple cellular processes in C. albicans.

The general amino acid permease (Gap1p) of Saccharomyces cerevisiae is a broad range, low affinity permease that imports amino acids in cells growing on poor nitrogen sources. This permease also signals the presence of amino acids through the fermentable growth medium pathway allowing the cell to respond to new sources of nitrogen in the surrounding medium. Yeast with an activated Ras2/cAMP pathway show many phenotypes indicative of altered nitrogen uptake and metabolism; sensitivity to nitrogen starvation, low amino acid pools. We have shown that Gap1p activity is lowered in cells with an activated RAS2(val19) allele or elevated cAMP levels whereas cells with inactive ras2 allele lose ammonia repression of Gap1p-mediated transport. This regulation is through a post-transcriptional mechanism; transcription of GAP1 is not affected by cAMP level. A mechanism by which the Ras2/cAMP/PKA pathway controls the ubiquitin-dependent degradation of Gap1p is most consistent with the data.

Previously, we reported that Rck1 up-regulates Ras2 and pseudohyphal growth of Saccharomyces cerevisiae. Here, we further investigate the involvement of Rck1 in the activation of pseudohyphal growth. Rck1 activated phosphorylation of the deubiquitinase Ubp3 through a direct protein interaction between Rck1 and Ubp3. The N-terminal Bre5 binding region of Ubp3 physically interacted with Rck1, and Ubp3 and Rck1 co-precipitated. Overexpression of UBP3 using a high-copy plasmid resulted in the upregulation of Ras2, and deletion of UBP3 blocked the upregulation of Ras2 by RCK1 overexpression. Treatment with the proteasome inhibitor MG132 resulted in accumulation of Ras2, indicating that Rck1 is involved in Ras2 degradation in a proteasome-dependent manner. Furthermore, deletion of UBP3 blocked the upregulation of FLO11, a flocculin required for pseudohyphal and invasive growth induced by RCK1 overexpression in S. cerevisiae. Taken together, these results demonstrate that Rck1 promotes S. cerevisiae pseudohyphal growth via the activation of Ubp3 phosphorylation.

Osmotin is a stress responsive antifungal protein belonging to the pathogenesis-related (PR)-5 family that confers tolerance to both biotic and abiotic stresses in plants. Protective efforts of osmotin in plants range from high temperature to cold and salt to drought. It lyses the plasma membrane of the pathogens. It is widely distributed in fruits and vegetables. It is a differentially expressed and developmentally regulated protein that protects the cells from osmotic stress and invading pathogens as well, by structural or metabolic alterations. During stress conditions, osmotin helps in the accumulation of the osmolyte proline, which quenches reactive oxygen species and free radicals. Osmotin expression results in the accumulation of storage reserves and increases the shelf-life of fruits. It binds to a seven-transmembrane-domain receptor-like protein and induces programmed cell death in Saccharomyces cerevisiae through RAS2/cAMP signaling pathway. Adiponectin, produced in adipose tissues of mammals, is an insulin-sensitizing hormone. Strangely, osmotin acts like the mammalian hormone adiponectin in various in vitro and in vivo models. Adiponectin and osmotin, the two receptor binding proteins do not share sequence similarity at the amino acid level, but interestingly they have a similar structural and functional properties. In experimental mice, adiponectin inhibits endothelial cell proliferation and migration, primary tumor growth, and reduces atherosclerosis. This retrospective work examines the vital role of osmotin in plant defense and as a potential targeted therapeutic drug for humans.

Neurofibromatosis type 1 (NF1) is among the most common genetic disorders of humans and is caused by loss of neurofibromin, a large and highly conserved protein whose only known function is to serve as a GTPase-Activating Protein (GAP) for Ras. However, most Drosophila NF1 mutant phenotypes, including an overall growth deficiency, are not readily modified by manipulating Ras signaling strength, but are rescued by increasing signaling through the cAMP-dependent protein kinase A pathway. This has led to suggestions that NF1 has distinct Ras- and cAMP-related functions. Here we report that the Drosophila NF1 growth defect reflects a non-cell-autonomous requirement for NF1 in larval neurons that express the R-Ras ortholog Ras2, that NF1 is a GAP for Ras1 and Ras2, and that a functional NF1-GAP catalytic domain is both necessary and sufficient for rescue. Moreover, a Drosophila p120RasGAP ortholog, when expressed in the appropriate cells, can substitute for NF1 in growth regulation. Our results show that loss of NF1 can give rise to non-cell-autonomous developmental defects, implicate aberrant Ras-mediated signaling in larval neurons as the primary cause of the NF1 growth deficiency, and argue against the notion that neurofibromin has separable Ras- and cAMP-related functions.

Class I phosphoinositide 3-kinases (PI(3)Ks) are activated through associated adaptor molecules in response to G protein-coupled and tyrosine kinase receptor signalling1. They contain Ras-binding domains (RBDs) and can also be activated through direct association with active GTP-bound Ras2,3,4,5,6,7,8,9,10. The ability of Ras to activate PI(3)K has been established in vitro and by overexpression analysis, but its relevance for normal PI(3)K function in vivo is unknown. The Drosophila class I PI(3)K, Dp110, is activated by nutrient-responsive insulin signalling and modulates growth, oogenesis and metabolism11,12,13,14,15. To investigate the importance of Ras-mediated PI(3)K activation for normal PI(3)K function, we replaced Dp110 with Dp110RBD, which is unable to bind to Ras but otherwise biochemically normal. We found that Ras-mediated Dp110 regulation is dispensable for viability. However, egg production, which requires large amounts of growth, is dramatically lowered in Dp110RBD flies. Furthermore, insulin cannot maximally activate PI(3)K signalling in Dp110RBD imaginal discs and Dp110RBD flies are small. Thus, Dp110 integrates inputs from its phosphotyrosine-binding adaptor and Ras to achieve maximal PI(3)K signalling in specific biological situations.

The yeast Saccharomyces cerevisiae deploys two different types of glucose sensors on its cell surface that operate in distinct glucose signaling pathways: the glucose transporter-like Snf3 and Rgt2 proteins and the Gpr1 receptor that is coupled to Gpa2, a G-protein alpha subunit. The ultimate target of the Snf3/Rgt2 pathway is Rgt1, a transcription factor that regulates expression of HXT genes encoding glucose transporters. We have found that the cAMP-dependent protein kinase A (PKA), which is activated by the Gpr1/Gpa2 glucose-sensing pathway and by a glucose-sensing pathway that works through Ras1 and Ras2, catalyzes phosphorylation of Rgt1 and regulates its function. Rgt1 is phosphorylated in vitro by all three isoforms of PKA, and this requires several serine residues located in PKA consensus sequences within Rgt1. PKA and the consensus serine residues of Rgt1 are required for glucose-induced removal of Rgt1 from the HXT promoters and for induction of HXT expression. Conversely, overexpression of the TPK genes led to constitutive expression of the HXT genes. The PKA consensus phosphorylation sites of Rgt1 are required for an intramolecular interaction that is thought to regulate its DNA binding activity. Thus, two different glucose signal transduction pathways converge on Rgt1 to regulate expression of glucose transporters.

Expression of mitochondrial thioredoxin peroxidase (Prx1p) from Saccharomyces cerevisiae is subjected to complex transcriptional regulation and is responsive to the levels of several compounds such as glucose and peroxides. We have previously shown that glucose represses the expression of mitochondrial thioredoxin peroxidase gene (PRX1) in a process mediated by cAMP/protein kinase A (PKA) and Msn2/4p. Here, we show by northern blot and reporter gene (b-galactosidase) assays that deletion of genes encoding Tor1p and Ras2p resulted in increased PRX1 expression, indicating that these proteins are also mediators of the glucose repression effect. We also identified the position of the stress transcription responsive element (STRE) in the PRX1 promoter, which is recognized by Msn2p and Msn4p activators. Mutation of AGGGG sequence at position � 116 to � 112 caused a high drop in PRX1 expression under respiratory conditions and in strains containing deletions of TOR1 or RAS2, confirming the finding that this sequence is a STRE. 2004 Published by Elsevier B.V. on behalf of the Federation of European Microbiological Societies.

In Saccharomyces cerevisiae , Ras proteins connect nutrient availability to cell growth through regulation of protein kinase A (PKA) activity. Ras proteins also have PKA-independent functions in mitosis and actin repolarization. We have found that mutations in MOB2 or CBK1 confer a slow-growth phenotype in a ras2 Δ background. The slow-growth phenotype of mob2 Δ ras2 Δ cells results from a G 1 delay that is accompanied by an increase in size, suggesting a G 1 /S role for Ras not previously described. In addition, mob2 Δ strains have imprecise bud site selection, a defect exacerbated by deletion of RAS2 . Mob2 and Cbk1 act to properly localize Ace2, a transcription factor that directs daughter cell-specific transcription of several genes. The growth and budding phenotypes of the double-deletion strains are Ace2 independent but are suppressed by overexpression of the PKA catalytic subunit, Tpk1. From these observations, we conclude that the PKA pathway and Mob2/Cbk1 act in parallel to determine bud site selection and promote cell cycle progression.

Mutations in RAS2, CYR1, and SCH9 extend the chronological life span in Saccharomyces cerevisiae by activating stress-resistance transcription factors and mitochondrial superoxide dismutase (Sod2). Here we show that mutations in CYR1 and SCH9 also extend the replicative life span of individual yeast mother cells. However, the triple deletion of stress-resistance genes MSN2/MSN4 and RIM15, which causes a major decrease in chronological life span, extends replicative life span. Similarly, the overexpression of superoxide dismutases, which extends chronological survival, shortens the replicative life span and prevents budding in 30–40% of virgin mother cells. These results suggest that stress-resistance transcription factors Msn2/Msn4 negatively regulate budding and the replicative life span in part by increasing SOD2 expression. The role of superoxide dismutases and of other stress-resistance proteins in extending the chronological life span of yeast, worms, and flies indicates that the negative effect of Sod2, Msn2/Msn4/Rim15 on the replicative life span of S. cerevisiae is independent of aging.

We selected for increased phenotypic expression of a synthetic cox2::arg8m-G66S reporter gene inserted into Saccharomyces cerevisiae mtDNA in place of COX2. Recessive mutations in ras2 and cyr1, as well as elevated dosage of PDE2, allowed cox2::arg8m-G66S to support Arg prototrophy. Each of these genetic alterations should decrease cellular cAMP levels. The resulting signal was transduced through redundant action of the three cAMP-dependent protein kinases, TPK1, TPK2, and TPK3. ras2 had little or no effect on the level of wild-type Arg8p encoded by cox2::ARG8m, but did increase Arg8p activity, as judged by growth phenotype. ras2 also caused increased fluorescence in cells carrying the synthetic cox3::GFPm reporter in mtDNA, but had little effect on the steady-state level of GFP polypeptide detected immunologically. Thus, decreased cAMP levels did not affect the synthesis of mitochondrially coded protein reporters in glucose-grown cells, but rather elevated activities in the matrix that promote efficient folding. Furthermore, we show that when Arg8p is synthesized in the cytoplasm and imported into mitochondria, it has greater activity than when it is synthesized in the matrix. Thus, mitochondrially synthesized proteins may not have the same access to matrix chaperones as cytoplasmically synthesized proteins emerging from the import apparatus.

The RAS2(val19) allele, which renders the cAMP-PKA pathway constitutively active and decreases the replicative life-span of yeast cells, is demonstrated to increase production of reactive oxygen species (ROS) and to elevate oxidative protein damage. Mitochondrial respiration in the mutant is locked in a non-phosphorylating mode prone to generate ROS but this phenotype is not linked to a constitutively active PKA pathway. In contrast, providing RAS2(val19) cells with the mammalian uncoupling protein UCP1 restores phosphorylating respiration and reduces ROS levels, but does not correct for PKA-dependent defects. Thus, the RAS2(val19) allele acts like a double-edged sword with respect to oxidation management: (i). it diminishes expression of STRE element genes required for oxidative stress defenses in a PKA-dependent fashion, and (ii). it affects endogenous ROS production and the respiratory state in a PKA-independent way. The effect of the oncogenic RAS allele on the replicative life-span is primarily asserted via the PKA-dependent pathway since Pde2p, but not UCP1, overproduction suppressed premature aging of the RAS2(val19) mutant.

The cyclic AMP (cAMP)-signaling pathway regulates cell morphology and plays a crucial role during pathogenic development of the plant-pathogenic fungus Ustilago maydis . Strains lacking components of this signaling pathway, such as the Gα-subunit Gpa3 or the adenylyl cyclase Uac1, are nonpathogenic and grow filamentously. On the other hand, strains exhibiting an activated cAMP pathway due to a dominant-active allele of gpa3 display a glossy colony phenotype and are unable to proliferate in plant tumors. Here we present the identification of sql2 as a suppressor of the glossy colony phenotype of a gpa3 Q206L strain. sql2 encodes a protein with similarity to CDC25-like guanine nucleotide exchange factors, which are known to act on Ras proteins. Overexpression of sql2 leads to filamentous growth that cannot be suppressed by exogenous cAMP, suggesting that Sql2 does not act upstream of Uac1. To gain more insight in signaling processes regulated by Sql2, we isolated two genes encoding Ras proteins. Expression of dominant active alleles of ras1 and ras2 showed that Ras2 induces filamentous growth while Ras1 does not affect cell morphology but elevates pheromone gene expression. These results indicate that Ras1 and Ras2 fulfill different functions in U. maydis . Moreover, observed similarities between the filaments induced by sql2 and ras2 suggest that Sql2 is an activator of Ras2. Interestingly, sql2 deletion mutants are affected in pathogenic development but not in mating, indicating a specific function of sql2 during pathogenesis.

Signal transduction pathways inactivated during periods of starvation are implicated in the regulation of longevity in organisms ranging from yeast to mammals, but the mechanisms responsible for life-span extension are poorly understood. Chronological life-span extension in S. cerevisiae cyr1 and sch9 mutants is mediated by the stress-resistance proteins Msn2/Msn4 and Rim15. Here we show that mitochondrial superoxide dismutase (Sod2) is required for survival extension in yeast. Deletion of SOD2 abolishes life-span extension in sch9Δ mutants and decreases survival in cyr1:mTn mutants. The overexpression of Sods—mitochondrial Sod2 and cytosolic CuZnSod (Sod1)—delays the age-dependent reversible inactivation of mitochondrial aconitase, a superoxide-sensitive enzyme, and extends survival by 30%. Deletion of the RAS2 gene, which functions upstream of CYR1, also doubles the mean life span by a mechanism that requires Msn2/4 and Sod2. These findings link mutations that extend chronological life span in S. cerevisiae to superoxide dismutases and suggest that the induction of other stress-resistance genes regulated by Msn2/4 and Rim15 is required for maximum longevity extension.

We observed that the transcription of Saccharomyces cerevisiae cytoplasmic thiol peroxidase type II (cTPx II) (YDR453C) is regulated in response to various stresses (e.g. oxidative stress, carbon starvation, and heat-shock). It has been suggested that both transcription-activating proteins, Yap1p and Skn7p, regulate the transcription of cTPx II upon exposure to oxidative stress. However, a dramatic loss of transcriptional response to various stresses in yeast mutant strains lacking both Msn2p and Msn4p suggests that the transcription factors act as a principal transcriptional activator. In addition to two Yap1p response elements (YREs), TTACTAA and TTAGTAA, the presence of two stress response elements (STREs) (CCCCT) in the upstream sequence of cTPx II also suggests that Msn2p/Msn4p could control stress-induced expression of cTPx II. Analysis of the transcriptional activity of site-directed mutagenesis of the putative STREs (STRE1 and STRE2) and YREs (TRE1 and YRE2) in terms of the activity of a lacZ reporter gene under control of the cTPx II promoter indicates that STRE2 acts as a principal binding element essential for transactivation of the cTPx II promoter. The transcriptional activity of the cTPx II promoter was exponentially increased after postdiauxic growth. The transcriptional activity of the cTPx II promoter is greatly increased by rapamycin. Deletion of Tor1, Tor2, Ras1, and Ras2 resulted in a considerable induction when compared with their parent strains, suggesting that the transcription of cTPx II is under negative control of the Ras/cAMP and target of rapamycin signaling pathways. Taken together, these results suggest that cTPx II is a target of Msn2p/Msn4p transcription factors under negative control of the Ras-protein kinase A and target of rapamycin signaling pathways. Furthermore, the accumulation of cTPx II upon exposure to oxidative stress and during the postdiauxic shift suggests an important antioxidant role in stationary phase yeast cells.

The RPI1 gene of Saccharomyces cerevisiae was identified initially as a dosage suppressor of the heat shock sensitivity associated with overexpression of RAS2 (J. Kim and S. Powers, Mol. Cell. Biol. 11:3894–3904, 1991). Based on its failure to suppress mutationally activated RAS2 , RPI1 was proposed to be a negative regulator of the Ras/cyclic AMP (cAMP) pathway that functions at a point upstream of Ras. We isolated RPI1 as a high-copy-number suppressor of the cell lysis defect associated with a null mutation in the MPK1 gene, which encodes the mitogen-activated protein kinase of the cell wall integrity-signaling pathway. Although the sequence of Rpi1 is not informative about its function, we present evidence that this protein resides in the nucleus, possesses a transcriptional activation domain, and affects the mRNA levels of several cell wall metabolism genes. In contrast to the previous report, we found that RPI1 overexpression suppresses defects associated with mutational hyperactivation of the Ras/cAMP pathway at all points including constitutive mutations in the cAMP-dependent protein kinase. We present additional genetic and biochemical evidence that Rpi1 functions independently of and in opposition to the Ras/cAMP pathway to promote preparations for the stationary phase. Among these preparations is a fortification of the cell wall that is antagonized by Ras pathway activity. This observation reveals a novel link between the Ras/cAMP pathway and cell wall integrity. Finally, we propose that inappropriate expression of RPI1 during log phase growth drives fortification of the cell wall and that this behavior is responsible for suppression of the mpk1 cell lysis defect.

Recent studies have revealed the role of actin dynamics in the regulation of yeast aging. Although the target of rapamycin (TOR) complex, serine/threonine kinase Sch9, and Ras2 have been shown to play important roles in aging for a long time, the relationship between these regulators and actin has not yet been reported. In this study we investigated the roles of actin polarization in tor1Δ, sch9Δ, and ras2Δ mutant cells. We found that the actin structures in tor1Δ, sch9Δ, and ras2Δ mutant cells were more dynamic than those in the wild type. Destruction of the actin structures with jasplakinolide decreased the life span of tor1Δ, sch9Δ, and ras2Δ mutants. Furthermore, deletion of SLA1 in tor1Δ, sch9Δ, and ras2Δ mutants inhibited the actin dynamics and life span. In addition, we found that the actin cytoskeleton of the long-lived mutant sch9Δ, depended on the transcription factors RIM15 and MSN2/4, but not GIS1, while the actin skeleton of the tor1Δ and ras2Δ mutants depended on RIM15 as expected. Our data suggest that the longevity of tor1Δ, sch9Δ, and ras2Δ mutants is dependent on actin dynamics.

Upon starvation for glucose or any other core nutrient, yeast cells exit from the mitotic cell cycle and acquire a set of G0-specific characteristics to ensure long-term survival. It is not well understood whether or how cell cycle progression is coordinated with the acquisition of different G0-related features during the transition to stationary phase (SP). Here, we identify the yeast GSK-3 homologue Mck1 as a key regulator of G0 entry and reveal that Mck1 acts in parallel to Rim15 to activate starvation-induced gene expression, the acquisition of stress resistance, the accumulation of storage carbohydrates, the ability of early SP cells to exit from quiescence, and their chronological lifespan. FACS and microscopy imaging analyses indicate that Mck1 promotes mother-daughter cell separation and together with Rim15, modulates cell size. This indicates that the two kinases coordinate the transition-phase cell cycle, cell size and the acquisition of different G0-specific features. Epistasis experiments place MCK1, like RIM15, downstream of RAS2 in antagonising cell growth and activating stress resistance and glycogen accumulation. Remarkably, in the ras2∆ cells, deletion of MCK1 and RIM15 together, compared to removal of either of them alone, compromises respiratory growth and enhances heat tolerance and glycogen accumulation. Our data indicate that the nutrient sensor Ras2 may prevent the acquisition of G0-specific features via at least two pathways. One involves the negative regulation of the effectors of G0 entry such as Mck1 and Rim15, while the other likely to involve its functions in promoting respiratory growth, a phenotype also contributed by Mck1 and Rim15., Author Summary The vast majority of eukaryotic cells exist in a non-proliferating state known as G0. However, how cells transit into, and survive during, the G0 state is poorly understood. Dysregulation of the G0 state leads to age-related diseases such as Alzheimer’s or cancers. We have revealed that the yeast Mck1 and Rim15 kinases, which function downstream of the PKA and/or TOR signaling pathways, coordinate cell cycle progression, cell size homeostasis, and the acquisition of a variety of G0-specific characteristics during the transition into stationary phase. Failure of this coordination compromises the ability of early stationary-phase cells to exit from quiescence and severely shortens their chronological lifespan. Further genetic analyses suggest that the nutrient sensor Ras2 may antagonize G0 entry via at least two pathways, one through the negative regulation of the G0-specific effectors (Mck1 and Rim15) and the other possibly involving its functions in promoting respiratory growth, a phenotype also intricately modulated by Mck1 and Rim15. As Ras2 and Rim15 have homolog in both insects and/or mammals, the identification of the GSK-3 homologue Mck1 and the characterisation of its relationship with Rim15 and Ras2 in G0 entry could provide important clues to the regulation of these processes in more complex organisms.

The yeast Saccharomyces cerevisiae undergoes a dramatic growth transition from its unicellular form to a filamentous state, marked by the formation of pseudohyphal filaments of elongated and connected cells. Yeast pseudohyphal growth is regulated by signaling pathways responsive to reductions in the availability of nitrogen and glucose, but the molecular link between pseudohyphal filamentation and glucose signaling is not fully understood. Here, we identify the glucose-responsive Sks1p kinase as a signaling protein required for pseudohyphal growth induced by nitrogen limitation and coupled nitrogen/glucose limitation. To identify the Sks1p signaling network, we applied mass spectrometry-based quantitative phosphoproteomics, profiling over 900 phosphosites for phosphorylation changes dependent upon Sks1p kinase activity. From this analysis, we report a set of novel phosphorylation sites and highlight Sks1p-dependent phosphorylation in Bud6p, Itr1p, Lrg1p, Npr3p, and Pda1p. In particular, we analyzed the Y309 and S313 phosphosites in the pyruvate dehydrogenase subunit Pda1p; these residues are required for pseudohyphal growth, and Y309A mutants exhibit phenotypes indicative of impaired aerobic respiration and decreased mitochondrial number. Epistasis studies place SKS1 downstream of the G-protein coupled receptor GPR1 and the G-protein RAS2 but upstream of or at the level of cAMP-dependent PKA. The pseudohyphal growth and glucose signaling transcription factors Flo8p, Mss11p, and Rgt1p are required to achieve wild-type SKS1 transcript levels. SKS1 is conserved, and deletion of the SKS1 ortholog SHA3 in the pathogenic fungus Candida albicans results in abnormal colony morphology. Collectively, these results identify Sks1p as an important regulator of filamentation and glucose signaling, with additional relevance towards understanding stress-responsive signaling in C. albicans., Author Summary Eukaryotic cells respond to nutritional and environmental stress through complex regulatory programs controlling cell metabolism, growth, and morphology. In the budding yeast Saccharomyces cerevisiae, conditions of limited nitrogen and/or glucose can initiate a dramatic growth transition wherein the yeast cells form extended multicellular filaments resembling the true hyphal tubes of filamentous fungi. The formation of these pseudohyphal filaments is governed by core regulatory pathways that have been studied for decades; however, the mechanism by which these signaling systems are integrated is less well understood. We find that the protein kinase Sks1p contributes to the integration of signals for nitrogen and/or glucose limitation, resulting in pseudohyphal growth. We implemented a mass spectrometry-based approach to profile phosphorylation events across the proteome dependent upon Sks1p kinase activity and identified phosphorylation sites important for mitochondrial function and pseudohyphal growth. Our studies place Sks1p in the regulatory context of a well-known pseudohyphal growth signaling pathway. We further find that SKS1 is conserved and required for stress-responsive colony morphology in the principal opportunistic human fungal pathogen Candida albicans. Thus, Sks1p is part of the mechanism integrating glucose-responsive cell signaling and pseudohyphal growth, and its function is required for colony morphology linked with virulence in C. albicans.

Data in literature suggest that budding yeast adenylate cyclase forms a membrane-associated complex with the upstream components of the cAMP/PKA pathway. Here we provide evidences that adenylate cyclase (Cyr1p) acts as a scaffold protein keeping Ras2 available for its regulatory factors. We show that in a strain with deletion of the CYR1 gene (cyr1Δ pde2Δ msn2Δ msn4Δ) the basal Ras2-GTP level is very high and this is independent on the lack of feedback inhibition that could result from the absence of adenylate cyclase activity. Moreover, strains effected either in the intrinsic adenylate cyclase activity (fil1 strain) or in the stimulation of adenylate cyclase activity by active G-proteins (lcr1 strain) had a normal basal and glucose-induced Ras2-GTP level, indicating that adenylate cyclase activity does not influence the Ras2 activation state and suggesting that Cyr1 protein is required for the proper interaction between Ras2 and the Ira proteins. We also provide evidence that the two Ras-binding sites mapped on Cyr1p are required for the signalling complex assembly. In fact, we show that the cyr1Δ strain expressing CYR1 alleles lacking either the LRR region or the C-terminal domain still have a high basal and glucose-induced Ras2-GTP level. In contrast, a mutant expressing a Cyr1 protein only missing the N-terminal domain showed a normal Ras2 activation pattern. Likewise, the Ras2-GTP levels are comparable in the wild type strain and the srv2Δ strain, supporting the hypothesis that Cap is not essential for the Ras-adenylate cyclase interaction.

The CWI pathway cross-talks with TOR and RAS in both the oxidative and glucose starvation responses. Mtl1 is the cell-wall protein in charge of sensing and regulating this response. Rom2 and Rho1, which are the upper elements in the pathway, mediate this signal. Several outputs are involved and required for this response, one of which, ribosomal gene expression, seems to be regulated by Sfp1, amongst other possible transcription factors. Moreover, cross-talk also occurs in a reverse flow from TOR and RAS to the CWI pathway. Thus Tor1 and Ras2 inhibition also activates Slt2 in the absence of the Mtl1 protein and assures the proper adaptive response to oxidation and glucose deprivation.

In budding yeast, the Ras/cAMP pathway is involved in the coordination of cell growth and cell division. Glucose-rich medium stimulates Ras/cAMP signaling, which causes an increase in the critical cell size for cell cycle entry. Here we show that glucose and activated Ras proteins also influence the function of the anaphase-promoting complex (APC/C), a ubiquitin-protein ligase required for sister chromatid separation and mitotic exit. We found that apc10-22 and other mutants defective in the APC/C are suppressed by reduced Ras signaling activity, by a deletion of the RAS2 gene, by a cdc25 mutation, by elevated levels of PDE2, or by growth without glucose. Viability of these mutants is also enhanced by decreased Cdk1 activity. In contrast, a constitutively activated RAS2Val19 allele or shifts to glucose medium are deleterious to apc10-22 mutants. Remarkably, cdc34-2 mutants, which are impaired in SCF function, are differently affected with respect to Ras activity. Viability of cdc34-2 mutants at elevated temperatures is dependent on glucose and the RAS2 gene. We conclude that glucose and Ras proteins influence the APC/C and the SCF complex in an opposite manner. These ubiquitin ligases might represent novel targets for modulating cell division in response to growth conditions.

The cAMP-protein kinase A (PKA) pathway in the yeast Saccharomyces cerevisiae plays a major role in the control of metabolism, proliferation and stress resistance. Derepressed cells show a rapid increase in the cAMP level (within 1 min) after addition of glucose or after intracellular acidification. A specific mutation in adenylate cyclase, the enzyme that catalyzes the synthesis in cAMP, largely prevents both cAMP responses. The responsible mutation was originally called lcr1 (for lack of cAMP responses); lcr1 was later identified as allelic with CYR1/CDC35. The mutation was introduced into the CYR1 gene of a W303-1A wild type strain, which resulted in a large decrease in cAMP signalling. Furthermore, there was a strong reduction in GTP/Mg2+-stimulated but not in Mn2+-stimulated adenylate cyclase activity in isolated plasma membranes, which is consistent with the absence of signalling through adenylate cyclase in vivo. Glucose-induced activation of trehalase was reduced and mobilization of trehalose and glycogen and loss of stress resistance were delayed in the lcr1 mutant. Because of the absence of cAMP signalling during exponential growth on glucose, it was concluded that glucose-induced cAMP signalling is restricted to the transition from gluconeogenic/respiratory to fermentative growth. Activation of the PKA pathway is mediated by a G protein (either Ras1/Ras2 or Gpa2). Constitutive activation of the pathway by Ras2(val19) or Gpa2(val132) has a negative effect on glycogen and trehalose accumulation and heat shock survival. The lcr1 mutation partially suppresses this effect indicating that the target sites of the two G-proteins on adenylate cyclase might have at least a part in common. Copyright (C) 2000 Elsevier Science B.V.

The hypoxic SRP1/TIR1 gene encodes a stress-response cell wall mannoprotein, which is shown to be necessary for yeast growth at acidic pH in the presence of sodium dodecyl sulfate. However, the hypoxic expression of SRP1 is shown to be downregulated at acidic pH. The stress-responsive HOG pathway appeared necessary to maintain hypoxic SRP1 expression, but only at acidic pH. However, unlike known HOG pathway-dependent genes, SRP1 was under positive cAMP control and was positively modulated by protein kinase A at neutral and acidic pH. In addition, the HOG-independent hypoxic HEM13 gene was also positively regulated by cAMP levels. Therefore, the positive cAMP control of the hypoxic SRP1 and HEM13 genes was uncoupled from the HOG pathway. Surprisingly, this positive cAMP control was found to be mediated by GPA2 but not by RAS2, so the Gpa2p requirement appears critical at acidic pH. Although RAS2 is not involved in the regulation of SRP1 expression, the guanine nucleotide exchange factor Cdc25, which is known to control the GTP/GDP ratio on the Ras proteins, was nevertheless required for hypoxic SRP1 expression. Furthermore, the Ras proteins did not compensate for Gpa2p requirement in a delta gpa2 mutated strain. These results suggest that the Cdc25 factor might also control Gpa2p.

Ras proteins are small guanine nucleotide-binding proteins that cycle between the active GTP-bound and the inactive GDP-bound states. They are conserved from yeasts to mammals and play pivotal signaling roles in the regulation of cell growth and differentiation. Ras undergoes posttranslational lipid modifications, which are crucial for its membrane anchoring as well as for biological functions, at its C terminus (7, 8). In mammals, a serine/threonine kinase Raf-1 and its isoforms B-Raf and A-Raf are major effectors of Ras (9). In addition, recent searches have identified a number of mammalian Ras effectors and effector candidates, such as Ral guanine nucleotide dissociation stimulator (RalGDS), phosphoinositide 3-kinase, and protein kinase Cζ, all of which associate directly with the GTP-bound Ras (6, 26). However, the molecular mechanism of effector regulation by Ras is still unclear. It is established that the association with the posttranslationally modified Ras induces translocation of Raf-1 to the plasma membrane, where it is activated by membrane-bound factors (30, 44). The membrane recruitment role of Ras is also implicated in activation of RalGDS and phosphoinositide 3-kinase (33, 40). However, several recent studies have indicated that the mechanism of Raf activation by association with Ras seems to involve a number of complex processes in addition to the simple membrane recruitment (21, 36, 42, 46). In any of these cases, unavailability of in vitro systems, which could reconstitute the Ras-dependent effector regulation with the purified components only, hampered full elucidation of the Ras function other than the membrane recruitment. On the other hand, in the budding yeast Saccharomyces cerevisiae, it has been established that adenylyl cyclase (CYR1) is a major downstream effector of RAS1 and RAS2, which are structural, functional, and biochemical homologues of mammalian Ras (3, 17). The Ras-CYR1 pathway has been implicated in transduction of a glucose-triggered signal to an intracellular environment where a protein phosphorylation cascade is initiated by cyclic AMP (cAMP). Since the Ras-dependent CYR1 activation could be reconstituted in vitro with the purified components only, the Ras-CYR1 system provided a unique opportunity to examine molecular mechanisms underlying Ras-dependent regulation of effector activities. CYR1 consists of 2,026-amino-acid residues that comprise at least four domains: the N-terminal, middle repetitive, catalytic, and C-terminal domains (23, 52). The middle repetitive domain is composed of a repetition of 23-amino-acid amphipathic leucine-rich motifs that have homology to the leucine-rich repeat (LRR) family of proteins (27). Genetic and biochemical studies demonstrated that the LRR domain contains a binding site for the GTP-bound Ras (35, 43, 49). Mammalian Ras can substitute for yeast RAS to activate CYR1 (4, 24). CYR1 forms a complex with the 60-kDa adenylyl cyclase-associated protein (CAP) (11, 12). CAP is a bifunctional protein: its N-terminal region binds to the C-terminal region of CYR1, and this association appears to be required for proper in vivo response to Ras, while its C-terminal region binds actin monomer and is somehow involved in the regulation of the actin cytoskeleton (13, 14, 16, 50). These two functions appear to be separable from each other (14). Although the mechanism of regulation of the Ras-CYR1 pathway by CAP was unknown, we have recently found a possible link between CAP and the CYR1 activation by the modified Ras. By using an in vitro reconstituted system, we previously demonstrated that the posttranslational modification, in particular farnesylation, of Ras is required for efficient activation of CYR1 by Ras (28). However, we next observed that the posttranslational modification of Ras did not affect the association of Ras with the LRR domain of CYR1 and that the association with the CAP N-terminal region is essential for the efficient activation of CYR1 by the modified Ras (43). The effect of CAP was successfully reconstituted in vitro by the purified components only. These findings suggested that CAP might mediate the stimulatory effect of the modified Ras on CYR1 activation and reminded us of our past observation on mammalian Raf-1. We and others identified the cysteine-rich domain (CRD) of Raf-1 as another Ras-binding site than the primary binding site, the Ras-binding domain (5, 15, 21). The association of Ras with CRD is dependent on the posttranslational modification of Ras (21, 31) and is required for the efficient in vivo activation of Raf-1 by Ras (10, 21, 31, 38, 46). This led us to hypothesize that the associated CAP might constitute a Ras-binding site of CYR1, which mediates the CYR1 activation, other than the primary Ras-binding site LRR domain. Here we report that in fact CAP in complex with the CYR1 C-terminal region is capable of direct association with the posttranslationally modified Ras. By isolating mutants of CAP which are defective in association with Ras, we provide genetic evidence for the importance of this binding in the Ras-dependent CYR1 activation both in vivo and in vitro. Further, evidence is presented for the importance of the strength of this binding in the CYR1 activation.

In Saccharomyces cerevisiae pseudohyphae formation may be triggered by nitrogen deprivation and is stimulated by cAMP. It was observed that even in a medium with an adequate nitrogen supply, cAMP can induce pseudohyphal growth when S. cerevisiae uses ethanol as carbon source. This led us to investigate the effects of the carbon source and of a variety of stresses on yeast morphology. Pseudohyphae formation and invasive growth were observed in a rich medium (YP) with poor carbon sources such as lactate or ethanol. External cAMP was required for the morphogenetic transition in one genetic background, but was dispensable in strain Σ1278b which has been shown to have an overactive Ras2/cAMP pathway. Pseudohyphal growth and invasiveness also took place in YPD plates when the yeast was subjected to different stresses: a mild heat-stress (37 °C), an osmotic stress (1 m NACl), or addition of compounds which affect the lipid bilayer organization of the cell membrane (aliphatic alcohols at 2%) or alter the glucan structure of the cell wall (Congo red). We conclude that pseudohyphal growth is a physiological response not only to starvation but also to a stressful environment; it appears to require the coordinate action of a MAP kinase cascade and a cAMP-dependent pathway.

The number of revertants with restored ability to form colony increases in a time-dependent manner during long-term selective starvation of dense mutant microbial cultures. This is due to starvation-associated (also called adaptive) mutations that arise in a replication independent manner. Here we report that in Saccharomyces cerevisiae the frequency of starvation-associated reversions of mutant genes whose products are necessary for amino acids biosynthesis are influenced by Ras2/cAMP signaling pathway. This signaling pathway is a yeast general regulatory pathway involved in nutritional sensing, UV response, sporulation control and life span control and its changes are manifested in both, cell cycle and life cycle. Inactivation of the RAS2 gene causes an increase in number of starvation-associated revertants in comparison to an isogenic wild type strain and a strain with constitutively activated Ras2/cAMP signaling pathway. Therefore, we suggest that starvation-associated mutagenesis is different from spontaneous mutagenesis and is related to the cellular capacity to adopt distinct physiological states in response to environmental signals.

Haploid yeast cells are capable of invading agar when grown on rich media. Cells of the Sigma1278b genetic background manifest this property, whereas other laboratory strains are incapable of invasive growth. We show that disruption of the RAS2 gene in the Sigma1278b background significantly reduces invasive growth but that expression of a constitutively active Ras2p (Ras2(Val19)p) in this strain has a minimal effect on its invasiveness. On the other hand, expression of Ras2(Val19)p in another laboratory strain, SP1, rendered it invasive. These results suggest that a hyperactive Ras2 pathway induces invasive growth and that this pathway might be overactive in the Sigma1278b genetic background. Indeed, cells of the Sigma1278b are defective in the induction of stress-responsive genes, while their Gcn4 target genes are constitutively transcribed. This pattern of gene expression was previously shown to be associated with an active Ras/cyclic AMP (cAMP) pathway. We show that suppression of stress-related genes in Sigma1278b cells is a result of their inability to activate transcription through the stress response element (STRE). Disruption of RAS2, which abolished invasiveness, induced an increase in STRE activity. Further, in the SP1 genetic background, disruption of either the MSN2/4 genes (encoding activators of STRE) or the yAP-1 gene was sufficient to restore invasive growth in ras2Delta cells. We conclude that Ras2-mediated suppression of the stress response is sufficient to induce invasiveness. Accordingly, the fact that the stress response is suppressed in Sigma1278b background explains its invasiveness. It seems that invasiveness is a phenotype related to unregulated growth and is therefore manifested by cells harboring an overactive Ras/cAMP cascade. In this respect, invasiveness in yeast is reminiscent of the property of ras-transformed fibroblasts to invade soft agar.

A novel gene designated moc2, which encodes a putative RNA helicase, was isolated from Schizosaccharomyces pombe on the basis of its suppression of the sterility of two different mutant strains, one of which had elevated levels of cAMP and the other deregulated Ras functioning as a result of an ectopic expression of dominant negative RAS2. Moc2 is highly homologous to the RNA helicase DED1 of Saccharomyces cerevisiae (58% identity) and PL10 of mouse (50% identity). Disruption of the moc2 gene indicated that moc2 is essential for cell growth. The moc2 gene seems to have roles in both sexual differentiation and cell growth.

In response to nitrogen limitation, diploid cells of Saccharomyces cerevisiae undergo a dimorphic transition to a filamentous growth form referred to as pseudohyphal differentiation (14, 19). This filamentous growth form represents a dramatic change in the cellular program in which the cells elongate, adopt a unipolar budding pattern, remain physically connected in chains, and invade the agar (14, 23). This alternative growth form may enable this nonmotile species to forage for nutrients under adverse conditions. Two signaling pathways that regulate yeast filamentous growth have been defined. The first involves components of the mitogen-activated protein (MAP) kinase pathway that also functions during mating in haploid cells (8, 28, 35). These components include the kinases Ste20, Ste11, Ste7, and Kss1 and the transcription factor Ste12. In addition, the transcription factor Tec1 forms heterodimers with Ste12 that regulate expression of Tec1 itself and additional targets, such as the cell surface flocculin Flo11 required for invasive and filamentous growth (3, 11, 31, 34). Early elements of the pheromone response pathway, including the pheromones, their receptors, and the coupled heterotrimeric G protein, are not expressed in diploid cells and are not required for filamentous differentiation (28). Instead, the MAP kinase pathway is activated by Cdc42, Ras2, and the 14-3-3 proteins Bmh1 and Bmh2 (38, 39, 49), possibly in response to the Sho1 osmosensing receptor (46). A second signaling pathway functions in parallel with the MAP kinase pathway to regulate pseudohyphal differentiation. This pathway involves a novel G protein-coupled receptor, Gpr1, which is required for both pseudohyphal differentiation (33) and, in conjunction with Ras2, vegetative growth (63). The Gpr1 ligand has not yet been identified. The Gpr1 receptor is coupled to a heterotrimeric G protein α subunit, Gpa2, which is also required for pseudohyphal differentiation and plays a role in nutrient sensing (26, 32). Early studies suggested Gpa2 might stimulate cyclic AMP (cAMP) production by adenylyl cyclase (41). Consistent with this, cAMP stimulates pseudohyphal differentiation and suppresses the filamentation defects of gpr1 and gpa2 mutant strains (26, 32, 33). A recent study has confirmed that Gpa2 regulates cAMP production by adenylyl cyclase in response to nutritional signals (7). Dominant activated Gpa2 mutants or cAMP suppresses the pseudohyphal defect of mutant strains lacking MAP kinase cascade components (32). In summary, a second signaling pathway comprised of the Gpr1 receptor, the Gpa2 Gα protein, and cAMP regulates pseudohyphal growth in parallel to and independently from the MAP kinase pathway. The target of cAMP in yeast is the cAMP-dependent protein kinase, protein kinase A (PKA). The yeast PKA kinase is similar to mammalian PKA and consists of a regulatory subunit encoded by a single gene, BCY1, and three catalytic subunits encoded by the TPK1, TPK2, and TPK3 genes (6, 57, 58). In both yeast and mammals, PKA in resting cells is an inactive tetramer composed of two regulatory subunits bound to two active subunits. In response to external signals that increase intracellular cAMP levels, cAMP binds to the regulatory subunit and triggers conformational changes that release the active catalytic subunits. Hydrolysis of cAMP by cAMP phosphodiesterases, the products of the PDE1 and PDE2 genes in yeast, restores PKA to the resting, inactive state (43, 54). The yeast cAMP-dependent protein kinase is required for vegetative growth (58). Triple mutants lacking the Tpk1, Tpk2, and Tpk3 catalytic subunits are inviable, whereas mutant strains expressing any one of the three Tpk subunits are all viable. These findings led to the model that the three PKA catalytic subunits are largely redundant for function. The PKA catalytic subunits share a conserved C-terminal kinase domain attached to unique N-terminal regions. Tpk1 and Tpk3 share 88% identity in the kinase domain, whereas Tpk2 is more divergent (77 and 75% identity with Tpk1 and -3, respectively). Several candidate PKA targets for vegetative growth have recently been identified. For example, the Msn2 and Msn4 transcription factors are regulated by PKA and repress expression of genes that regulate vegetative growth (4, 18, 56). The Rim15 protein kinase is also phosphorylated and inhibited by PKA and regulates entry into meiosis and stationary phase (48, 60). In parallel, studies of PKA constitutively activated by cAMP, bcy1 mutation, or activated Ras2 revealed roles in regulating stationary phase, meiosis, and sporulation (5, 59). Activation of PKA prevents glycogen accumulation, heat shock resistance, and survival during nutrient limitation, all hallmarks of entry into stationary phase. Similarly, activation of PKA inhibits sporulation. Thus, activation of PKA promotes vegetative growth in response to nutrients, whereas inactivation of PKA in response to nutrient limitation regulates sporulation and entry into stationary phase. Several observations suggest PKA might also regulate yeast pseudohyphal differentiation. The dominant active Ras2val19 mutant protein enhances filamentous growth (14), whereas overexpression of the cAMP phosphodiesterase Pde2 inhibits filamentation (62). In addition, exogenous cAMP enhances filamentous growth (32). Here we report that the cAMP-dependent protein kinase regulates yeast pseudohyphal differentiation. First, we show that mutation of the PKA regulatory subunit Bcy1 enhances filamentous growth. Second, we demonstrate that the PKA catalytic subunits play distinct roles in regulating filamentous growth: the Tpk2 subunit activates filamentous growth, whereas the Tpk1 and Tpk3 subunits primarily inhibit filamentous growth. The unique activating function of the Tpk2 subunit is linked to structural differences in the catalytic region of the kinase and not to differences in gene regulation or the unique amino-terminal region of the protein. Genetic epistasis experiments support a model in which Tpk2 functions downstream of the Gpr1 receptor and the Gα protein Gpa2. Importantly, activation of PKA by mutation of the Bcy1 regulatory subunit restores pseudohyphal growth in mutants lacking elements of the MAP kinase pathway, including ste12, tec1, and ste12 tec1 mutant strains. Thus, the MAP kinase and PKA pathways independently regulate filamentous growth. Further analysis reveals that the PKA pathway regulates the switch to unipolar budding and invasion, whereas the MAP kinase pathway is required for cell elongation and invasion. Finally, our studies define a role for the PKA pathway in activating pseudohyphal growth via transcriptional regulation of the cell surface flocculin Flo11 by the Flo8 transcription factor, and both Flo11 and Flo8 were previously shown to be required for pseudohyphal growth (27, 29, 31). Taken together, our studies reveal an intimate role for the cAMP-dependent kinase in the regulation of yeast dimorphism and suggest this role has been evolutionarily conserved in diverse yeast species and fungi, including pathogens of both plants and animals.

The two highly conserved RAS genes of the budding yeast Saccharomyces cerevisiae are redundant for viability. Here we show that haploid invasive growth development depends on RAS2 but not RAS1. Ras1p is not sufficiently expressed to induce invasive growth. Ras2p activates invasive growth using either of two downstream signaling pathways, the filamentation MAPK (Cdc42p/Ste20p/MAPK) cascade or the cAMP-dependent protein kinase (Cyr1p/cAMP/PKA) pathway. This signal branch point can be uncoupled in cells expressing Ras2p mutant proteins that carry amino acid substitutions in the adenylyl cyclase interaction domain and therefore activate invasive growth solely dependent on the MAPK cascade. Both Ras2p-controlled signaling pathways stimulate expression of the filamentation response element-driven reporter gene depending on the transcription factors Ste12p and Tec1p, indicating a crosstalk between the MAPK and the cAMP signaling pathways in haploid cells during invasive growth.

Replicative capacity, which is the number of times an individual cell divides, is the measure of longevity in the yeast Saccharomyces cerevisiae. In this study, a process that involves signaling from the mitochondrion to the nucleus, called retrograde regulation, is shown to determine yeast longevity, and its induction resulted in postponed senescence. Activation of retrograde regulation, by genetic and environmental means, correlated with increased replicative capacity in four different S. cerevisiae strains. Deletion of a gene required for the retrograde response, RTG2, eliminated the increased replicative capacity. RAS2, a gene previously shown to influence longevity in yeast, interacts with retrograde regulation in setting yeast longevity. The molecular mechanism of aging elucidated here parallels the results of genetic studies of aging in nematodes and fruit flies, as well as the caloric restriction paradigm in mammals, and it underscores the importance of metabolic regulation in aging, suggesting a general applicability.

Ras proteins are conserved from yeasts to mammals and implicated in regulation of the actin cytoskeleton. The flightless-1 (fli-1) gene of Drosophila melanogaster and its homologs in Caenorhabditis elegans and humans encode proteins (FLI-1) comprising a fusion of a leucine-rich repeats (LRRs) domain and a gelsolin-like domain. This LRRs domain is highly homologous to those of three proteins involved in Ras-mediated signaling; Saccharomyces cerevisiae adenylyl cyclase, C. elegans SUR-8, and mammalian RSP-1. Here we report that the LRRs domain of C. elegans FLI-1 (Ce-FLI-1) associates directly with Ras (Kd = 11 nM) and, when overexpressed, suppresses the heat shock sensitive phenotype of yeast cells bearing the activated RAS2 gene (RAS2(Val-19)). Further, the gelsolin-like domain of Ce-FLI-1 is shown to possess a Ca2+-independent G-actin-binding activity as well as F-actin-binding and -severing activities. FLI-1 may be involved in regulation of the actin cytoskeleton through Ras.

The 2818 amino acids of neurofibromin, the product of the human NF1 gene, include a 230 amino acid Ras-GAP related domain (GRD). Functions which may be associated with the rest of the protein remain unknown. However, many NF1 mutations in neurofibromatosis 1 patients are found downstream of the GRD, suggesting that the C-terminal region of the protein is also functionally important. Since the C-terminal region of neurofibromin encompassing these mutations is homologous with the corresponding regions in the two Saccharomyces cerevisiae Ras-GAPs, Ira1p and Ira2p, we chose yeast as a model system for functional exploration of this region (Ira-C region). Three missense mutations that affect the Ira-C region of NF1 were used as a model for the mutagenesis of IRA1. The yeast phenotypes of heat shock sensitivity, iodine staining, sporulation efficiency, pseudohyphae formation, and GAP activity were scored. Even though none of the mutations directly affected the Ira1p-GRD, mutations at two of the three sites resulted in a decrease in the GAP activity present in ira1 cells. The third mutation appeared to disassociate the phenotypes of sporulation ability and GAP activity. This and other evidence suggest an effector function for Ira1p.

The genetic analysis of aging in the yeast Saccharomyces cerevisiae has revealed the importance of metabolic capacity, resistance to stress, integrity of gene regulation, and genetic stability for longevity. A balance between these life maintenance processes is sustained by the RAS2 gene, which channels cellular resources among them. This gene cooperates with mitochondria and PHB1 in metabolic adjustments important for longevity. It also modulates stress responses. Transcriptional silencing of heterochromatic regions of the genome is lost during aging, suggesting that gene dysregulation accompanies the aging process. There is evidence that this age change plays a causal role. Aging possesses features of a nonlinear process, and it is likely that application of nonlinear system methodology to aging will be productive.

The yeast Saccharomyces cerevisiae has a limited life span that can be measured by the number of times individual cells divide. Several genetic manipulations have been shown to prolong the yeast life span. However, environmental effects that extend longevity have been largely ignored. We have found that mild, nonlethal heat stress extended yeast life span when it was administered transiently early in life. The increased longevity was due to a reduction in the mortality rate that persisted over many cell divisions (generations) but was not permanent. The genes RAS1 and RAS2 were necessary to observe this effect of heat stress. The RAS2 gene is consistently required for maintenance of life span when heat stress is chronic or in its extension when heat stress is transient or absent altogether. RAS1, on the other hand, appears to have a role in signaling life extension induced by transient, mild heat stress, which is distinct from its life-span-curtailing effect in the absence of stress and its lack of involvement in the response to chronic heat stress. This distinction between the RAS genes may be partially related to their different effects on growth-promoting genes and stress-responsive genes. The ras2 mutation clearly hindered resumption of growth and recovery from stress, while the ras1 mutation did not. The HSP104 gene, which is largely responsible for induced thermotolerance in yeast, was necessary for life extension induced by transient heat stress. An interaction between mitochondrial petite mutations and heat stress was found, suggesting that mitochondria may be necessary for life extension by transient heat stress. The results raise the possibility that the RAS genes and mitochondria may play a role in the epigenetic inheritance of reduced mortality rate afforded by transient, mild heat stress.

The PKC1–MPK1 pathway in yeast functions in the maintenance of cell wall integrity and in the stress response. We have identified a family of genes that are putative regulators of this pathway. WSC1 , WSC2 , and WSC3 encode predicted integral membrane proteins with a conserved cysteine motif and a WSC1–green fluorescence protein fusion protein localizes to the plasma membrane. Deletion of WSC results in phenotypes similar to mutants in the PKC1–MPK1 pathway and an increase in the activity of MPK1 upon a mild heat treatment is impaired in a wsc Δ mutant. Genetic analysis places the function of WSC upstream of PKC1 , suggesting that they play a role in its activation. We also find a genetic interaction between WSC and the RAS–cAMP pathway. The RAS–cAMP pathway is required for cell cycle progression and for the heat shock response. Overexpression of WSC suppresses the heat shock sensitivity of a strain in which RAS is hyperactivated and the heat shock sensitivity of a wsc Δ strain is rescued by deletion of RAS2 . The functional characteristics and cellular localization of WSC suggest that they may mediate intracellular responses to environmental stress in yeast.

Many fungi undergo a morphological transition to filamentous growth in response to limiting nutrient conditions. Constitutively elongated Saccharomyces cerevisiae mutants (elm) have been isolated; the ELM1 gene encodes a putative serine/threonine protein kinase. A novel allele, elm1-15, has been isolated in an S288C-derived strain, which causes a pleiotropic phenotype, including media-specific growth effects, abnormal morphology and altered stress response, in cells that are auxotrophic for tryptophan. elm1-15 trp1 cells cannot use many nitrogen sources, are sensitive to amino acid analogues, have very low general amino acid permease activity and do not accumulate trehalose. In contrast, haploid elm1-15 TRP1 cells grow well in budding form on all media, are stress resistant and overaccumulate trehalose. Several lines of evidence suggest that Elm1 acts on functions related to the RAS/cAMP pathway. Overexpression of Elm1 partially rescues the ts phenotype of cdc25 and cyr1 mutants. Deletion of ELM1 in low PKA activity mutants increased the severity of their phenotypes, and activation of Ras2 decreases the cell elongation phenotype of elm1 mutants. A 'signal integration' model for the complex relationship of Elm1 and the RAS/cAMP pathway in controlling morphogenesis in response to nutrients is proposed.

Les auteurs ont examine les consequences phenotypiques chez le Saccharomyces cerevisiae d'un allele de dislocation (ras2::LEU2) et d'une forme mutante dominante (RAS2 ala18.val19 ) du RAS2. En plus des phenotypes decrits plus tot pour ces mutants, les auteurs ont observe une petite augmentation de la duree de vie pour l'allele de dislocation et une diminution drastique de la duree de vie pour la forme mutante dominante, comparativement au type sauvage isogenique. Ceci a ete constate en analysant ces alleles dans deux ensembles genetiques differents avec a peu pres les memes resultats. Les durees de vie ont ete determinees en manipulant les cellules meres et en comptant les generations jusqu'a l'arret des divisions cellulaires. Les auteurs ont effectue une analyse morphologique des phenotypes terminaux sur de tres vieilles cellules meres qui a montre des cellules agrandies et arrondies et dans certains cas des bourgeons allonges, dont certains etaient difficiles a separer de la cellule mere. Ceci a ete observe dans les cellules de type sauvage, aussi bien que chez les cellules mutantes. Cependant, le mutant dominant RAS2 (mais pas le type sauvage ou les cellules mutantes ras2::LEU2), apres 2 jours sur des milieux complexes, montrent des phenotypes similaires au phenotype terminal des vieilles cellules meres. Une importante proportion des cellules sont agrandies et generent des bourgeons allonges, perdent la coloration des cicatrices des bourgeons au Calcofluor, la surface cellulaire semble ridee, le cytosquelette d'actine est aberrant, et les fuseaux mitotiques ainsi que les microtubules cytoplasmiques sont defectueux quant a leur orientation propre, ce qui conduit a des mitoses aberrantes et des bourgeons vides. Ces caracteristiques phenotypiques de la mutation RAS2 ala18.val19 pourraient etre la cause de la perte rapide de viabilite de ces cellules en phase stationnaire.