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The yeast Saccharomyces cerevisiae has long been used to produce alcohol from glucose and other sugars. While much is known about glucose metabolism, relatively little is known about the receptors and signaling pathways that indicate glucose availability. Here we compare the two glucose receptor systems in S. cerevisiae. The first is a heterodimer of transporter-like proteins (transceptors), while the second is a seven-transmembrane receptor coupled to a large G protein (Gpa2) and two small G proteins (Ras1 and Ras2). Through comprehensive measurements of glucose-dependent transcription and metabolism, we demonstrate that the two receptor systems have distinct roles in glucose signaling: the G protein-coupled receptor directs carbohydrate and energy metabolism, while the transceptors regulate ancillary processes such as ribosome, amino acids, cofactor and vitamin metabolism. The large G protein transmits the signal from its cognate receptor, while the small G protein Ras2 (but not Ras1) integrates responses from both receptor pathways. Collectively, our analysis reveals the molecular basis for glucose detection and the earliest events of glucose-dependent signal transduction in yeast.

Ras proteins are highly conserved small GTPases in eukaryotes. GTP-bound Ras binds to effectors to trigger signaling cascades. In order to understand how extensive is the functional homology between the highly homologous proteins, S. cerevisiae Ras2 and C. albicans Ras1, we examined whether ScRas2 could functionally complement CaRas1 in activating hyphal morphogenesis as well as GPI anchor biosynthesis. We show that ScRas2 functionally complements CaRas1 in rescuing growth as well as activating hyphal growth, a process that involves plasma membrane localized Ras activating cAMP/PKA signaling via Cyr1. However, ScRas2 is unable to activate the GPI-N-acetylglucosaminyl transferase (GPI-GnT) which catalyzes the first step of GPI biosynthesis. That CaRas1 alone activates GPI-GnT and not ScRas2 suggests that this process is cAMP independent. Interestingly, CaRas1 transcriptionally activates CaGPI2, encoding a GPI-GnT subunit that has been shown to interact with CaRas1 physically. In turn, CaGPI2 downregulates CaGPI19, encoding another GPI-GnT subunit. This has direct consequences for expression of CaERG11, encoding the target of azole antifungals. This effect too is specific to CaRas1 and ScRas2 is unable to replicate it.

The ability of the yeast Saccharomyces cerevisiae to adapt to the changing environment of industrial processes lies in the activation and coordination of many molecular pathways. The most relevant ones are nutrient signaling pathways because they control growth and stress response mechanisms as a result of nutrient availability or scarcity and, therefore, leave an ample margin to improve yeast biotechnological performance. A standardized grape juice fermentation assay allowed the analysis of mutants for different elements of many nutrient signaling pathways under different conditions (low/high nitrogen and different oxygenation levels) to allow genetic-environment interactions to be analyzed. The results indicate that the cAMP-dependent PKA pathway is the most relevant regardless of fermentation conditions, while mutations on TOR pathways display an effect that depends on nitrogen availability. The production of metabolites of interest, such as glycerol, acetic acid and pyruvate, is controlled in a coordinated manner by the contribution of several components of different pathways. Ras GTPase Ras2, a stimulator of cAMP production, is a key factor for achieving fermentation, and is also relevant for sensing nitrogen availability. Increasing cAMP concentrations by deleting an enzyme used for its degradation, phosphodiesterase Pde2, proved a good way to increase fermentation kinetics, and offered keys for biotechnological improvement. Surprisingly glucose repression protein kinase Snf1 and Nitrogen Catabolite Repression transcription factor Gln3 are relevant in fermentation, even in the absence of starvation. Gln3 proved essential for respiration in several genetic backgrounds, and its presence is required to achieve full glucose de-repression. Therefore, most pathways sense different types of nutrients and only their coordinated action can ensure successful wine fermentation., This work was funded by a grant from the Spanish Ministry of Science (AGL2017-83254-R) to EM and AA. BV was supported by an F.P.U. fellowship from the Spanish Ministry of Education., We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

Accumulating evidence from both experimental and clinical investigations indicates a tight interaction between metabolism and circadian timekeeping; however, knowledge of the underlying mechanism is still incomplete. Metabolic compensation allows circadian oscillators to run with a constant speed at different substrate levels and, therefore, is a substantial criterion of a robust rhythm in a changing environment. Because previous data have suggested a central role of RAS2-mediated signaling in the adaptation of yeast to different nutritional environments, we examined the involvement of RAS2 in the metabolic regulation of the clock in the circadian model organism Neurospora crassa. We show that, in a ras2-deficient strain, the period is longer than in the control. Moreover, unlike in the WT, in Δras2, operation of the circadian clock was affected by glucose; compared with starvation conditions, the period was longer and the oscillation of expression of the frequency (frq) gene was dampened. In constant darkness, the delayed phosphorylation of the FRQ protein and the long-lasting accumulation of FRQ in the nucleus were in accordance with the longer period and the less robust rhythm in the mutant. Although glucose did not affect the subcellular distribution of FRQ in the WT, highly elevated FRQ levels were detected in the nucleus in Δras2. RAS2 interacted with the RAS-binding domain of the adenylate cyclase in vitro, and the cAMP analogue 8-bromo-cyclic AMP partially rescued the circadian phenotype in vivo. We therefore propose that RAS2 acts via a cAMP-dependent pathway and exerts significant metabolic control on the Neurospora circadian clock.

The ability of Candida albicans to switch between yeast to hyphal form is a property that is primarily associated with the invasion and virulence of this human pathogenic fungus. Several glycosylphosphatidylinositol (GPI)-anchored proteins are expressed only during hyphal morphogenesis. One of the major pathways that controls hyphal morphogenesis is the Ras-signaling pathway. We examine the cross-talk between GPI anchor biosynthesis and Ras signaling in C. albicans. We show that the first step of GPI biosynthesis is activated by Ras in C. albicans. This is diametrically opposite to what is reported in Saccharomyces cerevisiae. Of the two C. albicans Ras proteins, CaRas1 alone activates GPI–GnT activity; activity is further stimulated by constitutively activated CaRas1. CaRas1 localized to the cytoplasm or endoplasmic reticulum (ER) is sufficient for GPI–GnT activation. Of the six subunits of the GPI–N-acetylglucosaminyltransferase (GPI–GnT) that catalyze the first step of GPI biosynthesis, CaGpi2 is the key player involved in activating Ras signaling and hyphal morphogenesis. Activation of Ras signaling is independent of the catalytic competence of GPI–GnT. This too is unlike what is observed in S. cerevisiae where multiple subunits were identified as inhibiting Ras2. Fluorescence resonance energy transfer (FRET) studies indicate a specific physical interaction between CaRas1 and CaGpi2 in the ER, which would explain the ability of CaRas1 to activate GPI–GnT. CaGpi2, in turn, promotes activation of the Ras-signaling pathway and hyphal morphogenesis. The Cagpi2 mutant is also more susceptible to macrophage-mediated killing, and macrophage cells show better survival when co-cultured with Cagpi2.

Appressorium formation and invasive growth are two important steps in the infection cycle of Magnaporthe oryzae that are regulated by the Mst11-Mst7-Pmk1 mitogen-activated protein kinase (MAPK) pathway. However, the molecular mechanism involved in the activation of Mst11 MAPK kinase kinase is not clear in the rice blast fungus. In this study, we functionally characterized the regulatory region of Mst11 and its self-inhibitory binding. Deletion of the middle region of Mst11, which contains the Ras-association (RA) domain and two conserved phosphorylation sites (S453 and S458), blocked Pmk1 activation and appressorium formation. However, the MST11ΔRA transformant MRD-2 still formed appressoria, although it was reduced in virulence. Interestingly, over 50% of its germ tubes branched and formed two appressoria by 48 h, which was suppressed by treatments with exogenous cAMP. The G18V dominant active mutation enhanced the interaction of Ras2 with Mst11, suggesting that Mst11 has stronger interactions with the activated Ras2. Furthermore, deletion and site-directed mutagenesis analyses indicated that phosphorylation at S453 and S458 of Mst11 is important for appressorium formation and required for the activation of Pmk1. We also showed that the N-terminal region of Mst11 directly interacted with its kinase domain, and the S789G mutation reduced their interactions. Expression of the MST11S789G allele rescued the defect of the mst11 mutant in plant infection and resulted in the formation of appressoria on hydrophilic surfaces, suggesting the gain-of-function effect of the S789G mutation. Overall, our results indicate that the interaction of Mst11 with activated Ras2 and phosphorylation of S453 and S458 play regulatory roles in Mst11 activation and infection-related morphogenesis, possibly by relieving its self-inhibitory interaction between its N-terminal region and the C-terminal kinase domain. In addition, binding of Mst11 to Ras2 may be involved in the feedback inhibition of cAMP signaling and further differentiation of germ tubes after appressorium formation.

Various surface signals are recognized by Magnaporthe oryzae to activate the Pmk1 MAP kinase that is essential for appressorium formation and invasive growth. One of upstream sensors of the Pmk1 pathway is the MoMsb2 signalling mucin. However, the activation of MoMsb2 and its relationship with other sensors is not clear. In this study, we showed that the cleavage and transmembrane domains are essential for MoMsb2 functions. Cleavage of MoMsb2 was further confirmed by western blot analysis, and five putative cleavage sites were functionally characterized. Expression of the extracellular region alone partially rescued the defects of Momsb2 in appressorium formation and virulence. The cytoplasmic region of MoMsb2, although dispensable for appressorium formation, was more important for penetration and invasive growth. Interestingly, the Momsb2 cbp1 double mutant deleted of both mucin genes was blocked in Pmk1 activation. It failed to form appressoria on artificial surfaces and was non-pathogenic. In addition, we showed that MoMsb2 interacts with Ras2 but not with MoCdc42 in co-immunoprecipitation assays. Overall, results from this study indicated that the extracellular and cytoplasmic regions of MoMsb2 have distinct functions in appressorium formation, penetration and invasive growth, and MoMsb2 has overlapping functions with Cbp1 in recognizing environmental signals for Pmk1 activation.

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.

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.

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.

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.

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.

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 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.

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.

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.

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 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 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.

We report the identification of the PPS1 gene of Saccharomyces cerevisiae The deduced amino acid sequence of PPS1p shows similarity with protein-tyrosine phosphatases (PTPases) and is most closely related to a subfamily of PTPases that are capable of dephosphorylating phosphoseryl and phosphothreonyl residues as well as phosphotyrosyl residues. Analysis of the predicted amino acid sequence suggests that the protein consists of an active phosphatase domain, an inactive phosphatase-like domain, and an NH2-terminal extension. Mutation of the catalytic cysteinyl residue in the active phosphatase domain reduced the in vitro activity of the mutant protein to less than 0.5% of wild type activity, while mutation of the corresponding cysteinyl residue of the inactive phosphatase-like domain had no effect on in vitro activity. The PPS1 protein was expressed in Escherichia coli, and the protein was shown to catalyze the hydrolysis of p-nitrophenyl phosphate, dephosphorylate phosphotyrosyl, and phosphothreonyl residues in synthetic diphosphorylated peptides and to inactivate the human ERK1 protein. PPS1 transcript abundance is coregulated with that of the divergently transcribed DPB3 gene, which codes for a subunit of DNA polymerase II, with both transcripts showing peak abundance in S phase. pps1Δ mutant strains did not differ from PPS1 strains under any of the conditions tested, but overexpression of the PPS1 protein in S. cerevisiae led to synchronous growth arrest and to aberrant DNA synthesis. A screen for suppressors of this growth arrest identified the RAS2 gene as a multicopy suppressor of the PPS1p overexpression arrest. The arrest was not suppressed by the presence of multicopy RAS1, TPK2, or TPK3 genes or by the presence of 5 mM cAMP in the growth medium, suggesting that PPS1 functions in a pathway involving RAS2, but not TPK kinases or adenylate cyclase.

Ras proteins were highly conserved during evolution. They function as a point of convergence for different signalling pathways in eukaryotes and are involved in a wide range of cellular responses (shift from gluconeogenic to fermentative growth, breakdown of storage carbohydrates, stress resistance, growth control and determination of life span, morphogenesis and development, and others). These proteins are members of the small GTPase superfamily, which are active in the GTP-bound form and inactive in the GDP-bound form. Given the importance of studies on the Ras protein activation state to understand the detailed mechanism of Ras-mediated signal transduction, we provide here a simple, sensitive, and reliable method, based on the high affinity interaction of Ras-GTP with the Ras binding domain (RBD) of Raf1, to measure the level of Ras2-GTP on total Ras2 in Saccharomyces cerevisiae. Moreover, to study the localization of Ras-GTP in vivo in single S. Cerevisiae cells, we expressed a probe consisting of a GFP fusion with a trimeric Ras Binding Domain of Raf1 (eGFP-RBD3), which was proven to be a useful live-cell biosensor for Ras-GTP in mammalian cells. © 2014 Springer Science+Business Media, LLC.

Individual cells of the budding yeast, Saccharomyces cerevisiae, have a limited division capacity and undergo characteristic changes as they senesce, primarily increasing both their cell size and cell cycle time. The mortality curve for ageing yeast cells can be described by the Gompertz equation, the classical definition for an ageing population. Recent work from several laboratories has demonstrated that genes can determine the yeast lifespan. Studies with the UTH genes have implicated changes in transcriptional silencing during yeast ageing, but the roles of the RAS2, LAG1 and PHB1 genes in regulating yeast longevity are still unclear. What is becoming clearer, however, is that yeast ageing is more than just a bud scar phenomenon.

Spontaneous mutations in the gene which encodes the regulatory subunit of cAMP-dependent protein kinase (PKA) of Saccharomyces cerevisiae (BCY1) have been isolated previously [Cannon, J. F., Gibbs, J. B. & Tatchell, K. (1986) Genetics 113, 247-264] by selection of ras2::LEU2 revertants that grew on non-fermentable carbon sources. The revertants were placed into groups of increasing severity based on the number of PKA-dependent traits affected [Cannon, J. F., Gitan, R. & Tatchell, K. (1990) J. Biol. Chem. 265, 11897-11904]. In this work the ras2 mutation has been crossed out in each bcy1 allele and the phenotypes of these mutants have been assessed. The order of severity of the mutants in both genetic backgrounds is maintained but the severity of each mutant in the normal background is higher than in the ras2::LEU2 background. Total catalytic-subunit and regulatory-subunit activities were measured in crude extracts of the bcy1 ras2::LEU2 mutants. With one exception (bcy1-6) the calculated regulatory subunit/catalytic subunit ratios of the bcy1 mutants relative to that of wild-type cells were greater than one. The dependence of PKA activity on cAMP was measured in permeabilized cells. The strains show an activity ratio in the absence and presence of cAMP in the range 0.5-1 for Kemptide phosphorylation. Overexpression of the high-affinity cAMP phosphodiesterase gene (PDE2) in the bcy1 ras2::LEU2 strains did not alter their PKA-dependent phenotypes. However, transformants were not observed from the parental ras2::LEU2 strain and the bcy1-6 ras2::LEU2 strain. The results are discussed with respect to a hypothesis for the molecular mechanism of the differential reversal of ras2 phenotypes by the bcy1 alleles. Mutations in the regulatory subunit are predicted to affect the structure of the holoenzyme such that the catalytic subunit is capable of maintaining an active catalytic state, without the need to dissociate from the regulatory subunit.

Schizosaccharomyces pombe contains single ras oncogene homologue, ras1, that functions in the signal transduction pathway conducting the cell's mating processes. To understand the biochemical basis of yeast ras proteins, we have purified the ras1 protein and compared the major biochemical constants with those of RAS2 protein from Saccharomyces cerevisiae and mammalian ras proteins. The purified ras1 protein showed a remarkably high Kd value for GDP binding (178 nM) and for binding with ATP. In contrast, the Kd value for GTP binding and the rate of GTPase activity were 64 nM and 77 x 10(-6) s-1 at 37 degrees C, respectively; both were higher than normal p21ras protein, but at the same level as the RAS2 protein. We directly measured rate of GTP binding and GDP binding which were 3.9 x 10(-3) s-1 and 1.8 x 10(-3) s-1 at 30 degrees C, respectively. On the other hand, exchange rates between bound and free nucleotides remained almost constant throughout the tested combination of GTP and GDP, and were several-fold lower than the binding rate. These results suggest that the release of the guanine nucleotide is the rate-limiting step in the ras-GTP/GDP cycle. As a whole, the biochemical properties of the ras1 protein are close to those of the RAS2 protein, although these two proteins function differently in the signal transduction pathway in the cells.

The ability of Ras proteins to initiate eukaryotic cell proliferation requires the post-translational attachment of a farnesyl group, an isoprenoid lipid moiety derived from mevalonate, to the carboxyl-terminus of the protein. This modification is essential for the subsequent processing and intracellular targeting of the Ras protein. Here we report that mevalonate is also required for the efficient synthesis of Ras proteins in Saccharomyces cerevisiae. Depletion of intracellular mevalonate resulted in decreased steady-state levels of Ras1p and Ras2p, an effect that was mediated at the level of mRNA accumulation. The sequences controlling the response of RAS2 mRNA level to mevalonate availability, mapped to the coding region of the RAS2 gene. Mevalonate starvation also had a significant effect on the expression of some, but not all, genes encoding prenylated proteins. The regulatory effect on RAS2 mRNA did not require a functional farnesyl transferase. These results uncover a novel regulatory role for mevalonate-derived products and expand the potential for inhibitors of mevalonate metabolism as anti-cancer agents.

In the yeast Saccharomyces cerevisiae, the Ras/cAMP/PKA pathway plays a major role in the regulation of metabolism, stress resistance and cell cycle progression. We extend here a mechanistic model of the Ras/cAMP/PKA pathway that we previously defined by describing the molecular interactions and post-translational modifications of proteins, and perform a computational analysis to investigate the dynamical behaviors of the components of this pathway, regulated by different control mechanisms. We carry out stochastic simulations to consider, in particular, the effect of the negative feedback loops on the activity of both Ira2 (a Ras-GAP) and Cdc25 (a Ras-GEF) proteins. Our results show that stable oscillatory regimes for the dynamics of cAMP can be obtained only through the activation of these feedback mechanisms, and when the amount of Cdc25 is within a specific range. In addition, we highlight that the levels of guanine nucleotides pools are able to regulate the pathway, by influencing the transition between stable steady states and oscillatory regimes.

The presence of adenylate cyclase activity was first demonstrated in membrane fractions from the budding yeast Kluyveromyces marxianus. The enzyme showed a Mn(2+)- and Mg(2+)-dependent activity, with optimal pH at around 6 as observed in other yeast species. As in Saccharomyces cerevisiae, where adenylate cyclase is regulated by RAS1 and RAS2, we detected a guanyl nucleotide-dependent activity. Interestingly Y13-259 monoclonal antibody, raised against mammalian p21Ha-ras, inhibited Mg2+ plus GTP-gamma-S-dependent cAMP production, suggesting that the GTP binding proteins involved in adenylate cyclase regulation could be Ras proteins. The same antibody recognized on Western blot and immunoprecipitated a 40 kDa polypeptide from K. marxianus crude membranes. This polypeptide was not detected by an anti-RAS2 polyclonal antibody raised against S. cerevisiae RAS2 protein, suggesting that Ras proteins from the two species could be structurally different.

Ras proteins appear to have two distinct downstream effectors, adenylyl cyclase in Saccharomyces cerevisiae and a product of raf-1 protooncogene in higher organisms. We found that in vitro activation of adenylyl cyclase by yeast Ras2 and human H-Ras proteins is subject to competitive inhibition by its leucine-rich repeats domain and by the N-terminal regulatory domain of human Raf-1 protein. Kinetic analyses of the inhibition patterns enabled us to determine exact dissociation constants (Kd) of the two polypeptides for Ras2 and H-Ras. The leucine-rich repeats domain bound to the posttranslationally modified Ras2 with the Kd of approximately 13 nM, which was close to the value (7 nM) of the whole adenylyl cyclase. The Kd of Raf-1 for the modified H-Ras, 3.5 nM, was significantly lower than that for Ras2, 24 nM, whereas adenylyl cyclase bound preferentially to Ras2. Similar inhibition was also observed in vivo by suppression of RAS2Val-19-dependent heat shock sensitivity and of Ras-dependent cAMP response to glucose upon overexpression of Raf-1 in yeast. These results indicate that the leucine-rich repeats domain contains the Ras protein-binding site and that Raf-1 and adenylyl cyclase, sharing no structural homology with each other, bind to a similar, if not identical, region of Ras with comparable affinities.

Individual cells of the yeast Saccharomyces cerevisiae have a limited replicative life-span. The role of the genes RAS1 and RAS2 in yeast longevity was examined. Over-expression of RAS2 led to a 30% increase in the life-span on average and postponed the senescence-related increase in generation time seen during yeast aging. No life-span extension was obtained by overexpression of RAS1. However, deletion of RAS1 prolonged the life-span. These results suggest that RAS1 and RAS2 play reciprocal roles in determining yeast longevity. RAS1 and RAS2 mRNA and protein levels declined with replicative age, suggesting a diminishing impact on yeast longevity. The major known pathway through which Ras proteins function in yeast involves stimulation of adenylate cyclase. No evidence for a life-span-extending effect of elevated intracellular cAMP was found. Indeed, high intracellular cAMP was associated with curtailed life-span. A similar decrease in life-span was found on disruption of BCY1, which codes for the regulatory subunit of protein kinase A, the downstream target of cAMP. Importantly, overexpression of an effector domain mutant of RAS2, defective in stimulation of adenylate cyclase, prolonged life-span to the same extent as the wild-type gene, suggesting that the cAMP pathway is neither sufficient nor necessary for increased longevity.

We have studied the interaction of the yeast RAS2 protein with guanine nucleotides using energetic parameters for the dissociation of RAS·nucleotide complexes. The results indicated that a Gly → Ser substitution at position 82 led to an altered interaction with GppNHp and, to a lesser extent, also with GDP. It was also possible to conclude that structural perturbation of Gly82 can stimulate nucleotide release by decreasing the energetic barrier for nucleotide dissociation. This, together with the observation that residues 80 and 81 are involved in the response of RAS to nucleotide exchange factors without affecting GDP binding per se, suggests a potential mechanism for exchange factor-stimulated GDP release.

We have explored the role of the distal switch II region of the yeast RAS2 protein in determining the response to the nucleotide exchange factor CDC25. We first constructed yeast tester strains in which the deletion of the chromosomal CDC25, RAS1, and RAS2 genes, in combination with the chromosomal suppressor CRI4, resulted in detectable phenotypes in vivo and in vitro. Phenotypes included impaired growth at 37 degrees C, defective glucose-induced cyclic AMP signaling, and low adenylyl cyclase activity of membrane preparations. Tester strains were subsequently used for the reintroduction of various combinations of wild-type and mutated RAS2 and CDC25 genes by genetic techniques, as well as for in vitro reconstitution assays with the corresponding proteins. CDC25 restored both growth and glucose-induced cyclic AMP signaling in the presence, but not in the absence of wild-type RAS2. A gene encoding a RAS2 protein with a mutationally altered switch II region was expressed but was ineffective in reintegrating exchange factor-dependent responses in vivo. Wild-type, but not mutagenically altered, RAS2 proteins were stimulated by exchange factors in vitro. We conclude that the conserved distal switch II region is required for CDC25-dependent activation of RAS.

The Saccharomyces cerevisiae SLK1 protein is implicated in nutrient sensing and growth control. Under nutrient-limiting conditions, slk1 mutants fail to undergo cell cycle arrest. The role of the SLK1 protein in nutrient sensing was examined with respect to the cAMP-dependent protein kinase (PKA) pathway, which has a well characterized role in growth control in yeast, and by the analysis of dominant SLK1 alleles that affect the nutrient response of wild-type cells. Interactions with the PKA pathway were examined by phenotypic analysis of double mutants of slk1 and various PKA pathway mutants. Combining the slk1-delta mutation with a mutation that is thought constitutively activate the PKA pathway, pde2, resulted in enhanced growth control defects. The combination of slk1-delta with mutations that inhibit the PKA pathway, cdc25 and ras1, ras2, failed to alleviate the slk1 cell cycle arrest defect and lowered the permissive temperature for growth. Furthermore bcy1 tpk1 tpk2 tpk3w (bcy1 tpkw) mutants, which have constitutive, low-level, cAMP-independent kinase activity, exhibit nutrient sensing, which is eliminated in the slk1 bcy1 tpkw mutants. These results implicated SLK1 in PKA-independent growth control in yeast. The amino-terminal, noncatalytic region of the SLK1 protein may be important in the regulation of SLK1 function in growth control. Overexpression of this region caused starvation sensitivity in wild-type cells by interfering with SLK1 protein function.

Two C-terminal fragments (334 and 509 amino acid residues) of CDC25, a Saccharomyces cerevisiae GDP/GTP exchange factor, and the RAS2 protein were purified from E. coli, using the pGEX system. With this method it was possible to avoid in part the proteolytic phenomena that usually convert full-length RAS2 (42kDa) into 37 and 30kDa forms. Of the two CDC25 fragments containing the conserved catalytic domain, only CDC25-509 could enhance the guanine nucleotide exchange on RAS2. Comparison of the activities of RAS2-42/37kDa and RAS2-30kDa showed that the C-terminal region (112 residues) influences neither the intrinsic GDP/GTP exchange nor its stimulation by CDC25-509. RAS2-42/37kDa was somewhat more effective in enhancing the adenylylcyclase activity of a yeast membrane reconstituted system. CDC25-509 displayed a higher specific activity than the catalytic domains of the two CDC25-like proteins: S. cerevisiae SDC25 and mouse CDC25Mm.

Glucose deprivation causes the yeast Saccharomyces cerevisiae to enter a stationary phase and accumulate proteins and mRNAs in cytoplasmic foci called P bodies, which are thought to store these macromolecules until needed. Glucose stimulates the activity of the cyclic adenosine monophosphate–dependent protein kinase (PKA) in S. cerevisiae , leading Ramachandran et al. to investigate the role of this kinase in P body assembly. Genetic manipulations that increased PKA activity, such as expression of an allele encoding a mutant form of an upstream activator ( RAS2 val19 ), resulted in the formation of fewer P bodies in response to glucose deprivation. Conversely, genetic manipulations that decreased PKA activity resulted in P body formation in the presence of glucose. Pat1, a scaffold for P body assembly that is also involved in repressing translation and stimulating mRNA decay, was phosphorylated at Ser 456 and Ser 457 by PKA in vitro and in cells, and glucose starvation reduced phosphorylation of Pat1 at these residues. Expression of a Pat1 mutant that could not be phosphorylated by PKA (Pat1-AA) rescued the defect in P body formation caused by increased PKA signaling. A Pat1 mutant that mimicked the PKA-phosphorylated form (Pat1-EE) showed reduced association with Dhh1, which has roles in repressing translation and stimulating mRNA decay, and expression of this mutant reduced the amount of various P body proteins, including Dhh1, in these foci. S. cerevisiae with constitutive PKA activity, such as those expressing RAS2 val19 , do not enter the stationary phase and do not survive nutrient deprivation, and loss of viability in RAS2 val19 stationary cultures was prevented by expression of Pat1-AA. Thus, phosphorylation of Pat1 by PKA may prevent P body assembly in cells experiencing nutrient-rich conditions. V. Ramachandran, K. H. Shah, P. K. Herman, The cAMP-dependent protein kinase signaling pathway is a key regulator of P body foci formation. Mol. Cell 43 , 973–981 (2011). [PubMed]