The S. cerevisiae CDC25 gene product regulates the RAS/adenylate cyclase pathway

The gene corresponding to the S. cerevisiae cell division cycle mutant cdc25 has been cloned and sequenced, revealing an open reading frame encoding a protein of 1589 amino acids that contains no significant homologies with other known proteins. Cells lacking CDC25 have low levels of cyclic AMP and decreased levels of Mg2+-dependent adenylate cyclase activity. The lethality resulting from disruption of the CDC25 gene can be suppressed by the presence of the activated RAS2val19 gene, but not by high copy plasmids expressing a normal RAS2 or RAS1 gene. These results suggest that normal RAS is dependent on CDC25 function. Furthermore, mutationally activated alleles of CDC25 are capable of inducing a set of phenotypes similar to those observed in strains containing a genetically activated RAS/adenylate cyclase pathway, suggesting that CDC25 encodes a regulatory protein. We propose that CDC25 regulates adenylate cyclase by regulating the guanine nucleotide bound to RAS proteins.     

SDC25, a CDC25-like gene which contains a RAS-activating domain and is a dispensable gene of Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the CDC25 gene product activates adenylate cyclase through RAS1 and RAS2 gene products. We have recently described the cloning of a DNA fragment which suppresses the cdc25 mutation but not ras1, ras2, or cdc35 mutations. This fragment contains a 5'-truncated open reading frame which shares 47% identity with the C-terminal part of the CDC25 gene. We named the entire gene SDC25. In this paper, we report the cloning, sequencing, and characterization of the complete SDC25 gene. The SDC25 gene is located on the chromosome XII close to the centromere. It is transcribed into a 4-kb-long mRNA that contains an open reading frame of 1,251 codons. Homology with the CDC25 gene extends in the N-terminal part, although the degree of similarity is lower than in the C-terminal part. In contrast with the C-terminal part, the complete SDC25 gene was found not to suppress the CDC25 gene defect. A deletion in the N-terminal part restored the suppressing activity, a result which suggests the existence of a regulatory domain. The SDC25 gene was found to be dispensable for cell growth under usual conditions. No noticeable phenotype was found in the deleted strain.     

Mutants of H-ras that interfere with RAS effector function in Saccharomyces cerevisiae.

We report a class of interfering mutants of the human H-ras gene capable of inhibiting phenotypes arising from the expression of the activated RAS2 gene, RAS2val19, in the yeast Saccharomyces cerevisiae. All these mutants encode unprocessed H-ras proteins that remain in the cytoplasm. One of the mutants, H-rasarg186, was examined in detail. H-rasarg186 protein is a competitive inhibitor of RAS2val19 protein. It does not interfere with processing and membrane localization of RAS2val19, nor does it appear to compete with RAS protein for its proposed regulator, the CDC25 protein. By several criteria the RAS2val19 adenylate cyclase interaction is unaffected by H-rasarg186. We infer from our results that H-rasarg186 protein interferes with an alternative function of RAS2val19.     

The activation of adenylate cyclase by guanyl nucleotides in Saccharomyces cerevisiae is controlled by the CDC25 start gene product.

In the thermosensitive cdc25 start mutant of Saccharomyces cerevisiae, the regulation of adenylate cyclase by guanyl nucleotides was rapidly nullified when the enzyme was prepared from nonsynchronized cells shifted to the restrictive temperature. In agreement with previous in vivo complementation studies, this biochemical defect was fully suppressed by the expression of either the whole cloned CDC25 gene or its C-terminal portion. Moreover, membranes prepared from cdc25(Ts) cells grown at the permissive temperature evinced an altered regulation of adenylate cyclase by guanyl nucleotides. These results indicate that the CDC25 protein, together with RAS, is involved in the regulation of adenylate cyclase by guanyl nucleotides and raise the possibility that adenylate cyclase might form a ternary complex with RAS and CDC25.     

Interactions between adenylate cyclase and the yeast GTPase-activating protein IRA1.

The adenylate cyclase system of the yeast Saccharomyces cerevisiae contains many proteins, including the CYR1 polypeptide, which is responsible for catalyzing the formation of cyclic AMP from ATP, RAS1 and RAS2 polypeptides, which mediate stimulation of cyclic AMP synthesis by guanine nucleotides, and the yeast GTPase-activating protein analog IRA1. We have previously reported that adenylate cyclase is only peripherally bound to the yeast membrane. We have concluded that IRA1 is a strong candidate for a protein involved in anchoring adenylate cyclase to the membrane. We base this conclusion on the following criteria: (i) a disruption of the IRA1 gene produced a mutant with very low membrane-associated levels of adenylate cyclase activity, (ii) membranes made from these mutants were incapable of binding adenylate cyclase in vitro, (iii) IRA1 antibodies inhibit binding of adenylate cyclase to the membrane, and (iv) IRA1 and adenylate cyclase comigrate on Sepharose 4B.     

An adenylate cyclase from Saccharomyces cerevisiae that is stimulated by RAS proteins with effector mutations.

Conservative amino acid substitutions were introduced into the proposed effector regions of both mammalian Ha-ras (residues 32 to 40) and Saccharomyces cerevisiae RAS2 (residues 39 to 47) proteins. The RAS2[Ser 42] protein had reduced biological function in the yeast S. cerevisiae. A S. cerevisiae strain with a second-site suppressor mutation, SSR2-1, was isolated which could grow on nonfermentable carbon sources when the endogenous RAS2 protein was replaced by the RAS2[Ser 42] protein. The SSR2-1 mutation was mapped to the structural gene for adenylate cyclase (CYR1), and the gene containing SSR2-1 was cloned and sequenced. SSR2-1 corresponded to a point mutation that would create an amino acid substitution of a tyrosine residue for an aspartate residue at position 1547. The SSR2-1 gene encodes an adenylate cyclase that is dependent on ras proteins for activity, but is stimulated by Ha-ras and RAS2 mutant proteins that are unable to stimulate wild-type adenylate cyclase.     

Anti-Cdc25 antibodies inhibit guanyl nucleotide-dependent adenylyl cyclase of Saccharomyces cerevisiae and cross-react with a 150-kilodalton mammalian protein.

The CDC25 gene product of the yeast Saccharomyces cerevisiae has been shown to be a positive regulator of the Ras protein. The high degree of homology between yeast RAS and the mammalian proto-oncogene ras suggests a possible resemblance between the mammalian regulator of Ras and the regulator of the yeast Ras (Cdc25). On the basis of this assumption, we have raised antibodies against the conserved C-terminal domain of the Cdc25 protein in order to identify its mammalian homologs. Anti-Cdc25 antibodies raised against a beta-galactosidase-Cdc25 fusion protein were purified by immunoaffinity chromatography and were shown by immunoblotting to specifically recognize the Cdc25 portion of the antigen and a truncated Cdc25 protein, also expressed in bacteria. These antibodies were shown both by immunoblotting and by immunoprecipitation to recognize the CDC25 gene product in wild-type strains and in strains overexpressing Cdc25. The anti-Cdc25 antibodies potently inhibited the guanyl nucleotide-dependent and, approximately 3-fold less potently, the Mn(2+)-dependent adenylyl cyclase activity in S. cerevisiae. The anti-Cdc25 antibodies do not inhibit cyclase activity in a strain harboring RAS2Val-19 and lacking the CDC25 gene product. These results support the view that Cdc25, Ras2, and Cdc35/Cyr1 proteins are associated in a complex. Using these antibodies, we were able to define the conditions to completely solubilize the Cdc25 protein. The results suggest that the Cdc25 protein is tightly associated with the membrane but is not an intrinsic membrane protein, since only EDTA at pH 12 can solubilize the protein. The anti-Cdc25 antibodies strongly cross-reacted with the C-terminal domain of the Cdc25 yeast homolog, Sdc25. Most interestingly, these antibodies also cross-reacted with mammalian proteins of approximately 150 kDa from various tissues of several species of animals. These interactions were specifically blocked by the beta-galactosidase-Cdc25 fusion protein.     

A dominant activating mutation in the effector region of RAS abolishes IRA2 sensitivity.

Previously described mutations in RAS genes that cause a dominant activated phenotype affect the intrinsic biochemical properties of RAS proteins, either decreasing the intrinsic GTPase or reducing the affinity for guanine nucleotides. In this report, we describe a novel activating mutation in the RAS2 gene of Saccharomyces cerevisiae that does not alter intrinsic biochemical properties of the mutant RAS2 protein. Rather, this mutation, RAS2-P41S (proline 41 to serine), which lies in the effector region of RAS, is shown to abolish the ability of the IRA2 protein to stimulate the GTPase activity of the mutant RAS protein. This mutation also modestly reduced the ability of the mutant protein to stimulate the target adenylate cyclase in an in vitro assay, although in vivo the phenotypes it induced suggest that it retains potency in stimulation of adenylate cyclase. Our results demonstrate that although the effector region of RAS appears to be important for interaction with both target effector and negative regulators of RAS, it is possible to eliminate negative regulator responsiveness and retain potency in effector stimulation.     

SRV2, a gene required for RAS activation of adenylate cyclase in yeast

We have identified a gene, SRV2, mutations of which alleviate stress sensitivity in strains carrying an activated RAS gene. Epistasis analysis suggests that the gene affects accumulation of cAMP in the cell. Direct assays of cAMP accumulation indicate that mutations of the gene diminish the rate of in vivo production of cAMP following stimulation by an activated RAS allele. Null mutations of srv2 result in lethality, which cannot be suppressed by mutational activation of the cAMP-dependent protein kinase. The sequence of the gene indicates that it encodes an adenylate cyclase-associated protein. These results demonstrate that SRV2 protein is required for RAS-activated adenylate cyclase activity, but that it participates in other essential cellular functions as well.     

Control of Saccharomyces cerevisiae catalase T gene (CTT1) expression by nutrient supply via the RAS-cyclic AMP pathway.

In Saccharomyces cerevisiae, lack of nutrients triggers a pleiotropic response characterized by accumulation of storage carbohydrates, early G1 arrest, and sporulation of a/alpha diploids. This response is thought to be mediated by RAS proteins, adenylate cyclase, and cyclic AMP (cAMP)-dependent protein kinases. This study shows that expression of the S. cerevisiae gene coding for a cytoplasmic catalase T (CTT1) is controlled by this pathway: it is regulated by the availability of nutrients. Lack of a nitrogen, sulfur, or phosphorus source causes a high-level expression of the gene. Studies with strains with mutations in the RAS-cAMP pathway and supplementation of a rca1 mutant with cAMP show that CTT1 expression is under negative control by a cAMP-dependent protein kinase and that nutrient control of CTT1 gene expression is mediated by this pathway. Strains containing a CTT1-Escherichia coli lacZ fusion gene have been used to isolate mutants with mutations in the pathway. Mutants characterized in this investigation fall into five complementation groups. Both cdc25 and ras2 alleles were identified among these mutants.     

In yeast, RAS proteins are controlling elements of adenylate cyclase

S. cerevisiae strains containing RAS2val19, a RAS2 gene with a missense mutation analogous to one that activates the transforming potential of mammalian ras genes, have growth and biochemical properties strikingly similar to yeast strains carrying IAC or bcy1. Yeast strains carrying the IAC mutation have elevated levels of adenylate cyclase activity. bcy1 is a mutation that suppresses the lethality in adenylate cyclase deficient yeast. Yeast strains deficient in RAS function exhibit properties similar to adenylate cyclase deficient yeast. bcy1 suppresses lethality in ras1- ras2- yeast. Compared to wild-type yeast strains, intracellular cyclic AMP levels are significantly elevated in RAS2val19 strains, significantly depressed in ras2- strains, and virtually undetectable in ras1- ras2- bcy1 strains. Membranes from ras1- ras2- bcy1 yeast lack the GTP-stimulated adenylate cyclase activity present in membranes from wild-type cells, and membranes from RAS2val19 yeast strains have elevated levels of an apparently GTP-independent adenylate cyclase activity. Mixing membranes from ras1- ras2- yeast with membranes from adenylate cyclase deficient yeast reconstitutes a GTP-dependent adenylate cyclase.     

Yeast mutants temperature-sensitive for growth after random mutagenesis of the chromosomal RAS2 gene and deletion of the RAS1 gene.

Saccharomyces cerevisiae strains with a disrupted RAS1 gene and with an intact RAS2 gene (ras1- RAS2 strains) grew well on both fermentable and nonfermentable carbon sources. By constructing isogenic mutants having a disrupted RAS1 locus and a randomly mutagenized chromosomal RAS2 gene, we obtained yeast strains with specific growth defects. The strain TS1 was unable to grow on nonfermentable carbon sources and galactose at 37 degrees C, while it could grow on glucose at the same temperature. The mutated RAS2 gene in TS1 cells encoded a protein with the glycines at positions 82 and 84 replaced by serine and arginine respectively. Both mutations were necessary for temperature sensitivity. We also isolated a mutant yeast that was unable to grow on nonfermentable carbon sources both at 30 and 37 degrees C, while growing on glucose at both temperatures. This phenotype was caused by a single chromosomal mutation, leading to the replacement of aspartic acid 40 of the RAS2 protein by asparagine. A ras1- yeast strain with a chromosomal RAS2 gene harbouring the three mutations together did not grow at any temperature using non-fermentable carbon sources, but it was able to grow on glucose at 30 degrees C, and not at 37 degrees C. The mutated proteins were much less effective than the wild-type RAS2 protein in the stimulation of adenylate cyclase, but were efficiently expressed in vivo. The possible roles of residues 40, 82 and 84 of the RAS2 protein in the regulation of adenylate cyclase are discussed.     

Suppression of defective RAS1 and RAS2 functions in yeast by an adenylate cyclase activated by a single amino acid change.

We have constructed the yeast strain TS1, with the RAS2 gene replaced by mutant allele encoding a partially defective gene product, and with an inactive RAS1 gene. TS1 cells accumulate as unbudded cells upon temperature shift from 30 to 37 degrees C, thus showing that the RAS1 and RAS2 gene functions are important for progression through the G1 phase of the cell cycle. After the isolation of revertants able to grow at the nonpermissive temperature, we have found that a chromosomal point mutation can bypass the G1 arrest of TS1 and cdc25 cells, and the lethality of ras1 ras2 mutants. The mutation predicts the replacement of threonine by isoleucine at position 1651 of yeast adenylate cyclase. The RAS-independent, as well as the RAS-dependent adenylate cyclase activity, is increased by the mutation. Like the wild-type enzyme, the RAS-dependent activity of the mutant adenylate cyclase is turned on by the GTP-bound form of the RAS2 protein. The amino acid sequence surrounding the threonine 1651 shows similarity with protein kinase substrates. Possible implications for the function of adenylate cyclase are discussed.     

Evidence for adenylate cyclase as a scaffold protein for Ras2–Ira interaction in Saccharomyces cerevisie

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.     

Isolation and Characterization of Temperature-Sensitive Mutations in the Ras2 and Cyr1 Genes of Saccharomyces Cerevisiae

The yeast Saccharomyces cerevisiae contains two ras homologues, RAS1 and RAS2, whose products have been shown to modulate the activity of adenylate cyclase encoded by the CYR1 gene. To isolate temperature-sensitive mutations in the RAS2 gene, we constructed a plasmid carrying a RAS2 gene whose expression is under the control of the galactose-inducible GAL1 promoter. A ras1 strain transformed with this plasmid was subjected to ethyl methanesulfonate mutagenesis and nystatin enrichment. Screening of approximately 13,000 mutagenized colonies for galactose-dependent growth at a high temperature (37 degrees) yielded six temperature-sensitive ras2 (ras2ts) mutations and one temperature-sensitive cyr1 (cyr1ts) mutation that can be suppressed by overexpression or increased dosage of RAS2. Some ras2ts mutations were shown to be suppressed by an extra copy of CYR1. Therefore increased dosage of either RAS2 or CYR1 can suppress the temperature sensitivity caused by a mutation in the other. ras1 ras2ts and ras1 cyr1ts mutants arrested in the G1 phase of the cell cycle at the restrictive temperature, and showed pleiotropic phenotypes to varying degrees even at a temperature permissive for growth (25 degrees), including slow growth, sporulation on rich media, increased accumulation of glycogen, impaired growth on nonfermentable carbon sources, heat-shock resistance, impaired growth on low concentrations of glucose, and lithium sensitivity. Of these, impaired growth on low concentrations of glucose and sensitivity to lithium are new phenotypes, which have not been reported for mutants defective in the cAMP pathway.     

Suppressors of the ras2 Mutation of SACCHAROMYCES CEREVISIAE

Saccharomyces cerevisiae contains two members of the ras gene family. Strains with disruptions of the RAS2 gene fail to grow efficiently on nonfermentable carbon sources. This growth defect can be suppressed by extragenic mutations called sra. We have isolated 79 independent suppressor mutations, 68 of which have been assigned to one of five loci. Eleven additional dominant mutations have not been assigned to a specific locus. Some sra1 and SRA4 and all SRA3 mutations were RAS independent, allowing growth of yeast cells that lack a functional RAS gene. Mutations in sra1, SRA3, SRA4 and sra6 are linked to his6, ino1, met3 and ade6, respectively. Some sra mutants have pleiotropic phenotypes that affect glycogen accumulation, sporulation, viability, respiratory capacity and suppression of two cell-division-cycle mutations, cdc25 and cdc35. The proposed functions of many of the suppressor genes are consistent with the model in which RAS activates adenylate cyclase.     

Adenylate cyclase in Saccharomyces cerevisiae is a peripheral membrane protein.

The adenylate cyclase system of the yeast Saccharomyces cerevisiae contains the CYR1 polypeptide, responsible for catalyzing formation of cyclic AMP (cAMP) from ATP, and two RAS polypeptides, which mediate stimulation of cAMP synthesis of guanine nucleotides. By analogy to the mammalian enzyme, models of yeast adenylate cyclase have depicted the enzyme as a membrane protein. We have concluded that adenylate cyclase is only peripherally bound to the yeast membrane, based on the following criteria: (i) substantial activity was found in cytoplasmic fractions; (ii) activity was released from membranes by the addition of 0.5 M NaCl; (iii) in the presence of 0.5 M NaCl, activity in detergent extracts had hydrodynamic properties identical to those of cytosolic or NaCl-extracted enzyme; (iv) antibodies to yeast adenylate cyclase identified a full-length adenylate cyclase in both membrane and cytosol fractions; and (v) activity from both cytosolic fractions and NaCl extracts could be functionally reconstituted into membranes lacking adenylate cyclase activity. The binding of adenylate cyclase to the membrane may have regulatory significance; the fraction of activity associated with the membrane increased as cultures approached stationary phase. In addition, binding of adenylate cyclase to membranes appeared to be inhibited by cAMP. These results indicate the existence of a protein anchoring adenylate cyclase to the membrane. The identity of this protein remains unknown.     

Differential activation of yeast adenylate cyclase by wild-type and mutant RAS proteins

In these experiments we demonstrate that purified RAS proteins, whether derived from the yeast RAS1 or RAS2 or the human H-ras genes, activate yeast adenylate cyclase in the presence of guanine nucleotides. These results confirm the prediction of earlier genetic and biochemical data and for the first time provide a complete biochemical assay for RAS protein function. Furthermore, we observe a biochemical difference between the RAS2 and RAS2val19 proteins in their ability to activate adenylate cyclase after preincubation with GTP.