Regulatory function of the Saccharomyces cerevisiae RAS C-terminus.

Activating mutations (valine 19 or leucine 68) were introduced into the Saccharomyces cerevisiae RAS1 and RAS2 genes. In addition, a deletion was introduced into the wild-type gene and into an activated RAS2 gene, removing the segment of the coding region for the unique C-terminal domain that lies between the N-terminal 174 residues and the penultimate 8-residue membrane attachment site. At low levels of expression, a dominant activated phenotype, characterized by low glycogen levels and poor sporulation efficiency, was observed for both full-length RAS1 and RAS2 variants having impaired GTP hydrolytic activity. Lethal CDC25 mutations were bypassed by the expression of mutant RAS1 or RAS2 proteins with activating amino acid substitutions, by expression of RAS2 proteins lacking the C-terminal domain, or by normal and oncogenic mammalian Harvey ras proteins. Biochemical measurements of adenylate cyclase in membrane preparations showed that the expression of RAS2 proteins lacking the C-terminal domain can restore adenylate cyclase activity to cdc25 membranes.     

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.     

A new RAS mutation that suppresses the CDC25 gene requirement for growth of Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the activation of adenylate cyclase requires the products of the RAS genes and of CDC25. We isolated several dominant extragenic suppressors of the yeast cdc25 mutation. They did not suppress a thermosensitive allele of the adenylate cyclase gene (CDC35). One of these suppressors was a mutated RAS2 gene in which the transition C/G----T/A at position 455 resulted in replacement of threonine 152 by isoleucine in the protein. The same mutation in a v-Ha-ras gene reduces the affinity of p21 for guanine nucleotides (L.A. Feig, B. Pan, T.M. Roberts, and G.M. Cooper, Proc. Natl. Acad. Sci. USA 83:4607-4611, 1986). These results support a model in which the CDC25 gene product is the GDP-GTP exchange factor regulating the activity of the RAS gene product.     

The ras oncogene--an important regulatory element in lower eucaryotic organisms.

The ras proto-oncogene in mammalian cells encodes a 21-kilodalton guanosine triphosphate (GTP)-binding protein. This gene is frequently activated in human cancer. As one approach toward understanding the mechanisms of cellular transformation by ras, the function of this gene in lower eucaryotic organisms has been studied. In the yeast Saccharomyces cerevisiae, the RAS gene products serve as essential function by regulating cyclic adenosine monophosphate metabolism. Stimulation of adenylyl cyclase is dependent not only on RAS protein complexed to GTP, but also on the CDC25 and IRA gene products, which appear to control the RAS GTP-guanosine diphosphate cycle. Although analysis of RAS biochemistry in S. cerevisiae has identified mechanisms central to RAS action, RAS regulation of adenylyl cyclase appears to be strictly limited to this particular organism. In Schizosaccharomyces pombe, Dictyostelium discoideum, and Drosophila melanogaster, ras-encoded proteins are not involved with regulation of adenylyl cyclase, similar to what is observed in mammalian cells. However, the ras gene product in these other lower eucaryotes is clearly required for appropriate responses to extracellular signals such as mating factors and chemoattractants and for normal growth and development of the organism. The identification of other GTP-binding proteins in S. cerevisiae with distinct yet essential functions underscores the fundamental importance of G-protein regulatory processes in normal cell physiology.     

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.     

In vitro reconstitution of cdc25 regulated S. cerevisiae adenylyl cyclase and its kinetic properties.

The attenuated GTP regulation adenylyl cyclase (CDC35) lysates or membranes prepared from cells of a cdc25ts strain is enhanced 2.5- to 6-fold by mixing these lysates or membranes with lysates or membranes from a cdc35ts strain harboring wild-type CDC25. The kinetics of activation of the Saccharomyces cerevisiae adenylyl cyclase in vitro is first order, as is the activation of mammalian adenylyl cyclase. The rate of enzyme activation in the presence of non-hydrolysable analogs of GTP increases with the number of CDC25 gene copies present in the cell. When GppNHp was used the rate of activation of the cyclase in a strain harboring a multicopy plasmid of CDC25 was 7.0-fold higher than the rate in an isogenic strain with the cdc25-2 mutation. The rate of adenylyl cyclase activation from a strain with a disrupted CDC25 gene is 14.7-fold lower than the rate in an isogenic strain containing the CDC25 gene on a multicopy plasmid. The reconstitution experiments described provide direct biochemical evidence for the role of the CDC25 protein in regulating the RAS dependent adenylyl cyclase in S.cerevisiae. The reconstitution experiments and the kinetic experiments may also provide a biochemical assay for the CDC25 protein and can form the basis for its characterization. In this study we also show that adenylyl cyclase activity in ras1ras2byc1 cells is found in the soluble fraction, whereas in wild-type strain it is found in the membrane fraction. Overexpression of the gene CDC25 in the ras1ras2bcy1 strain relocalizes adenylyl cyclase activity to the membrane fraction. This finding suggests a biochemical link between CDC25 and CDC35 in the absence of RAS, in addition to its role in regulating RAS dependent adenylyl cyclase.     

Yeast cdc35 mutants are defective in adenylate cyclase and are allelic with cyr1 mutants while CAS1, a new gene, is involved in the regulation of adenylate cyclase

Newly isolated temperature-sensitive cdc35 mutants of Saccharomyces cerevisiae have been characterized. They show the morphology, growth and conjugation characteristics typical of class-A or class-II start mutants. The cdc35 mutation induces a significant decrease of the intracellular cAMP level and produces a thermolabile adenylate cyclase. By classical genetic criteria the CDC35 gene is identical with the structural gene of adenylate cyclase, CYR1. The results of the mutant selection, the kinetics of macromolecule accumulation and the cell-density change of cdc35 mutants at the restrictive temperature, indicate that CDC35 function may not be cell cycle-specific. A new mutation, cas1, was isolated and partially characterized. It mediates the suppression by external cAMP of the unlinked cdc35 mutation. It causes a slight increase of the intracellular cAMP level and has strong effects on the adenylate cyclase activities, especially on the Mg2+ dependent activity. The data suggest that the CAS1 protein is a controlling element of adenylated cyclase. The CAS1 locus is different from the RAS1 and RAS2 loci.     

Different kinetic properties of the two mutants, RAS2Ile152 and RAS2Val19, that suppress the CDC25 requirement in RAS/adenylate cyclase pathway in Saccharomyces cerevisiae

The properties of RAS2Gly19----Val and RAS2Thr152----Ile, two mutants suppressing the CDC25 requirement for the activation of adenylate cyclase in Saccharomyces cerevisiae, were compared with the properties of wild-type RAS2. We examined (a) the guanine nucleotide interaction, (b) the intrinsic GTPase (EC 3.6.1-) activity, and (c) the ability to activate adenylate cyclase in vitro. The low GTPase of RAS2Val19 is associated with an increased stability of the GTP complex. By contrast, RAS2Ile152 shows a strong destabilization of the GDP complex (the dissociation rate constants of the RAS2Ile152.GDP complex is enhanced almost 50 times) and an increased GTPase activity. Remarkably, all the parameters of the interaction with GDP and GTP as well as the catalytic activity are modified by the two mutations in an opposite manner. Our kinetic results show that the functional modifications of RAS2 compensating for the CDC25 inactivation can not only be associated with the presence of a long-lived RAS2.GTP complex, but also with a rapid GDP to GTP exchange reaction. As a striking result, the functional modifications induced by Thr152----Ile activate the adenylate cyclase in vitro much more efficiently than those induced by Gly19----Val. This stresses the importance of a rapid regeneration of the RAS2.GTP complex for the activation of the adenylate cyclase pathway.     

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.     

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.     

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.     

cAMP- and RAS-independent nutritional regulation of plasma-membrane H+-ATPase activity in Saccharomyces cerevisiae

The plasma-membrane ATPase of Saccharomyces cerevisiae is a proton pump whose activity, essential fro proliferation, is subject to regulation by nutritional signals. The previous finding that the CDC25 gene product is required for the glucose-induced H+-ATPase activation suggested that H+-ATPase activity is regulated by cAMP. Analysis of starvation-induced inactivation and glucose-induced activation of the H+-ATPase in mutants affected in activity of the RAS proteins, adenylyl cyclase or cAMP-dependent protein kinase showed that nutritional regulation of H+-ATPase activity does not depend directly on any of these factors. We conclude that adenlyl cyclase does not mediate all nutritional responses. This also indicates that the specific CDC25 requirement for the glucose-induced activation of the H+-ATPase identifies a new function for the CDC25 gene product, a function that appears to be independent of CDC25-mediated modulation of the RAS/adenylyl cyclase/cAMP pathway.     

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.     

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.     

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.     

Guanine nucleotide activation of, and competition between, RAS proteins from Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, yeast RAS proteins are potent activators of adenylate cyclase. In the present work we measured the activity of adenylate cyclase in membranes from Saccharomyces cerevisiae which overexpress this enzyme. The response of the enzyme to added RAS2 proteins bound with various guanine nucleotides and their analogs suggests that RAS2 proteins are active in their GTP-bound form and are virtually inactive in their GDP-bound form. Also, active RAS2 protein is not inhibited by inactive RAS2, suggesting that the inactive form does not compete with the active form in binding to its effector.     

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.