Analysis of the role of the hypervariable region of yeast Ras2p and its farnesylation in the interaction with exchange factors and adenylyl cyclase.

Ras proteins from Saccharomyces cerevisiae differ from mammalian Ha-Ras in their extended C-terminal hypervariable region. We have analyzed the function of this region and the effect of its farnesylation with respect to the action of the GDP/GTP exchange factors (GEFs) Cdc25p and Sdc25p and the target adenylyl cyclase. Whereas Ras2p farnesylation had no effect on the interaction with purified GEFs from the Cdc25 family, this modification became a strict requirement for stimulation of the nucleotide exchange on Ras using reconstituted cell-free systems with GEFs bound to the cell membrane. Determination of GEF effects showed that in cell membrane the Cdc25p dependent activity on Ras2p was predominant over that of Sdc25p. In contrast to full-length GEFs, a membrane-bound C-terminal region containing the catalytic domain of Cdc25p was still able to react productively with unfarnesylated Ras2p. These results indicate that in membrane-bound full-length GEF the N-terminal moiety regulates the interaction between catalytic domain and farnesylated Ras2p.GDP. Differently from GEF, full activation of adenylyl cyclase did not require farnesylation of Ras2p.GTP, even if this step of maturation was found to facilitate the interaction. The use of Ha-Ras/Ras2p chimaeras of different length emphasized the key role of the hypervariable region of Ras2p in inducing maximum activation of adenylyl cyclase and for a productive interaction with membrane-bound GEF.

Distal switch II region of Ras2p is required for interaction with guanine nucleotide exchange factor.

The interaction of Saccharomyces cerevisiae Ras2p with the catalytic domain of the GDP/GTP exchange factors (GEFs) mouse CDC25(Mm), yeast Cdc25p, and Sdc25p was analyzed by introducing the substitution R80D/N81D into Ras2p S24N, a mutant that is shown to interfere with the Ras2p wild type (wt)-GEF interaction by forming a stable complex. The triple mutant, like Ras2p R80D/N81D, did not interfere with the action of GEF on Ras2p wt (or H-Ras p21) and was unable to form a stable complex with GEF. The GEF stimulation of the nucleotide dissociation of the triple mutant was virtually abolished and strongly decreased with the double mutant. The affinity of Ras2p S24N/R80D/N81D for GDP and GTP was decreased 3 and 4 orders of magnitude, respectively, like that of Ras2p S24N, whereas the double mutant behaved as Ras2p wt. Like Ras2p S24N and unlike Ras2p R80D/N81D, the GTP-bound triple mutant did not activate adenylyl cyclase. Thus, the triple mutant and Ras2p S24N have opposite properties toward the binding to GEF but similarly modified behaviors toward GDP, GTP, and adenylyl cyclase. This work emphasizes the determinant role of the distal switch II region of Ras2p for the interaction with GEF and the different structural background of the interaction with adenylyl cyclase.

Properties of the catalytic domain of sdc25p, a yeast GDP/GTP exchange factor of Ras proteins. Complexation with wild-type Ras2p, [S24N]Ras2p and [R80D, N81D]Ras2p.

The catalytic domain of the Saccharomyces cerevisiae SDC25 gene product, including the last 550 C-terminal residues (Sdc25p-C), was produced as an Escherichia coli recombinant protein fused with glutathione S-transferase. The highly purified (greater than 95%) stable fusion protein, obtained by affinity chromatography, was very active in enhancing the dissociation rate or the GDP/GTP exchange of the GDP complex of Ras2p or human H-ras p21. This activity was further increased (three times) by glutathione S-transferase cleavage with thrombin. The stimulation of the guanine nucleotide release by Sdc25p-C was stronger for Ras2p.GDP than Ras2p.GTP, an effect that was less pronounced in the case of the p21 complexes. The association rate of the Ras2p.GDP (GTP) complex was also enhanced by Sdc25p-C. Monovalent and divalent salts inhibit the nucleotide-releasing activity of Sdc25p-C. Retention phenomena occurring on gel-filtration chromatography hindered the use of highly purified Sdc25p-C to study the formation of stable complexes with Ras2p. For this purpose, Sdc25p-C was produced as a non-glutathione-S-transferase fusion protein via pTTQ19. Upon partial purification, this product yielded a 54-kDa truncated form of Sdc25p-C (truncated Sdc25p-C) showing the same specific activity as the 64-kDa Sdc25p-C protein. On gel filtration, truncated Sdc25p-C and nucleotide-free Ras2p (or p21) formed a stable 1:1 stoichiometric complex that was dissociated by increasing concentrations of GDP. The properties of this complex were analyzed by using the mutant [S24N]Ras2p, the homologue of [S17N]p21 known to induce a dominant negative phenotype, [R80D, N81D]Ras2p, a recessive negative mutant insensitive to the truncated form of Sdc25p-C in vitro. The complex with [S24N]Ras2p was greater than 100-fold less sensitive to the dissociating effect of GDP, whereas [R80D, N81D]Ras2p was unable to form a stable complex with truncated Sdc25p-C. These results strongly suggest that the residues R80 and N81 are situated in or closely associated with the Ras2p specific site binding Sdc25p.

Properties of the SDC25 C-domain, a GDP to GTP exchange factor of RAS proteins and in vitro modulation of adenylyl cyclase.

The SDC25 C-domain, the product encoded by the 3'-terminal part of the Saccharomyces cerevisiae SDC25 gene, acts as a GDP dissociation stimulator on RAS proteins (Créchet, J.B., Poullet, P., Mistou, M. Y., Parmeggiani, A., Camonis, J., Boy-Marcotte, E., Damak, F., and Jacquet, M. (1990b) Science 248, 866-868). To define further its role in the RAS-adenylyl cyclase pathway, an in vitro system was used, which utilized cell membranes from yeast strains with appropriate genotypes carrying alterations in the positive regulators of adenylyl cyclase activity. The SDC25 C-domain was able to stimulate the adenylyl cyclase activity of membranes from RAS2 cdc25 strains. Our results indicate that the SDC25 C-domain activates adenylyl cyclase by rapidly recycling the active RAS2. or RAS1.GTP complex from the respective GDP complex. This is also supported by the observation that the stimulation of adenylyl cyclase activity by RAS2T152I, a mutant characterized by a constitutively fast GDP to GTP exchange, was insensitive to the action of the SDC25 C-domain. No direct influence of this GDP dissociation stimulator on adenylyl cyclase was detected. Biochemical evidence was obtained, showing that in the presence of the functional target of RAS, the adenylyl cyclase, the effects of SDC25 C-domain and the catalytic domain of GTPase-activating protein are antagonistic. This in vitro system allowed a quantitative evaluation of the effects of positive and negative effectors of RAS on adenylyl cyclase and the biochemical analysis of conditions inducing a phenotype of permanently activated adenylyl cyclase.

RAS residues that are distant from the GDP binding site play a critical role in dissociation factor-stimulated release of GDP.

We have previously shown that a conserved glycine at position 82 of the yeast RAS2 protein is involved in the conversion of RAS proteins from the GDP- to the GTP-bound form. We have now investigated the role of glycine 82 and neighbouring amino acids of the distal switch II region in the physiological mechanism of activation of RAS. We have introduced single and double amino acid substitutions at positions 80-83 of the RAS2 gene, and we have investigated the interaction of the corresponding proteins with a yeast GDP dissociation stimulator (SDC25 C-domain). Using purified RAS proteins, we have found that the SDC25-stimulated conversion of RAS from the GDP-bound inactive state to the GTP-bound active state was severely impaired by amino acid substitutions at positions 80-81. However, the rate and the extent of conversion from the GDP- to the GTP-bound form in the absence of dissociation factor was unaffected. The insensitivity of the mutated proteins to the dissociation factor in vitro was paralleled by an inhibitory effect on growth in vivo. The mutations did not significantly affect the interaction of RAS with adenylyl cyclase. These findings point to residues 80-82 as important determinants of the response of RAS to GDP dissociation factors. This suggests a molecular model for the enhancement of nucleotide release from RAS by such factors.

Role of glycine-82 as a pivot point during the transition from the inactive to the active form of the yeast Ras2 protein.

Ras proteins bind either GDP or GTP with high affinity. However, only the GTP-bound form of the yeast Ras2 protein is able to stimulate adenylyl cyclase. To identify amino acid residues that play a role in the conversion from the GDP-bound to the GTP-bound state of Ras proteins, we have searched for single amino acid substitutions that selectively affected the binding of one of the two nucleotides. We have found that the replacement of glycine-82 of the Ras2 protein by serine resulted in an increased rate of dissociation of Gpp(NH)p, a nonhydrolysable analog of GTP, while the GDP dissociation rate was not significantly modified. Glycine-82 resides in a region that is highly conserved between the yeast and human proteins. However, this residue is structurally distant from residues that participate in the binding of the nucleotide, as determined from the crystal structure of the human H-ras gene product. Therefore, the ability of the nucleotide binding site to discriminate between GDP and GTP is dependent not only on residues that are spatially close to the nucleotide, but also on distant amino acids. This is in agreement with the role of glycine-82 as a pivot point during the transition from the GDP- to the GTP-bound form of the Ras proteins.

Enhancement of the GDP-GTP exchange of RAS proteins by the carboxyl-terminal domain of SCD25.

In Saccharomyces cerevisiae, the product of the CDC25 gene controls the RAS-mediated production of adenosine 3',5'-monophosphate (cAMP). In vivo the carboxyl-terminal third of the CDC25 gene product is sufficient for the activation of adenylate cyclase. The 3'-terminal part of SCD25, a gene of S. cerevisiae structurally related to CDC25, can suppress the requirement for CDC25. Partially purified preparations of the carboxy-terminal domain of the SCD25 gene product enhanced the exchange rate of guanosine diphosphate (GDP) to guanosine triphosphate (GTP) of pure RAS2 protein by stimulating the release of GDP. This protein fragment had a similar effect on the human c-H-ras-encoded p21 protein. Thus, the SCD25 carboxyl-terminal domain can enhance the regeneration of the active form of RAS proteins.

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.

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.

Characterization of the elongation factors from calf brain. 1. Purification, molecular and immunological properties.

This work describes a method for the purification of the elongation factors (EF) from calf brain. The elongation factor responsible for the binding of aminoacyl-tRNA to the ribosome is found in this organ as a light form (EF-1 alpha) and as a component of heavy, polydispersed aggregates (EF-1H). EF-1 beta, the factor enhancing the EF-1 alpha GDP/GTP exchange, is part of EF-1H and of smaller aggregates. The fraction of EF-1 alpha and EF-1 beta not associated with EF-1H, and EF-2 have been purified to homogeneity after several chromatographic steps. EF-1H consists of many proteins; among them, EF-1 alpha, EF-1 beta and an EF-1 gamma-like protein represent three of the major components. This conclusively shows that EF-1H from calf brain is not a polydispersed aggregate of only EF-1 alpha. EF-1 beta has also been purified to homogeneity from EF-1H. The property of EF-1 beta to aggregate with other proteins suggests that this factor plays an important role in the organization of EF-1H. The relative molecular mass of the purified factors have been determined as: EF-1 alpha, 50,000; EF-1 beta, 30,000; the EF-1 gamma-like component, 49,000; EF-2, 85,000. Some cross-reactivity with the antibodies against the prokaryotic counterparts has been shown for EF-1 alpha, EF-1 beta and EF-2 by functional and immuno-precipitation methods, suggesting the existence of structural homologies.

Characterization of the elongation factors from calf brain. 2. Functional properties of EF-1 alpha, the action of physiological ligands and kirromycin.

The properties of EF-1 alpha from calf brain have been investigated and compared with those of EF-Tu. EF-1 alpha binds GDP and GTP in a 1:1 stoichiometry, showing the same affinity for both nucleotides (K'd = 2-4 microM). EF-1 beta strongly enhances the dissociation rate of the EF-1 alpha X GDP complex and to a lesser extent of the EF-1 alpha X GTP complex. Aminoacyl-tRNA (aa-tRNA) stabilized EF-1 alpha X GTP much less efficiently than the EF-Tu X GTP complex. Unlike EF-Tu, EF-1 alpha sustains the binding of aa-tRNA to the ribosome also in the presence of GDP or in the absence of any nucleotide, though to a lesser degree than with GTP. Kirromycin enhances the dissociation rate of both EF-1 alpha X GTP and EF-1 alpha X GDP but especially that of the latter. This effect results in an increase of the exchange rate of the EF-1 alpha-bound nucleotide with free nucleotides. Although in this regard the effect of kirromycin mimics that of EF-1 beta, the antibiotic is incapable of increasing the EF-1 alpha X GDP/GTP exchange rate when aa-tRNA and ribosomes are present. Therefore, unlike EF-1 beta, kirromycin cannot enhance the rate of poly(Phe) synthesis. On the other hand, the failure of kirromycin to induce a GTP-like conformation of EF-1 alpha X GDP, as in the case of EF-Tu X GDP, explains its inability to inhibit peptide bond formation in the eukaryotic system.

Characterization of the elongation factors from calf brain. 3. Properties of the GTPase activity of EF-1 alpha and mode of action of kirromycin.

The GTPase activity of purified EF-1 alpha from calf brain has been studied under various experimental conditions and compared with that of EF-Tu. EF-1 alpha displays a much higher GTPase turnover than EF-Tu in the absence of aminoacyl-tRNA (aa-tRNA) and ribosomes (intrinsic GTPase activity); this is due to the higher exchange rate between bound GDP and free GTP. Also the intrinsic GTPase of EF-1 alpha is enhanced by increasing the concentration of monovalent cations, K+ being more effective than NH+4. Differently from EF-Tu, aa-tRNA is much more active than ribosomes in stimulating the EF-1 alpha GTPase activity. However, ribosomes strongly reinforce the aa-tRNA effect. In the absence of aa-tRNA the rate-limiting step of the GTPase turnover appears to be the hydrolysis of GTP, whereas in its presence the GDP/GTP exchange reaction becomes rate-limiting, since addition of EF-1 beta enhances turnover GTPase activity. Kirromycin moderately inhibits the intrinsic GTPase of EF-1 alpha; this effect turns into stimulation when aa-tRNA is present. Addition of ribosomes abolishes any kirromycin effect. The inability of kirromycin to affect the EF-1 alpha/guanine-nucleotide interaction in the presence of ribosomes shows that, differently from EF-Tu, the EF-1 alpha X GDP/GTP exchange reaction takes place on the ribosome.

Characterization of a kirromycin-resistant elongation factor Tu from Escherichia coli.

The Escherichia coli strain D2216 contains a kirromycin-resistant elongation factor Tu [EF-Tu(D2216); Fischer, E., Wolf, H., Hantke K., & Parmeggiani, A. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 4341-4345]. This stain grows much more slowly than wild-type E. coli strains and contains less than half the amount of EF-Tu. On isoelectric focusing, the whole cell lysate of strain D2216 as well as pure, crystalline EF-Tu(D2216) comprises only a single species indistinguishable from wild-type EF-Tu. In poly(uridylic acid)- [poly(U)] directed poly(phenylalanine) synthesis, enzymatic binding of aminoacyl transfer ribonucleic acid to the ribosome, and susceptibility to trypsin digestion, EF-Tu(D2216) behaves similarly to the EF-Tu from wild-type strains. Kirromycin, which increases the sensitivity to trypsinization of wild-type EF-Tu, has no effect on mutant EF-Tu. In poly(U)-directed poly(phenylalanine) synthesis, partially trypsinized EF-Tu(D2216) displays a 7-fold reduction of its kirromycin resistance as compared to the intact EF-Tu(D2216). This is approximately 300 times less sensitive to the antibiotic than wild-type EF-Tu. The EF-Tu(D2216), purified and crystallized, exhibits a guanosine 5'-triphosphatase activity in the absence of any other physiological effector or kirromycin. This activity is not a contaminant, since it can be selectively stimulated by ribosomes and is inactivated by temperature exactly in the same way as the guanosine 5'-diphosphate binding activity of Ef-Tu(D2216). We conclude that, as consequence of the mutation, the catalytic center of EF-Tu(D2216)-dependent guanosine 5'-triphosphate hydrolysis undergoes spontaneous activation.

Interaction of elongation factor Tu with the ribosome. A study using the antibiotic kirromycin.

Elongation factor Tu (EF-Tu) dependent GTP hydrolysis normally requires the presence of ribosomes and aminoacyl-tRNA (aa-tRNA). In the presence of the antibiotic kirromycin, the factor alone displays a GTPase activity that is enhanced by ribosomes and/or aa-tRNA [Wolf, H., Chinali, G., & Parmeggiani, A. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 4910-4914]. Using this system, we have found the following: (1) the 50S ribosomal subunit can substitute the 70S ribosome; (2) the 50S CsCl core a, b, and c particles [Sander, G., Marsh, R. C., Voigt, J., & Parmeggiani, A. (1975) Biochemistry 14, 1805-1814], lacking an increasing number of proteins, can induce ca. 65, 45, and 25%, respectively, of the EF-Tu-kirromycin GTPase activity of control 50S subunits, in the presence of 30S subunits and aa-tRNA; (3) addition of proteins L7/L12 with L10, but not of proteins L7/L12 free from L10, restored the activity of all the 50S CsCl cores in the EF-Tu-kirromycin-dependent GTPase to 70-90% of the control; (4) proteins L7/L12, with or without contaminating L10, did not induce any EF-Tu-dependent GTPase activity, in contrast to a recent report [Donner, D., Villems, R., Liljas, A., & Kurland, C. G. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 3192-3195], whether EF-Ts and/or kirromycin were present or not.