Activation state of the Ras2 protein and glucose-induced signaling in Saccharomyces cerevisiae

The activity of adenylate cyclase in the yeast Saccharomyces cerevisiae is controlled by two G-protein systems, the Ras proteins and the Galpha protein Gpa2. Glucose activation of cAMP synthesis is thought to be mediated by Gpa2 and its G-protein-coupled receptor Gpr1. Using a sensitive GTP-loading assay for Ras2 we demonstrate that glucose addition also triggers a fast increase in the GTP loading state of Ras2 concomitant with the glucose-induced increase in cAMP. This increase is severely delayed in a strain lacking Cdc25, the guanine nucleotide exchange factor for Ras proteins. Deletion of the Ras-GAPs IRA2 (alone or with IRA1) or the presence of RAS2Val19 allele causes constitutively high Ras GTP loading that no longer increases upon glucose addition. The glucose-induced increase in Ras2 GTP-loading is not dependent on Gpr1 or Gpa2. Deletion of these proteins causes higher GTP loading indicating that the two G-protein systems might directly or indirectly interact. Because deletion of GPR1 or GPA2 reduces the glucose-induced cAMP increase the observed enhancement of Ras2 GTP loading is not sufficient for full stimulation of cAMP synthesis. Glucose phosphorylation by glucokinase or the hexokinases is required for glucose-induced Ras2 GTP loading. These results indicate that glucose phosphorylation might sustain activation of cAMP synthesis by enhancing Ras2 GTP loading likely through inhibition of the Ira proteins. Strains with reduced feedback inhibition on cAMP synthesis also display elevated basal and induced Ras2 GTP loading consistent with the Ras2 protein acting as a target of the feedback-inhibition mechanism.     

Localization of Ras signaling complex in budding yeast

In Saccharomyces cerevisiae, cAMP/pKA pathway plays a major role in metabolism, stress resistance and proliferation control. cAMP is produced by adenylate cyclase, which is activated both by Gpr1/Gpa2 system and Ras proteins, regulated by Cdc25/Sdc25 guanine exchange factors and Ira GTPase activator proteins. Recently, both Ras2 and Cdc25 RasGEF were reported to localize not only in plasma membrane but also in internal membranes. Here, the subcellular localization of Ras signaling complex proteins was investigated both by fluorescent tagging and by biochemical cell membrane fractionation on sucrose gradients. Although a consistent minor fraction of Ras signaling complex components was found in plasma membrane during exponential growth on glucose, Cdc25 appears to localize mainly on ER membranes, while Ira2 and Cyr1 are also significantly present on mitochondria. Moreover, PKA Tpk1 catalytic subunit overexpression induces Ira2 protein to move from mitochondria to ER membranes. These data confirm the hypothesis that different branches of Ras signaling pathways could involve different subcellular compartments, and that relocalization of Ras signaling complex components is subject to PKA control.     

A mutation in Saccharomyces cerevisiae adenylate cyclase, Cyr1K1876M, specifically affects glucose- and acidification-induced cAMP signalling and not the basal cAMP level

In the yeast Saccharomyces cerevisiae, the addition of glucose to derepressed cells and intracellular acidification trigger a rapid increase in the cAMP level within 1 min. We have identified a mutation in the genetic background of several related 'wild-type' laboratory yeast strains (e.g. ENY.cat80-7A, CEN.PK2-1C) that largely prevents both cAMP responses, and we have called it lcr1 (for lack of cAMP responses). Subsequent analysis showed that lcr1 was allelic to CYR1/CDC35, encoding adenylate cyclase, and that it contained an A to T substitution at position 5627. This corresponds to a K1876M substitution near the end of the catalytic domain in adenylate cyclase. Introduction of the A5627T mutation into the CYR1 gene of a W303-1A wild-type strain largely eliminated glucose- and acidification-induced cAMP signalling and also the transient cAMP increase that occurs in the lag phase of growth. Hence, lysine1876 of adenylate cyclase is essential for cAMP responses in vivo. Lysine1876 is conserved in Schizosaccharomyces pombe adenylate cyclase. Mn2+-dependent adenylate cyclase activity in isolated plasma membranes of the cyr1met1876 (lcr1) strain was similar to that in the isogenic wild-type strain, but GTP/Mg2+-dependent activity was strongly reduced, 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 cyr1met1876 (lcr1) mutant. During exponential growth on glucose, there was little effect on these protein kinase A (PKA) targets, indicating that the importance of glucose-induced cAMP signalling is restricted to the transition from gluconeogenic/respiratory to fermentative growth. Inhibition of growth by weak acids was reduced, consistent with prevention of the intracellular acidification effect on cAMP by the cyr1met1876 (lcr1) mutation. The mutation partially suppressed the effect of RAS2val19 and GPA2val132 on several PKA targets. These results demonstrate the usefulness of the cyr1met1876 (lcr1) mutation for epistasis studies on the signalling function of the cAMP pathway.     

A Saccharomyces cerevisiae G-protein coupled receptor, Gpr1, is specifically required for glucose activation of the cAMP pathway during the transition to growth on glucose

In the yeast Saccharomyces cerevisiae the accumulation of cAMP is controlled by an elaborate pathway. Only two triggers of the Ras adenylate cyclase pathway are known. Intracellular acidification induces a Ras-mediated long-lasting cAMP increase. Addition of glucose to cells grown on a non-fermentable carbon source or to stationary-phase cells triggers a transient burst in the intracellular cAMP level. This glucose-induced cAMP signal is dependent on the G alpha-protein Gpa2. We show that the G-protein coupled receptor (GPCR) Gpr1 interacts with Gpa2 and is required for stimulation of cAMP synthesis by glucose. Gpr1 displays sequence homology to GPCRs of higher organisms. The absence of Gpr1 is rescued by the constitutively activated Gpa2Val-132 allele. In addition, we isolated a mutant allele of GPR1, named fil2, in a screen for mutants deficient in glucose-induced loss of heat resistance, which is consistent with its lack of glucose-induced cAMP activation. Apparently, Gpr1 together with Gpa2 constitute a glucose-sensing system for activation of the cAMP pathway. Deletion of Gpr1 and/or Gpa2 affected cAPK-controlled features (levels of trehalose, glycogen, heat resistance, expression of STRE-controlled genes and ribosomal protein genes) specifically during the transition to growth on glucose. Hence, an alternative glucose-sensing system must signal glucose availability for the Sch9-dependent pathway during growth on glucose. This appears to be the first example of a GPCR system activated by a nutrient in eukaryotic cells. Hence, a subfamily of GPCRs might be involved in nutrient sensing.     

The kelch proteins Gpb1 and Gpb2 inhibit Ras activity via association with the yeast RasGAP neurofibromin homologs Ira1 and Ira2

The G protein-coupled receptor Gpr1 and associated Galpha subunit Gpa2 govern dimorphic transitions in response to extracellular nutrients by signaling coordinately with Ras to activate adenylyl cyclase in the yeast Saccharomyces cerevisiae. Gpa2 forms a protein complex with the kelch Gbeta mimic subunits Gpb1/2, and previous studies demonstrate that Gpb1/2 negatively control cAMP-PKA signaling via Gpa2 and an unknown second target. Here, we define these targets of Gpb1/2 as the yeast neurofibromin homologs Ira1 and Ira2, which function as GTPase activating proteins of Ras. Gpb1/2 bind to a conserved C-terminal domain of Ira1/2, and loss of Gpb1/2 results in a destabilization of Ira1 and Ira2, leading to elevated levels of Ras2-GTP and unbridled cAMP-PKA signaling. Because the Gpb1/2 binding domain on Ira1/2 is conserved in the human neurofibromin protein, an analogous signaling network may contribute to the neoplastic development of neurofibromatosis type 1.     

Role of guanine nucleotides in the regulation of the Ras/cAMP pathway in Saccharomyces cerevisiae

The CDC25 gene product is a guanine nucleotide exchange factor for Ras proteins in yeast. Recently it has been suggested that the intracellular levels of guanine nucleotides may influence the exchange reaction. To test this hypothesis we measured the levels of nucleotides in yeast cells under different growth conditions and the relative amount of Ras2-GTP. The intracellular GTP/GDP ratio was found to be very sensitive to growth conditions: the ratio is high, close to that of ATP/ADP during exponential growth, but it decreases rapidly before the beginning of stationary phase, and it drops further under starvation conditions. The addition of glucose to glucose-starved cells causes a fast increase of the GTP/GDP ratio. The relative amount of Ras2-GTP changes in a parallel way suggesting that there is a correlation with the cytosolic GTP/GDP ratio. In addition 'in vitro' mixed-nucleotide exchange experiments done on purified Ras2 protein demonstrated that the GTP and GDP concentrations influence the extent of Ras2-GTP loading giving further support to their possible regulatory role.     

The RACK1 ortholog Asc1 functions as a G-protein beta subunit coupled to glucose responsiveness in yeast

According to the prevailing paradigm, G-proteins are composed of three subunits, an alpha subunit with GTPase activity and a tightly associated betagamma subunit complex. In the yeast Saccharomyces cerevisiae there are two known Galpha proteins (Gpa1 and Gpa2) but only one Gbetagamma, which binds only to Gpa1. Here we show that the yeast ortholog of RACK1 (receptor for activated protein kinase C1) Asc1 functions as the Gbeta for Gpa2. As with other known Gbeta proteins, Asc1 has a 7-WD domain structure, interacts directly with the Galpha in a guanine nucleotide-dependent manner, and inhibits Galpha guanine nucleotide exchange activity. In addition, Asc1 binds to the effector enzyme adenylyl cyclase (Cyr1), and diminishes the production of cAMP in response to glucose stimulation. Thus, whereas Gpa2 promotes glucose signaling through elevated production of cAMP, Asc1 has opposing effects on these same processes. Our findings reveal the existence of an unusual Gbeta subunit, one having multiple functions within the cell in addition to serving as a signal transducer for cell surface receptors and intracellular effectors.     

Rom2p, the Rho1 GTP/GDP exchange factor of Saccharomyces cerevisiae, can mediate stress responses via the Ras-cAMP pathway

The Ras-cyclic AMP pathway is connected to other nutrient-regulated signaling pathways and mediates the global stress responses of Saccharomyces cerevisiae. Here, we show that Rom2p, the Rho1 GTP/GDP exchange factor, can mediate stress responses and cell growth via the Ras-cAMP pathways. ROM2 was isolated as a suppresser of heat and NaCl sensitivity caused by the lack of the Ras-GTPase activator Ira2p or of cAMP phosphodiesterases. Subsequent analysis of strains with a rom2 deletion showed that Rom2p is essential for resistance to a variety of stresses caused by freeze-thawing, oxidants, cycloheximide, NaCl, or cobalt ions. Stress sensitivity and the growth defect caused by the rom2 deletion could be suppressed by depleting Ras or protein kinase A (PKA) activity or by overexpressing the high affinity cAMP phosphodiesterase Pde2p. In addition, overexpression of ROM2 could not rescue cells lacking the regulatory subunit of PKA, indicating that the Ras-adenylate, cyclase-PKA cascade is essential for Rom2p-mediated stress responses and cell growth. Deletion of IRA2 exacerbated the freeze-thaw sensitivity and growth defect of the rom2 mutant, indicating that Rom2p signaling may control Ras independently of IRA2. Increases in cAMP levels were detected in the rom2 deletion mutants, and these were comparable with the effects of an ira2 mutation. The effects of the deletion of ROM2 on sensitivity to hydrogen peroxide, paraquat, and cobalt ions, but not to caffeine, were reduced when a constitutive allele of RHO1 was introduced on a single copy plasmid. However, the effects of the deletion of ROM2 on sensitivity to diamide and NaCl were exacerbated. Taken together, our data indicate that Rom2p can regulate PKA activity by controlling cAMP levels via the Ras-cAMP pathway and that for those stresses related to oxidative stress, this cross-talk is probably mediated via the Rho1p-activated MAPK pathway.     

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.     

Phospholipase C binds to the receptor-like GPR1 protein and controls pseudohyphal differentiation in Saccharomyces cerevisiae.

The hormone receptor-like protein Gpr1p physically interacts with phosphatidylinositol-specific phospholipase C (Plc1p) and with the Galpha protein Gpa2p, as shown by two-hybrid assays and co-immune precipitation of epitope-tagged proteins. Plc1p binds to Gpr1p in either the presence or absence of Gpa2, whereas the Gpr1p/Gpa2p association depends on the presence of Plc1p. Genetic interactions between the null mutations plc1Delta, gpr1Delta, gpa2Delta, and ras2Delta suggest that Plc1p acts together with Gpr1p and Gpa2p in a growth control pathway operating in parallel to the Ras2p function. Diploid cells lacking Gpr1p, Plc1p, or Gpa2p fail to form pseudohyphae upon nitrogen depletion, and the filamentation defect of gpr1Delta and plc1Delta strains is rescued by activating a mitogen-activated protein kinase pathway via STE11-4 or by activating a cAMP pathway via overexpressed Tpk2p. Plc1p is also required for efficient expression of the FG(TyA)::lacZ reporter gene under nitrogen depletion. In conclusion, we have identified two physically interacting proteins, Gpr1p and Plc1p, as novel components of a nitrogen signaling pathway controlling the developmental switch from yeast-like to pseudohyphal growth. Our data suggest that phospholipase C modulates the interaction of the putative nutrient sensor Gpr1p with the Galpha protein Gpa2p as a downstream effector of filamentation control.     

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.     

Transient increase in Ca2+ influx in Saccharomyces cerevisiae in response to glucose: effects of intracellular acidification and cAMP levels

Influx of 45Ca2+ into Saccharomyces cerevisiae was measured under experimental conditions which enabled measurements of initial rate of transport across the plasma membrane, without interference by the vacuolar Ca2+ transport system. Addition of glucose or glycerol to the cells, after pre-incubation in glucose-free medium for 5 min, caused a rapid, transient increase in 45Ca2+ influx, reaching a peak at 3-5 min after addition of substrate. Ethanol, or glycerol added with antimycin A, had no effect on 45Ca2+ influx. We have shown previously that this increase is not mediated by an effect of the substrates on intracellular ATP levels. Changes in membrane potential accounted for only a part of the glucose-stimulated 45Ca2+ influx. The roles of intracellular acidification and changes in cellular cAMP in mediating the effects of glucose on 45Ca2+ influx were examined. After a short preincubation in glucose-free medium addition of glucose caused a decrease in the intracellular pH, [pH]i, which reached a minimum value after 3 min. A transient increase in the cellular cAMP level was also observed. Addition of glycerol also caused intracellular acidification, but ethanol or glycerol added with antimycin A had no effect on [pH]i. Artificial intracellular acidification induced by exposure to isobutyric acid or to CCCP caused a transient rise in Ca2+ influx but the extent of the increase was smaller than that caused by glucose, and the time-course was different. We conclude that intracellular acidification may be responsible for part of the glucose stimulation of Ca2+ influx.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glucose-induced cAMP signalling in yeast requires both a G-protein coupled receptor system for extracellular glucose detection and a separable hexose kinase-dependent sensing process

In Saccharomyces cerevisiae, glucose activation of cAMP synthesis requires both the presence of the G-protein-coupled receptor (GPCR) system, Gpr1-Gpa2, and uptake and phosphorylation of the sugar. In a hxt-null strain that lacks all physiologically important glucose carriers, glucose transport as well as glucose-induced cAMP signalling can be restored by constitutive expression of the galactose permease. Hence, the glucose transporters do not seem to have a regulatory function but are only required for glucose uptake. We established a system in which the GPCR-dependent glucose-sensing process is separated from the glucose phosphorylation process. It is based on the specific transport and hydrolysis of maltose providing intracellular glucose in the absence of glucose transport. Preaddition of a low concentration (0.7 mM) of maltose to derepressed hxt-null cells and subsequent addition of glucose restored the glucose-induced cAMP signalling, although there was no glucose uptake. Addition of a low concentration of maltose itself does not increase the cAMP level but enhances Glu6P and apparently fulfils the intracellular glucose phosphorylation requirement for activation of the cAMP pathway by extracellular glucose. This system enabled us to analyse the affinity and specificity of the GPCR system for fermentable sugars. Gpr1 displayed a very low affinity for glucose (apparent Ka = 75 mM) and responded specifically to extracellular alpha and beta D-glucose and sucrose, but not to fructose, mannose or any glucose analogues tested. The presence of the constitutively active Gpa2val132 allele in a wild-type strain bypassed the requirement for Gpr1 and increased the low cAMP signal induced by fructose and by low glucose up to the same intensity as the high glucose signal. Therefore, the low cAMP increases observed with fructose and low glucose in wild-type cells result only from the low sensitivity of the Gpr1-Gpa2 system and not from the intracellular sugar kinase-dependent process. In conclusion, we have shown that the two essential requirements for glucose-induced activation of cAMP synthesis can be fulfilled separately: an extracellular glucose detection process dependent on Gpr1 and an intracellular sugar-sensing process requiring the hexose kinases.     

Cyclic AMP-independent regulation of protein kinase A substrate phosphorylation by Kelch repeat proteins

Pseudohyphal and invasive growth in the yeast Saccharomyces cerevisiae is regulated by the kelch repeat-containing proteins Gpb1p and Gpb2p, which act downstream of the G protein alpha-subunit Gpa2p. Here we show that deletion of GPB1 and GPB2 causes increased haploid invasive growth in cells containing any one of the three protein kinase A (PKA) catalytic subunits, suggesting that Gpb1p and Gpb2p are able to inhibit each of these kinases. Cells containing gpb1Delta gpb2Delta mutations also display increased phosphorylation of the PKA substrates Sfl1p and Msn2p, indicating that Gpb1p and Gpb2p are negative regulators of PKA substrate phosphorylation. Stimulation of PKA-dependent signaling by gpb1Delta gpb2Delta mutations occurs in cells that lack both adenylyl cyclase and the high-affinity cyclic AMP (cAMP) phosphodiesterase. This effect is also seen in cells that lack the low-affinity cAMP phosphodiesterase. Given that these three enzymes control the synthesis and degradation of cAMP, these results indicate that the effect of Gpb1p and Gpb2p on PKA substrate phosphorylation does not occur by regulating the intracellular cAMP concentration. These findings suggest that Gpb1p and Gpb2p mediate their effects on the cAMP/PKA signaling pathway either by inhibiting the activity of PKA in a cAMP-independent manner or by activating phosphatases that act on PKA substrates.     

The Ras/cAMP/protein kinase A pathway regulates glucose-dependent assembly of the vacuolar (H+)-ATPase in yeast

Vacuolar (H+)-ATPases (V-ATPases) are ubiquitous, ATP-driven proton pumps that acidify organelles or the extracellular space. A rapid and effective mechanism for regulating V-ATPase activity involves reversible dissociation of the two functional domains of the pump, V1 and V0. This process is best characterized in yeast, where V-ATPases are reversibly disassembled in response to glucose depletion. To identify regulators that control this process in vivo, a genetic screen was performed in yeast to search for mutants that cannot disassemble their V-ATPases when grown in the absence of glucose. This screen identified IRA1 (inhibitory regulator of the Ras/cAMP pathway 1) and IRA2 as essential genes for regulating V-ATPase dissociation in vivo. IRA1 and IRA2 encode GTPase-activating proteins that negatively regulate Ras in nutrient-poor conditions. Down-regulation of Ras lowers cAMP levels by reducing adenylate cyclase activity. Decreased cAMP levels in turn lead to reduced activity of protein kinase A (PKA). Our results show that targeted deletion of IRA2 results in defective disassembly of the V-ATPase in response to glucose depletion, and reexpression of the gene rescues this phenotype. Glucose-dependent dissociation is also blocked in strains expressing the dominant active RAS2val19 allele or in strains deficient for the regulatory subunit of PKA, both of which lead to constitutively active PKA. These results reveal a role for PKA in controlling glucose-dependent V-ATPase assembly in yeast.     

The large N-terminal domain of Cdc25 protein of the yeast Saccharomyces cerevisiae is required for glucose-induced Ras2 activation

The Saccharomyces cerevisiae CDC25 gene encodes a guanine nucleotide exchange factor for Ras proteins whose catalytic domain is highly homologous to Ras-guanine nucleotide exchange factors from higher eukaryotes. In this study, glucose-induced Ras activation and cAMP response were investigated in mutants lacking the N-terminal domain of Cdc25 or where the entire CDC25 coding sequence was substituted by an expression cassette for a mammalian guanine nucleotide exchange factor catalytic domain. Our results suggest that an unregulated, low Ras guanine nucleotide exchange factor activity allows a normal glucose-induced cAMP signal that appears to be mediated mainly by the Gpr1/Gpa2 system, but it was not enough to sustain the glucose-induced increase of Ras2-GTP normally observed in a wild-type strain.     

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.