Properties of the exchange rate of guanine nucleotides to the novel rap-2B protein

Rap-2B is a novel ras-related protein that is 89% identical to rap-2 at the amino acid level. Based on its amino acid sequence, it is anticipated that rap-2B binds guanine nucleotides. Here we show that purified, bacterially expressed rap-2B does bind both GTP and GDP in a Mg2(+)-dependent fashion. The relative affinity of rap-2B for GTP is higher than that for GDP, both at low and high concentrations of Mg2+. This contrasts with N-ras p21 and could be of functional significance. Moreover, a polyclonal antiserum was raised against the recombinant rap-2B protein purified from E. coli lysates. This antiserum recognized a major protein of Mr approximately 21000 on Western blots of platelet membrane proteins, and immunoprecipitates rap-2B complexed with GTP or GDP.     

Analysis of rate constants governing the exchange of guanine nucleotides bound to EF-Tu catalysed by EF-Ts.

The kinetics of the heterologous exchange of GDP bound to EF-Tu by free GTP catalysed by EF-Ts have been analysed with a view to correlating results obtainable with different computational procedures. The affinity of EF-Ts for EF-Tu.GTP was found to be somewhat less than previously proposed by Romero et al. (Biochemistry 260, 6167:1985) though still greater than for EF-Tu.GDP. There is a close interrelationship between the constants for the binding of GTP to EF-Tu.EF-Ts and of EF-Ts to EF-Tu.GTP. The declining fractional rate of exchange observed by Romero et al. during displacement of GDP by GTP appears to be dependent on the ratio of the rate constants (k-1 + k-2)k4/k1k-2 as defined in the text, not on that of K4/K1 as they proposed.     

The CDC25 protein of Saccharomyces cerevisiae promotes exchange of guanine nucleotides bound to ras.

The product of the CDC25 gene of Saccharomyces cerevisiae, in its capacity as an activator of the RAS/cyclic AMP pathway, is required for initiation of the cell cycle. In this report, we provide an identification of Cdc25p, the product of the CDC25 gene, and evidence that it promotes exchange of guanine nucleotides bound to Ras in vitro. Extracts of strains containing high levels of Cdc25p catalyze both removal of GDP from and the concurrent binding of GTP to Ras. This same activity is also obtained with an immunopurified Cdc25p-beta-galactosidase fusion protein, suggesting that Cdc25p participates directly in the exchange reaction. This biochemical activity is consistent with previous genetic analysis of CDC25 function.     

Mutations in the GCD7 subunit of yeast guanine nucleotide exchange factor eIF-2B overcome the inhibitory effects of phosphorylated eIF-2 on translation initiation.

Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2 alpha) impairs translation initiation by inhibiting the guanine nucleotide exchange factor for eIF-2, known as eIF-2B. In Saccharomyces cerevisiae, phosphorylation of eIF-2 alpha by the protein kinase GCN2 specifically stimulates translation of GCN4 mRNA in addition to reducing general protein synthesis. We isolated mutations in several unlinked genes that suppress the growth-inhibitory effect of eIF-2 alpha phosphorylation catalyzed by mutationally activated forms of GCN2. These suppressor mutations, affecting eIF-2 alpha and the essential subunits of eIF-2B encoded by GCD7 and GCD2, do not reduce the level of eIF-2 alpha phosphorylation in cells expressing the activated GCN2c kinase. Four GCD7 suppressors were shown to reduce the derepression of GCN4 translation in cells containing wild-type GCN2 under starvation conditions or in GCN2c strains. A fifth GCD7 allele, constructed in vitro by combining two of the GCD7 suppressors mutations, completely impaired the derepression of GCN4 translation, a phenotype characteristic of deletions in GCN1, GCN2, or GCN3. This double GCD7 mutation also completely suppressed the lethal effect of expressing the mammalian eIF-2 alpha kinase dsRNA-PK in yeast cells, showing that the translational machinery had been rendered completely insensitive to phosphorylated eIF-2. None of the GCD7 mutations had any detrimental effect on cell growth under nonstarvation conditions, suggesting that recycling of eIF-2 occurs efficiently in the suppressor strains. We propose that GCD7 and GCD2 play important roles in the regulatory interaction between eIF-2 and eIF-2B and that the suppressor mutations we isolated in these genes decrease the susceptibility of eIF-2B to the inhibitory effects of phosphorylated eIF-2 without impairing the essential catalytic function of eIF-2B in translation initiation.     

Expression of mutant eukaryotic initiation factor 2 alpha subunit (eIF-2 alpha) reduces inhibition of guanine nucleotide exchange activity of eIF-2B mediated by eIF-2 alpha phosphorylation.

The inhibition of protein synthesis that occurs upon phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha) at serine 51 correlates with reduced guanine nucleotide exchange activity of eIF-2B in vivo and inhibition of eIF-2B activity in vitro, although it is not known if phosphorylation is the cause of the reduced eIF-2B activity in vivo. To characterize the importance of eIF-2 alpha phosphorylation in the regulation of eIF-2B activity, we studied the overexpression of mutant eIF-2 alpha subunits in which serine 48 or 51 was replaced by an alanine (48A or 51A mutant). Previous studies demonstrated that the 51A mutant was resistant to phosphorylation, whereas the 48A mutant was a substrate for phosphorylation. Additionally, expression of either mutant partially protected Chinese hamster ovary (CHO) cells from the inhibition of protein synthesis in response to heat shock treatment (P. Murtha-Riel, M. V. Davies, J. B. Scherer, S. Y. Choi, J. W. B. Hershey, and R. J. Kaufman, J. Biol. Chem. 268:12946-12951, 1993). In this study, we show that eIF-2B activity was inhibited in parental CHO cell extracts upon addition of purified reticulocyte heme-regulated inhibitor (HRI), an eIF-2 alpha kinase that phosphorylates Ser-51. Preincubation with purified HRI also reduced the eIF-2B activity in extracts from cells overexpressing wild-type eIF-2 alpha. In contrast, the eIF-2B activity was not readily inhibited in extracts from cells overexpressing either the eIF-2 alpha 48A or 51A mutant. In addition, eIF-2B activity was decreased in extracts prepared from heat-shocked cells overexpressing wild-type eIF-2 alpha, whereas the decrease in eIF-2B activity was less in heat-shocked cells overexpressing either mutant 48A or mutant 51A. While the phosphorylation at serine 51 in eIF-2 alpha impairs the eIF-2B activity, we propose that serine 48 acts to maintain a high affinity between phosphorylated eIF-2 alpha and eIF-2B, thereby inactivating eIF-2B activity. These findings support the hypothesis that phosphorylation of eIF-2 alpha inhibits protein synthesis directly through reducing eIF-2B activity and emphasize the importance of both serine 48 and serine 51 in the interaction with eIF-2B and regulation of eIF-2B activity.     

The effect of Mg2+ and guanine nucleotide exchange factor on the binding of guanine nucleotides to eukaryotic initiation factor 2

A major site of regulation of polypeptide chain initiation is the binding of Met-tRNA to 40 S ribosomal subunits which is mediated by eukaryotic initiation factor 2 (eIF-2). The formation of ternary complex, eIF-2.GTP.Met-tRNA, is potently inhibited by GDP. Measurement of the parameters for guanine nucleotide binding to eIF-2 is critical to understanding the control of protein synthesis by fluctuations in cellular energy levels. We have compared the dissociation constants (Kd) of eIF-2.GDP and eIF-2.GTP and find that GDP has a 400-fold higher affinity for GDP than GTP. The Kd for GDP is almost an order of magnitude less than has been reported previously. The difference between the Kd values for the two nucleotides is the result of a faster rate constant for GTP release, the rate constants for binding being approximately equal. This combination of rate constants and low levels of contaminating GDP in preparations of GTP can explain the apparently unstable nature of eIF-2.GTP observed by others. Mg2+ stabilizes binary complexes slowing the rates of release of nucleotide from both eIF-2.GDP and eIF-2.GTP. The competition between GTP and GDP for binding to eIF-2.guanine nucleotide exchange factor complex has been measured. A 10-fold higher GTP concentration than GDP is required to reduce [32P] GDP binding to eIF-2.guanine nucleotide exchange factor complex by 50%. The relevance of this competition to the regulation of protein synthesis by energy levels is discussed.     

Identification of guanine nucleotides bound to ras-encoded proteins in growing yeast cells

We have analyzed the guanine nucleotides bound to mammalian ras and yeast RAS proteins overexpressed in [32P]orthophosphate-labeled cultures of exponentially growing Saccharomyces cerevisiae cells. Whereas S. cerevisiae RAS1 and RAS2 proteins were immunoprecipitated bound entirely to GDP, mammalian Harvey ras was isolated with GTP and GDP bound in near-equimolar proportions. In a strain overexpressing a RAS2 variant where the RAS unique C-terminal domain was deleted, both GTP and GDP were detected in a ratio of 3:97. Increased amounts of GTP (16-75% of total guanine nucleotide) were observed bound to all ras proteins containing mutations that inhibit GTP hydrolytic activity. Increasing proportions of GTP bound to the various ras proteins correlated with increasing biological potency to bypass cdc25 lethality in yeast.     

Relationship between tubulin SH groups and bound guanine nucleotides.

Guanine nucleotides bound to both the non-exchangeable sites (N sites) and exchangeable sites (E sites) of tubulin were completely released after 7 moles of SH groups per tubulin subunit (55,000 molecular weight) had reacted with PCMPS. The blockage of 2 moles of SH groups in the glycerol-reassembly buffer or 1 mole of SH groups in glycerol-free reassembly buffer resulted in complete loss of tubulin polymerizability. However, under both sets of experimental conditions, the amount of guanine nucleotides released from the E sites was less than 8% and the loss of total guanine nucleotides was only 5%. Addition of GSH did not induce reassociation of released guanine nucleotides, although it restored tubulin polymerizability. These results indicate that the loss of tubulin polymerizability on blockage of the SH groups was not due to dissociation of bound guanine nucleotides and that the binding sites of the nucleotides were independent of the SH groups in tubulin required for polymerization. Furthermore, blockage of SH groups did not change the ratio of GTP to GDP bound to tubulin.     

Kinetic constants in the functioning of eIF-2 and eIF-2B

Minimum rate constants for reactions catalysed by the eukaryotic initiation factor eIF-2B in promoting formation of the ternary complex eIF-2.GTP.met-tRNAi from eIF-2.GDP are estimated from published data. The most plausible sequence of reactions in vivo is when eIF-2B remains bound to eIF-2.GTP.met-tRNA. Rate constants for reaction of eIF-2B and eIF-2.GDP are too large for protein:protein interactions at cellular concentrations in free solution. This finding suggests some form of sequestration of eIF-2 and eIF-2B in the cell to facilitate interaction, which may result in only a portion of cellular eIF-2 being actively engaged in initiation.     

Multidomain organization of eukaryotic guanine nucleotide exchange translation initiation factor eIF-2B subunits revealed by analysis of conserved sequence motifs.

Computer-assisted analysis of amino acid sequences using methods for database screening with individual sequences and with multiple alignment blocks reveals a complex multidomain organization of yeast proteins GCD6 and GCD1, and mammalian homolog of GCD6-subunits of the eukaryotic translation initiation factor eIF-2B involved in GDP/GTP exchange on eIF-2. It is shown that these proteins contain a putative nucleotide-binding domain related to a variety of nucleotidyltransferases, most of which are involved in nucleoside diphosphate-sugar formation in bacteria. Three conserved motifs, one of which appears to be a variant of the phosphate-binding site (P-loop) and another that may be considered a specific version of the Mg(2+)-binding site of NTP-utilizing enzymes, were identified in the nucleotidyltransferase-related domain. Together with the third unique motif adjacent to the the P-loop, these motifs comprise the signature of a new superfamily of nucleotide-binding domains. A domain consisting of hexapeptide amino acid repeats with a periodic distribution of bulky hydrophobic residues (isoleucine patch), which previously have been identified in bacterial acetyltransferases, is located toward the C-terminus from the nucleotidyltransferase-related domain. Finally, at the very C-termini of GCD6, eIF-2B epsilon, and two other eukaryotic translation initiation factors, eIF-4 gamma and eIF-5, there is a previously undetected, conserved domain. It is hypothesized that the nucleotidyltransferase-related domain is directly involved in the GDP/GTP exchange, whereas the C-terminal conserved domain may be involved in the interaction of eIF-2B, eIF-4 gamma, and eIF-5 with eIF-2.     

The association of eIF-2 with Met-tRNAi or eIF-2B alters the specificity of eIF-2 phosphatase

In unfractioned reticulocyte lysate, interaction of eukaryotic initiation factor 2 (eIF-2) with other components regulates the accessibility of phosphatases and kinases to phosphorylation sites on its alpha and beta subunits. Upon addition of eIF-2 phosphorylated on both alpha and beta subunits (eIF-2(alpha 32P, beta 32P) to lysate, the alpha subunit is rapidly dephosphorylated, but the beta subunit is not. In contrast, both sites are rapidly dephosphorylated by the purified phosphatase. The basis of this altered specificity appears to be the association of eIF-2 with other translational components rather than an alteration of the phosphatase. Formation of an eIF-2(alpha 32P,beta 32P) Met-tRNAi X GTP ternary complex prevents dephosphorylation of the beta subunit, but has no effect on the rate of alpha dephosphorylation. eIF-2B, a 280,000-dalton polypeptide complex required for GTP:GDP exchange, also protects the beta subunit phosphorylation site from the purified phosphatase. However, the dephosphorylation of eIF-2(alpha 32P) is inhibited by 75% while complexed with eIF-2B. The altered phosphatase specificity upon association of eIF-2 with eIF-2B also affects the access of protein kinases to these phosphorylation sites. In the eIF-2B X eIF-2 complex, the alpha subunit is phosphorylated at 30% the rate of free eIF-2. Under identical conditions, phosphorylation of eIF-2 beta can not be detected. These results illustrate the importance of substrate conformation and/or functional association with other components in determining the overall phosphorylation state allowed by alterations of kinase and phosphatase activities.     

Release of exchangeably bound guanine nucleotides from tubulin in a magnesium-free buffer

The number of moles of guanine nucleotides (denoted GXP), either guanosine 5'-triphosphate (GTP) or guanosine 5'-diphosphate (GDP), bound to a mole of phosphocellulose-purified tubulin after gel filtration into a variety of nucleotide-free buffers has been measured (H. B. Croom, J. J. Correia, and R. C. Williams, Jr., unpublished results). All buffers we have studied that promote reduction of the number of bound nucleotides to fewer than two per tubulin dimer also eventually cause irreversible loss of activity of the protein. However, in 0.1 M 1,4-piperazinediethanesulfonic acid (pH 6.9) and 2 mM dithioerythritol (with no Mg2+), tubulin rapidly releases approximately 0.4 mol of bound nucleotides during two successive gel filtrations requiring less than 0.5 h and regains the ability to polymerize when magnesium and GTP are immediately added to the buffer. No change in conformation detectable by circular dichroism or sedimentation velocity accompanies this reversible process. (Upon prolonged incubation in the buffer, however, tubulin undergoes irreversible changes according to apparent first-order kinetics with a half-life of approximately 8 h. These changes include the irreversible release of nucleotide, a loss of the ability to polymerize, and a decrease in molar ellipticity between 210 and 240 nm.) The nucleotide which is reversibly released in this buffer comes from that population which exchanges readily with [3H]GTP in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of periodate-oxidized guanine nucleotides to eukaryotic elongation factor 2

Periodate-oxidized guanine nucleotides (GTPox and GDPox) were shown to bind stoichiometrically to rat liver elongation factor 2 (EF-2). This binding was quantitatively inhibited in the presence of GTP. After binding, oxidized nucleotides remained on EF-2 despite extensive dialysis. They exchanged, however, with free quanine nucleotides in the course of prolonged (greater than 1 h) incubations. The prior reduction EF-2.GTPox with NaBH4 abolished, to a large extent, this slow exchange. Thus, a Schiff's base was implicated to be formed between EF-2 and oxidized guanine nucleotides. Mg2+ increased the GTPox concentration necessary for a stoichiometric binding to EF-2. EF-2-oxidized nucleotide conjugates bound in the presence of ribosomes a second molecule of GTP (or GTPox). GTPox bound to EF-2 in the presence of ribosomes appeared to exchange readily with free GTP. Moreover, GTPox proved to be active as substrate in EF-2 and ribosome-dependent GTPase reaction: Km values found for GTPox and GTP were 7.7 and 3.4 microM, respectively. The binding of GTPox to EF-2 inhibited only partially the subsequent ribosome-dependent GTP binding, and GTPase reaction or polyphenylalanine (polyPhe) synthesis. On the other hand, the binding of GuoPP[CH2]Pox to EF-2 inhibited all of these reactions strongly. The nature of the binding site involved in the direct interactions of EF-2 with guanine nucleotides is discussed in the light of these results.     

The conversion of eIF-2.GDP to eIF-2.GTP by eIF-2B requires Met-tRNA(fMet).

We have investigated why the recycling of eIF-2.GDP to eIF-2.GTP, mediated by the guanine nucleotide exchange factor eIF-2B, is rapid in rabbit reticulocyte lysate, reconstituted for optimal protein synthesis, but slow in an isolated reaction with purified eIF-2B. We have found that purified eIF-2B dissociates eIF-2.[3H]GDP as efficiently in the presence of GTP as it does in the presence of GDP provided Met-tRNA(fMet) is added. tRNA(fMet) is ineffective, and there is no Met-tRNA(fMet) requirement for exchange with GDP. Exchange of eIF-2 bound GDP for GTP is completely dependent upon Met-tRNA(fMet) in the presence of ATP, suggesting that under physiological conditions efficient recycling of eIF-2.GDP to eIF-2.GTP requires conversion of the latter, a relatively unstable complex, to a more stable Met-tRNA(fMet).eIF-2.GTP complex.     

The majority of the Saccharomyces cerevisiae septin complexes do not exchange guanine nucleotides

We show here that affinity-purified Saccharomyces cerevisiae septin complexes contain stoichiometric amounts of guanine nucleotides, specifically GTP and GDP. Using a (15)N-dilution assay read-out by liquid chromatography-tandem mass spectrometry, we determined that the majority of the bound guanine nucleotides do not turn over in vivo during one cell cycle period. In vitro, the isolated S. cerevisiae septin complexes have similar GTP binding and hydrolytic properties to the Drosophila septin complexes (Field, C. M., al-Awar, O., Rosenblatt, J., Wong, M. L., Alberts, B., and Mitchison, T. J. (1996) J. Cell Biol. 133, 605-616). In particular, the GTP turnover of septins is very slow when compared with the GTP turnover for Ras-like GTPases. We conclude that bound GTP and GDP play a structural, rather then regulatory, role for the majority of septins in proliferating cells as GTP does for alpha-tubulin.     

Reactions of tubulin-associated guanine nucleotides

Only exchangeably bound nucleotide (E-site) is involved in the reaction of the transplhosphorylase activity in microtubular protein. Contrary to earlier reports, we find that the nonexchangeable nucleotide (N-site) is not a substrate. This conclusion is based upon comparison of: (a) rates of hydrolysis of endogenous tubulin-associated GTP and added [32p]GTP: (b) hydrolysis rates for added [32p]GTP and [3h]GTP; (c) the 32P/3H ratio in bound and free GTP after reaction with [3h, 32p]gTP. During the course of the above studies we have made the unusual observation of a time dependent augmentation in the expected amount of GTP relative to GDP at the E-site; there is either a net conversion of E-site GDP to E-site GTP, or a means for providing additional E-site GTP from another source.     

Ric-8A catalyzes guanine nucleotide exchange on G alphai1 bound to the GPR/GoLoco exchange inhibitor AGS3

Microtubule pulling forces that govern mitotic spindle movement of chromosomes are tightly regulated by G-proteins. A host of proteins, including Galpha subunits, Ric-8, AGS3, regulators of G-protein signalings, and scaffolding proteins, coordinate this vital cellular process. Ric-8A, acting as a guanine nucleotide exchange factor, catalyzes the release of GDP from various Galpha.GDP subunits and forms a stable nucleotide-free Ric-8A:Galpha complex. AGS3, a guanine nucleotide dissociation inhibitor (GDI), binds and stabilizes Galpha subunits in their GDP-bound state. Because Ric-8A and AGS3 may recognize and compete for Galpha.GDP in this pathway, we probed the interactions of a truncated AGS3 (AGS3-C; containing only the residues responsible for GDI activity), with Ric-8A:Galpha(il) and that of Ric-8A with the AGS3-C:Galpha(il).GDP complex. Pulldown assays, gel filtration, isothermal titration calorimetry, and rapid mixing stopped-flow fluorescence spectroscopy indicate that Ric-8A catalyzes the rapid release of GDP from AGS3-C:Galpha(i1).GDP. Thus, Ric-8A forms a transient ternary complex with AGS3-C:Galpha(i1).GDP. Subsequent dissociation of AGS3-C and GDP from Galpha(i1) yields a stable nucleotide free Ric-8A.Galpha(i1) complex that, in the presence of GTP, dissociates to yield Ric-8A and Galpha(i1).GTP. AGS3-C does not induce dissociation of the Ric-8A.Galpha(i1) complex, even when present at very high concentrations. The action of Ric-8A on AGS3:Galpha(i1).GDP ensures unidirectional activation of Galpha subunits that cannot be reversed by AGS3.     

Evaluation and significance of kinetic parameters governing function of protein synthesis initiation factors eIF-2 and eIF-2B

Published data dealing with the formation of the ternary complex eIF-2 X GTP X met-tRNAi involved in eukaryotic initiation have been evaluated to calculate the expected inhibition by GDP and the role of eIF-2B in limiting this inhibition. It is concluded that cellular levels of GDP are unlikely seriously to inhibit ternary complex formation if the reaction can proceed to equilibrium. However, derivation of 'on' and 'off' rates for the interaction of GTP and GDP with eIF-2 demonstrates that these are too slow in the absence of eIF-2B to support active protein synthesis, particularly if eIF-2 is released from ribosomes as eIF-2 X GDP. Whilst eIF-2 X GDP and eIF-2 X GTP appear to dissociate equally slowly, it is concluded that GDP binds to eIF-2 100-times faster than GTP. Addition of eIF-2B has the effect of raising k-1 for both GDP and GTP several hundred-fold and k+1 50- and 7000-fold, respectively. Thus, a kinetic block can be relieved even if there is no change in the thermodynamic state. Phosphorylation of the alpha-subunit of eIF-2 appears to affect only those parameters influenced by eIF-2B. The reported rescue of inhibited lysates by addition of 1 mM GTP is not by mass action but by some other mechanism. Consideration of the kinetic parameters favours the formation of a ternary complex of eIF-2 X eIF-2B X GDP en route to eIF-2 X GTP as opposed to displacement of GDP from eIF-2 X GDP by eIF-2B.     

Binding of guanine nucleotides and Mg2+ to tubulin with a nucleotide-depleted exchangeable site

Binding of GTP and GDP to tubulin in the presence or absence of Mg2+ was measured following depletion of the exchangeable site--(E-site) nucleotide. The E-site nucleotide was displaced with a large molar excess of the nonhydrolyzable GTP analogue, GMPPCP, followed by the removal of the analogue. Using a micropartition assay, the equilibrium constant measured in 0.1 M 1.4-piperazinediethanesulfonic acid (Pipes), pH 6.9, 1 mM ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid, 1 mM dithiothreitol, and 1 mM MgSO4 at 4 degrees C was 9.1 x 10(6) M-1 for GTP and 4.4 x 10(6) M-1 for GDP. Removal of Mg2+ reduced the binding affinity of GTP by 160-fold while the affinity of GDP remained essentially unchanged. Similar values were obtained if 0.1 M Tris, pH 7.0, was used instead of Pipes. Binding of Mg2+ to tubulin containing GTP, GDP, or no nucleotide at the E-site was also examined by the micropartition method. Tubulin-GTP contained one high affinity Mg2+ site (K alpha = 1.2 x 10(6) M-1) in addition to the one occupied by Mg2+ as tubulin is isolated, while only weak Mg2+ binding to tubulin-GDP and to tubulin with a vacant E-site (K alpha = 10(3) M-1) was observed. It is suggested that Mg2+ binds to the beta and gamma phosphates of GTP, and only to the beta phosphate of GDP, as shown for the H. ras p21 protein.