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.     

Guanine nucleotide exchange factor for eukaryotic translation initiation factor 2 in Saccharomyces cerevisiae: interactions between the essential subunits GCD2, GCD6, and GCD7 and the regulatory subunit GCN3.

Phosphorylation of eukaryotic translation initiation factor 2 (eIF-2) in amino acid-starved cells of the yeast Saccharomyces cerevisiae reduces general protein synthesis but specifically stimulates translation of GCN4 mRNA. This regulatory mechanism is dependent on the nonessential GCN3 protein and multiple essential proteins encoded by GCD genes. Previous genetic and biochemical experiments led to the conclusion that GCD1, GCD2, and GCN3 are components of the GCD complex, recently shown to be the yeast equivalent of the mammalian guanine nucleotide exchange factor for eIF-2, known as eIF-2B. In this report, we identify new constituents of the GCD-eIF-2B complex and probe interactions between its different subunits. Biochemical evidence is presented that GCN3 is an integral component of the GCD-eIF-2B complex that, while dispensable, can be mutationally altered to have a substantial inhibitory effect on general translation initiation. The amino acid sequence changes for three gcd2 mutations have been determined, and we describe several examples of mutual suppression involving the gcd2 mutations and particular alleles of GCN3. These allele-specific interactions have led us to propose that GCN3 and GCD2 directly interact in the GCD-eIF-2B complex. Genetic evidence that GCD6 and GCD7 encode additional subunits of the GCD-eIF-2B complex was provided by the fact that reduced-function mutations in these genes are lethal in strains deleted for GCN3, the same interaction described previously for mutations in GCD1 and GCD2. Biochemical experiments showing that GCD6 and GCD7 copurify and coimmunoprecipitate with GCD1, GCD2, GCN3, and subunits of eIF-2 have confirmed that GCD6 and GCD7 are subunits of the GCD-eIF-2B complex. The fact that all five subunits of yeast eIF-2B were first identified as translational regulators of GCN4 strongly suggests that regulation of guanine nucleotide exchange on eIF-2 is a key control point for translation in yeast cells just as in mammalian cells.     

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.     

eIF-2B and the exchange of guanine nucleotides bound to eIF-2

Available data for the formation of the ternary complex eIF-2 X GTP X methionyl-tRNAi involved in eukaryotic initiation and of the inhibition of ternary complex formation by GDP have been examined with a view to determining the mechanism by which eIF-2B facilitates nucleotide exchange. Two mechanisms have been considered--first a displacement reaction in which eIF-2B displaces GDP and GTP in a manner analogous to a "ping-pong" enzyme mechanism, and secondly the possibility that binding of eIF-2B to eIF-2 nucleotide complexes enhances the rate of nucleotide exchange without itself inducing nucleotide displacement. Comparison has been made between the properties of eIF-2 and eIF-2B and of the bacterial elongation factors Tu and Ts. It seems most probable that, as previously suggested by others for Ts, eIF-2B effectively catalyses an exchange reaction through a "ping-pong" type mechanism. Possible explanations of data suggesting otherwise are put forward. Both eIF-2 and bacterial Tu are complex allosteric proteins subject to a variety of influences which in the case of eIF-2 include phosphorylation of the alpha subunit. This phosphorylation appears to change the equilibria in the reaction mechanism such that the transferred entity (eIF-2) becomes firmly bound to the catalyst (eIF-2B). Minimum rate constants for the formation of eIF-2 X eIF-2B from eIF-2 X GDP and eIF-2 X GTP and reverse reactions are derived. These values suggest that the initiation factors are likely to have to operate in a restricted environment if rates of protein synthesis seen in vivo are to be sustained.     

PKR and GCN2 kinases and guanine nucleotide exchange factor eukaryotic translation initiation factor 2B (eIF2B) recognize overlapping surfaces on eIF2alpha

Four stress-responsive protein kinases, including GCN2 and PKR, phosphorylate eukaryotic translation initiation factor 2alpha (eIF2alpha) on Ser51 to regulate general and gene-specific protein synthesis. Phosphorylated eIF2 is an inhibitor of its guanine nucleotide exchange factor, eIF2B. Mutations that block translational regulation were isolated throughout the N-terminal OB-fold domain in Saccharomyces cerevisiae eIF2alpha, including those at residues flanking Ser51 and around 20 A away in the conserved motif K79GYID83. Any mutation at Glu49 or Asp83 blocked translational regulation; however, only a subset of these mutations impaired Ser51 phosphorylation. Substitution of Ala for Asp83 eliminated phosphorylation by GCN2 and PKR both in vivo and in vitro, establishing the critical contributions of remote residues to kinase-substrate recognition. In contrast, mutations that blocked translational regulation but not Ser51 phosphorylation impaired the binding of eIF2B to phosphorylated eIF2alpha. Thus, two structurally distinct effectors of eIF2 function, eIF2alpha kinases and eIF2B, have evolved to recognize the same surface and overlapping determinants on eIF2alpha.     

An eIF5/eIF2 complex antagonizes guanine nucleotide exchange by eIF2B during translation initiation

In eukaryotic translation initiation, the eIF2.GTP/Met-tRNA(i)(Met) ternary complex (TC) binds the eIF3/eIF1/eIF5 complex to form the multifactor complex (MFC), whereas eIF2.GDP binds the pentameric factor eIF2B for guanine nucleotide exchange. eIF5 and the eIF2Bvarepsilon catalytic subunit possess a conserved eIF2-binding site. Nearly half of cellular eIF2 forms a complex with eIF5 lacking Met-tRNA(i)(Met), and here we investigate its physiological significance. eIF5 overexpression increases the abundance of both eIF2/eIF5 and TC/eIF5 complexes, thereby impeding eIF2B reaction and MFC formation, respectively. eIF2Bvarepsilon mutations, but not other eIF2B mutations, enhance the ability of overexpressed eIF5 to compete for eIF2, indicating that interaction of eIF2Bvarepsilon with eIF2 normally disrupts eIF2/eIF5 interaction. Overexpression of the catalytic eIF2Bvarepsilon segment similarly exacerbates eIF5 mutant phenotypes, supporting the ability of eIF2Bvarepsilon to compete with MFC. Moreover, we show that eIF5 overexpression does not generate aberrant MFC lacking tRNA(i)(Met), suggesting that tRNA(i)(Met) is a vital component promoting MFC assembly. We propose that the eIF2/eIF5 complex represents a cytoplasmic reservoir for eIF2 that antagonizes eIF2B-promoted guanine nucleotide exchange, enabling coordinated regulation of translation initiation.     

Identification of a regulatory subcomplex in the guanine nucleotide exchange factor eIF2B that mediates inhibition by phosphorylated eIF2.

Eukaryotic translation initiation factor 2B (eIF2B) is a five-subunit complex that catalyzes guanine nucleotide exchange on eIF2. Phosphorylation of the alpha subunit of eIF2 [creating eIF2(alphaP]) converts eIF2 x GDP from a substrate to an inhibitor of eIF2B. We showed previously that the inhibitory effect of eIF2(alphaP) can be decreased by deletion of the eIF2B alpha subunit (encoded by GCN3) and by point mutations in the beta and delta subunits of eIF2B (encoded by GCD7 and GCD2, respectively). These findings, plus sequence similarities among GCD2, GCD7, and GCN3, led us to propose that these proteins comprise a regulatory domain that interacts with eIF2(alphaP) and mediates the inhibition of eIF2B activity. Supporting this hypothesis, we report here that overexpression of GCD2, GCD7, and GCN3 specifically reduced the inhibitory effect of eIF2(alphaP) on translation initiation in vivo. The excess GCD2, GCD7, and GCN3 were coimmunoprecipitated from cell extracts, providing physical evidence that these three proteins can form a stable subcomplex. Formation of this subcomplex did not compensate for a loss of eIF2B function by mutation and in fact lowered eIF2B activity in strains lacking eIF2(alphaP). These findings indicate that the trimeric subcomplex does not possess guanine nucleotide exchange activity; we propose, instead, that it interacts with eIF2(alphaP) and prevents the latter from inhibiting native eIF2B. Overexpressing only GCD2 and GCD7 also reduced eIF2(alphaP) toxicity, presumably by titrating GCN3 from eIF2B and producing the four-subunit form of eIF2B that is less sensitive to eIF2(alphaP). This interpretation is supported by the fact that overexpressing GCD2 and GCD7 did not reduce eIF2(alphaP) toxicity in a strain lacking GCN3; however, it did suppress the impairment of eIF2B caused by the gcn3c-R104K mutation. An N-terminally truncated GCD2 protein interacted with other eIF2B subunits only when GCD7 and GCN3 were overexpressed, in accordance with the idea that the portion of GCD2 homologous to GCD7 and GCN3 is sufficient for complex formation by these three proteins. Together, our results provide strong evidence that GCN3, GCD7, and the C-terminal half of GCD2 comprise the regulatory domain in eIF2B.     

The alpha subunit of initiation factor 2 is phosphorylated in vivo in the yeast Saccharomyces cerevisiae.

The phosphorylation state of the alpha subunit of initiation factor 2 (eIF-2 alpha) in Saccharomyces cerevisiae has been determined by two-dimensional gel electrophoresis and autoradiography of lysates from cultures grown under a variety of conditions. The alpha subunit was maintained in a phosphorylated state during logarithmic growth on fermentable and nonfermentable carbon sources, during starvation for an essential amino acid, during heat shock, during stationary phase, and during sporulation. Only when cells were starved for a carbon source for 2 h in 1 M sorbitol was eIF-2 alpha isolated in the nonphosphorylated state. This is in contrast with the studies in rabbit reticulocyte lysates, in which arrested protein synthesis was correlated with a relative increase in the extent of phosphorylation of eIF-2 alpha.     

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.     

Interaction of eukaryotic initiation factor eIF-4B with a picornavirus internal translation initiation site.

We studied the interaction of cellular proteins with the internal ribosome entry site (IRES) of foot-and-mouth disease virus by UV cross-linking and observed specific binding of a 80-kDa protein contained in cytosolic HeLa cell extract and in rabbit reticulocyte lysate. Binding of the protein was dependent on the presence of ATP. Immunoprecipitation with eIF-4B antiserum revealed that the protein is identical to the initiation factor eIF-4B. Deletions in the 3' part, but not in the 5' part, of the IRES interfered with UV cross-linking, indicating that the binding site of eIF-4B is located close to the end of the element. Attempts to separate ribosome-associated from non-ribosome-associated protein fractions of cytosolic cell extracts led to the loss of cross-linking activity. This finding suggests that additional protein factors contribute to this interaction of eIF-4B with the IRES of foot-and-mouth disease virus.     

Identification of intersubunit domain interactions within eukaryotic initiation factor (eIF) 2B, the nucleotide exchange factor for translation initiation

In eukaryotic translation initiation, eIF2B is the guanine nucleotide exchange factor (GEF) required for reactivation of the G protein eIF2 between rounds of protein synthesis initiation. eIF2B is unusually complex with five subunits (α-ε) necessary for GEF activity and its control by phosphorylation of eIF2α. In addition, inherited mutations in eIF2B cause a fatal leukoencephalopathy. Here we describe experiments examining domains of eIF2Bγ and ε that both share sequence and predicted tertiary structure similarity with a family of phospho-hexose sugar nucleotide pyrophosphorylases. Firstly, using a genetic approach, we find no evidence to support a significant role for a potential nucleotide-binding region within the pyrophosphorylase-like domain (PLD) of eIF2Bε for nucleotide exchange. These findings are at odds with one mechanism for nucleotide exchange proposed previously. By using a series of constructs and a co-expression and precipitation strategy, we find that the eIF2Bε and -γ PLDs and a shared second domain predicted to form a left-handed β helix are all critical for interprotein interactions between eIF2B subunits necessary for eIF2B complex formation. We have identified extensive interactions between the PLDs and left-handed β helix domains that form the eIF2Bγε subcomplex and propose a model for domain interactions between eIF2B subunits.     

Regulation of polypeptide chain initiation in Chinese hamster ovary cells with a temperature-sensitive leucyl-tRNA synthetase. Changes in phosphorylation of initiation factor eIF-2 and in the activity of the guanine nucleotide exchange factor GEF

When cultures of the temperature-sensitive Chinese hamster ovary cell mutant tsH1 are shifted from 34 degrees C (permissive temperature) to 39.5 degrees C (nonpermissive temperature), protein synthesis is inhibited by more than 80%. This is due principally to a block in activity of polypeptide chain initiation factor eIF-2. In this paper we show that there is impairment of the ability of the guanine nucleotide exchange factor (GEF) to displace GDP from eIF-2 X GDP complexes in extracts from cells incubated at the nonpermissive temperature. Addition of GEF or of high concentrations of eIF-2 stimulates protein synthesis to the level observed in control cell extracts, suggesting that GEF is rate-limiting for eIF-2 activity and overall protein synthesis at the nonpermissive temperature. Analysis of eIF-2 by two-dimensional gel electrophoresis and immunoblotting reveals an increase in the proportion of the alpha subunit in the phosphorylated form from 5.5 +/- 2.4% to 17.2 +/- 3.9% on shifting tsH1 cells from 34 to 39.5 degrees C. No such effect is seen in wild-type cells, which do not exhibit temperature-sensitive protein synthetic activity. Since the primary lesion in tsH1 cells is in their leucyl-tRNA synthetase, these results suggest a role for eIF-2 phosphorylation and GEF activity in coupling the rate of polypeptide chain initiation to the activity of the chain elongation machinery.     

Protein synthesis in Drosophila melanogaster embryos. Two mechanisms for guanine nucleotide exchange on eukaryotic initiation factor 2

The mechanism for guanine nucleotide exchange with eukaryotic initiation factor-2 (eIF-2) from Drosophila melanogaster embryos was studied using the reaction eIF-2 X [3H]GDP + GDP (GTP) in equilibrium eIF-2 X GDP (GTP) + [3H]GDP. When highly purified eIF-2 is used the rate of nucleotide exchange is greatly reduced by Mg2+ and this reduction is overcome by the guanine-nucleotide-exchange factor (GEF) of rabbit reticulocytes. This GEF-dependent exchange is inhibited when Drosophila eIF-2 is either phosphorylated by the hemin-controlled inhibitor (HCI) of rabbit reticulocytes or treated with phosphatidylserine or a rabbit eIF-2 X phosphatidylserine complex. The Mg2+ impairment of guanine nucleotide exchange is less severe when highly purified eIF-2 is incubated at a higher temperature (37 degrees C) and is not observed at any temperature if partially purified eIF-2 is used instead of the highly purified factor. In the latter two cases the exchange is not inhibited by either phosphorylation with HCI or phospholipid treatment of Drosophila eIF-2, possibly suggesting that the observed exchange is not mediated by a GEF-like factor. Our data support two possible mechanisms for GDP/GTP exchange with Drosophila embryos eIF-2: a GEF-dependent exchange, similar to that described in rabbit reticulocytes, which may be regulated by phosphorylation of eIF-2, and a factor-independent exchange which appears to be insensitive to this type of control.     

A new yeast translation initiation factor suppresses a mutation in the eIF-4A RNA helicase.

We have isolated a gene, STM1, which encodes a new translation initiation factor from Saccharomyces cerevisiae. The gene acts, if present on a multicopy plasmid, as a suppressor of a temperature-sensitive mutation in eIF-4A. The single copy STM1 gene is not essential, but disruption causes a slow growth phenotype. Analysis of polysomes from a strain carrying a disrupted stm1 allele shows a clear defect in translation initiation as shown by a strong reduction in polysomes and an increase in the monosomes. Sequence analysis revealed interesting features of the putative Stm1 protein. Comparison of the entire protein sequence with databanks showed some similarity with the human eIF-4B protein. The Stm1 protein has potential RNP1 and RNP2 motifs characteristic for RNA-binding proteins. The protein also contains six highly conserved direct repeats of 21-26 amino acids and one partial repeat.     

Translational repression mediates activation of nuclear factor kappa B by phosphorylated translation initiation factor 2

Numerous stressful conditions activate kinases that phosphorylate the alpha subunit of translation initiation factor 2 (eIF2alpha), thus attenuating mRNA translation and activating a gene expression program known as the integrated stress response. It has been noted that conditions associated with eIF2alpha phosphorylation, notably accumulation of unfolded proteins in the endoplasmic reticulum (ER), or ER stress, are also associated with activation of nuclear factor kappa B (NF-kappaB) and that eIF2alpha phosphorylation is required for NF-kappaB activation by ER stress. We have used a pharmacologically activable version of pancreatic ER kinase (PERK, an ER stress-responsive eIF2alpha kinase) to uncouple eIF2alpha phosphorylation from stress and found that phosphorylation of eIF2alpha is both necessary and sufficient to activate both NF-kappaB DNA binding and an NF-kappaB reporter gene. eIF2alpha phosphorylation-dependent NF-kappaB activation correlated with decreased levels of the inhibitor IkappaBalpha protein. Unlike canonical signaling pathways that promote IkappaBalpha phosphorylation and degradation, eIF2alpha phosphorylation did not increase phosphorylated IkappaBalpha levels or affect the stability of the protein. Pulse-chase labeling experiments indicate instead that repression of IkappaBalpha translation plays an important role in NF-kappaB activation in cells experiencing high levels of eIF2alpha phosphorylation. These studies suggest a direct role for eIF2alpha phosphorylation-dependent translational control in activating NF-kappaB during ER stress.     

Identification of domains and residues within the epsilon subunit of eukaryotic translation initiation factor 2B (eIF2Bepsilon) required for guanine nucleotide exchange reveals a novel activation function promoted by eIF2B complex formation

Eukaryotic translation initiation factor 2B (eIF2B) is the guanine nucleotide exchange factor for protein synthesis initiation factor 2 (eIF2). Composed of five subunits, it converts eIF2 from a GDP-bound form to the active eIF2-GTP complex. This is a regulatory step of translation initiation. In vitro, eIF2B catalytic function can be provided by the largest (epsilon) subunit alone (eIF2Bepsilon). This activity is stimulated by complex formation with the other eIF2B subunits. We have analyzed the roles of different regions of eIF2Bepsilon in catalysis, in eIF2B complex formation, and in binding to eIF2 by characterizing mutations in the Saccharomyces cerevisiae gene encoding eIF2Bepsilon (GCD6) that impair the essential function of eIF2B. Our analysis of nonsense mutations indicates that the C terminus of eIF2Bepsilon (residues 518 to 712) is required for both catalytic activity and interaction with eIF2. In addition, missense mutations within this region impair the catalytic activity of eIF2Bepsilon without affecting its ability to bind eIF2. Internal, in-frame deletions within the N-terminal half of eIF2Bepsilon disrupt eIF2B complex formation without affecting the nucleotide exchange activity of eIF2Bepsilon alone. Finally, missense mutations identified within this region do not affect the catalytic activity of eIF2Bepsilon alone or its interactions with the other eIF2B subunits or with eIF2. Instead, these missense mutations act indirectly by impairing the enhancement of the rate of nucleotide exchange that results from complex formation between eIF2Bepsilon and the other eIF2B subunits. This suggests that the N-terminal region of eIF2Bepsilon is an activation domain that responds to eIF2B complex formation.     

Mechanism of the nucleotide exchange reaction in eukaryotic polypeptide chain initiation. Characterization of the guanine nucleotide exchange factor as a GTP-binding protein

Polypeptide chain initiation in mammalian systems is regulated at the level of the guanine nucleotide exchange factor (GEF). This multisubunit protein catalyzes the exchange of GDP bound to eukaryotic initiation factor 2 (eIF-2) for GTP. Although various models have been proposed for its mode of action, the exact sequence of events involved in nucleotide exchange is still uncertain. We have studied this reaction by three different experimental techniques: (a) membrane filtration assays to measure the release of [3H]GDP from the eIF-2.[3H]GDP binary complex, (b) changes in the steady-state polarization of fluorescamine-GDP during the nucleotide exchange reaction, and (c) sucrose gradient analysis of the total reaction. The results obtained do not support the reaction as written: eIF-2.GDP + GEF in equilibrium eIF-2.GEF + GDP. The addition of GEF alone does not result in the displacement of eIF-2-bound GDP. The release of bound GDP is dependent on the presence of both GTP and GEF, and this argues against the possibility of a substituted enzyme (ping-pong) mechanism for the guanine nucleotide exchange reaction. An important finding of the present study is the observation that GTP binds to GEF. The Kd value of 4 microM for GTP was estimated (a) by the extent of quenching of tryptophan fluorescence of GEF in the presence of GTP and (b) by the binding of [3H]GTP to GEF as measured on nitrocellulose membranes. The GEF-dependent release of eIF-2-bound GDP was studied at several constant concentrations of one substrate (GTP or eIF-2.GDP) while varying the second substrate concentration, and the results were then plotted according to the Lineweaver-Burk method. Taken together, the results of GTP and eIF-2.GDP binding to GEF and the pattern of the double-reciprocal plots strongly suggest that the guanine nucleotide exchange reaction follows a sequential mechanism.