A new function and complexity for protein translation initiation factor eIF2B

eIF2B is a multisubunit protein that is critical for protein synthesis initiation and its control. It is a guanine nucleotide exchange factor (GEF) for its GTP-binding protein partner eIF2. eIF2 binds initiator tRNA to ribosomes and promotes mRNA AUG codon recognition. eIF2B is critical for regulation of protein synthesis via a conserved mechanism of phosphorylation of eIF2, which converts eIF2 from a substrate to an inhibitor of eIF2B GEF. In addition, inherited mutations affecting eIF2B subunits cause the fatal disorder leukoencephalopathy with Vanishing White Matter (VWM), also called Childhood Ataxia with Central nervous system Hypomyelination (CACH). Here we review findings which reveal that eIF2B is a decameric protein and also define a new function for the eIF2B. Our results demonstrate that the eIF2Bγ subunit is required for eIF2B to gain access to eIF2•GDP. Specifically it displaces a third translation factor eIF5 (a dual function GAP and GDI) from eIF2•GDP/eIF5 complexes. Thus eIF2B is a GDI displacement factor (or GDF) in addition to its role as a GEF, prompting the redrawing of the eIF2 cycling pathway to incorporate the new steps. In structural studies using mass spectrometry and cross-linking it is shown that eIF2B is a dimer of pentamers and so is twice as large as previously thought. A binding site for GTP on eIF2B was also found, raising further questions concerning the mechanism of nucleotide exchange. The implications of these findings for eIF2B function and for VWM/CACH disease are discussed.     

Critical contacts between the eukaryotic initiation factor 2B (eIF2B) catalytic domain and both eIF2beta and -2gamma mediate guanine nucleotide exchange

Diverse guanine nucleotide exchange factors (GEFs) regulate the activity of GTP binding proteins. One of the most complicated pairs is eukaryotic initiation factor 2B (eIF2B) and eIF2, which function during protein synthesis initiation in eukaryotes. We have mutated conserved surface residues within the eIF2B GEF domain, located at the eIF2Bepsilon C terminus. Extensive genetic and biochemical characterization established how these residues contribute to GEF activity. We find that the universally conserved residue E569 is critical for activity and that even a conservative E569D substitution is lethal in vivo. Several mutations within residues close to E569 have no discernible effect on growth or GCN4 expression, but an alanine substitution at the adjacent L568 is cold sensitive and deregulates GCN4 activity at 15 degrees C. The mutation of W699, found on a separate surface approximately 40 A from E569, is also lethal. Binding studies show that W699 is critical for interaction with eIF2beta, while L568 and E569 are not. In contrast, all three residues are critical for interaction with eIF2gamma. These data show that multiple contacts between eIF2gamma and eIF2Bepsilon mediate nucleotide exchange.     

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.     

Crystal structure of the C-terminal domain of the ϵ subunit of human translation initiation factor eIF2B

Eukaryotic translation initiation factor eIF2B, the guanine nucleotide exchange factor (GEF) for eIF2, catalyzes conversion of eIF2·GDP to eIF2·GTP. The eIF2B is composed of five subunits, α, β, γ, δ and ε, within which the ε subunit is responsible for catalyzing the guanine exchange reaction. Here we present the crystal structure of the C-terminal domain of human eIF2Bε (eIF2Bε-CTD) at 2.0-Å resolution. The structure resembles a HEAT motif and three charge-rich areas on its surface can be identified. When compared to yeast eIF2Bε-CTD, one area involves highly conserved AA boxes while the other two are only partially conserved. In addition, the previously reported mutations in human eIF2Bε-CTD, which are related to the loss of the GEF activity and human VWM disease, have been discussed. Based on the structure, most of such mutations tend to destabilize the HEAT motif.     

Biochemical analysis of the eIF2beta gamma complex reveals a structural function for eIF2alpha in catalyzed nucleotide exchange

Eukaryotic translation initiation factor eIF2 is a heterotrimer that binds and delivers Met-tRNA(i)(Met) to the 40 S ribosomal subunit in a GTP-dependent manner. Initiation requires hydrolysis of eIF2-bound GTP, which releases an eIF2.GDP complex that is recycled to the GTP form by the nucleotide exchange factor eIF2B. The alpha-subunit of eIF2 plays a critical role in regulating nucleotide exchange via phosphorylation at serine 51, which converts eIF2 into a competitive inhibitor of the eIF2B-catalyzed exchange reaction. We purified a form of eIF2 (eIF2betagamma) completely devoid of the alpha-subunit to further study the role of eIF2alpha in eIF2 function. These studies utilized a yeast strain genetically altered to bypass a deletion of the normally essential eIF2alpha structural gene (SUI2). Removal of the alpha-subunit did not appear to significantly alter binding of guanine nucleotide or Met-tRNA(i)(Met) ligands by eIF2 in vitro. Qualitative assays to detect 43 S initiation complex formation and eIF5-dependent GTP hydrolysis revealed no differences between eIF2betagamma and the wild-type eIF2 heterotrimer. However, steady-state kinetic analysis of eIF2B-catalyzed nucleotide exchange revealed that the absence of the alpha-subunit increased K(m) for eIF2betagamma.GDP by an order of magnitude, with a smaller increase in V(max). These data indicate that eIF2alpha is required for structural interactions between eIF2 and eIF2B that promote wild-type rates of nucleotide exchange. We suggest that this function contributes to the ability of the alpha-subunit to control the rate of nucleotide exchange through reversible phosphorylation.     

Studies on the role of eukaryotic nucleotide exchange factor in polypeptide chain initiation

Interactions of eukaryotic 5-dimethylaminonaphthalene-1-sulfonyl-initiation factor 2 (eIF-2) from rabbit reticulocytes and the guanine nucleotide exchange factor ( GEF ), Met-tRNAf, GTP, and GDP were monitored by changes in fluorescence anisotropy and radioactive filtration assays. At 1 mM Mg2+, radioactive filtration assays demonstrate that GEF is necessary for nucleotide exchange. We did not observe a GDP dependence in the association reaction of eIF-2 X GEF for GDP concentrations from 0.01 to 20 microM. This is in disagreement with the model: eIF-2 X GDP + GEF in equilibrium eIF-2 X GEF + GDP. The addition of GTP caused a decrease in fluorescence anisotropy which is interpreted as a dissociation of eIF-2 X GEF . We propose an asymmetrical model of ternary complex (eIF-2 X GTP X Met-tRNAf) formation where 1) GDP does not displace GEF and 2) GTP replaces GEF and presumably GDP. For reticulocyte eIF-2, phosphorylation of the alpha subunit greatly inhibits protein synthesis. This inhibition derives neither from failure of GEF to bind to eIF-2(alpha P) nor from greatly enhanced binding of GEF . The inhibition results from the requirement of very high levels of GTP (100 microM) to dissociate the eIF-2(alpha P) X GEF complex.     

Purification and properties of the guanine nucleotide exchange factor (GEF) from HeLa cells and its role in the initiation of protein synthesis

A guanine nucleotide exchange factor (GEF), catalyzing the exchange of GDP bound to initiation factor eIF-2 for GTP, has been isolated from S3 HeLa cells as the eIF-2 X GEF complex and extensively purified by procedures originally developed for purification of GEF from rabbit reticulocytes. The HeLa cell factor resembles rabbit reticulocyte eIF-2 X GEF in polypeptide composition, catalytic activity, and inactivation by alpha-phosphorylated eIF-2.     

Toll-like receptor activation suppresses ER stress factor CHOP and translation inhibition through activation of eIF2B

Activation of Toll-like receptors (TLRs) induces the endoplasmic reticulum (ER) unfolded protein response (UPR) to accommodate essential protein translation. However, despite increased levels of phosphorylated eIF2α (p-eIF2α), a TLR-TRIF-dependent pathway assures that the cells avoid CHOP induction, apoptosis and translational suppression of critical proteins. As p-eIF2α decreases the functional interaction of eIF2 with eIF2B, a guanine nucleotide exchange factor (GEF), we explored the hypothesis that TLR-TRIF signalling activates eIF2B GEF activity to counteract the effects of p-eIF2α. We now show that TLR-TRIF signalling activates eIF2B GEF through PP2A-mediated serine dephosphorylation of the eIF2B ?-subunit. PP2A itself is activated by decreased Src-family-kinase-induced tyrosine phosphorylation of its catalytic subunit. Each of these processes is required for TLR-TRIF-mediated CHOP suppression in ER-stressed cells in vitro and in vivo. Thus, in the setting of prolonged, physiologic ER stress, a unique TLR-TRIF-dependent translational control pathway enables cells to carry out essential protein synthesis and avoid CHOP-induced apoptosis while still benefiting from the protective arms of the UPR.     

A GDP/GTP exchange factor essential for eukaryotic initiation factor 2 cycling in Ehrlich ascites tumor cells and its regulation by eukaryotic initiation factor 2 phosphorylation

Formation of the ternary complex Met-tRNAi X eukaryotic initiation factor (eIF) 2 X GTP from eIF-2 X GDP requires exchange of GDP for GTP. However, at physiological Mg2+ concentrations, GDP is released from eIF-2 exceedingly slowly (Clemens, M.J., Pain, V.M., Wong, S.T., and Henshaw, E.C. (1982) Nature (Lond.) 296, 93-95). However, GDP is released rapidly from impure eIF-2 preparations, indicating the presence of a GDP/GTP exchange factor. We have now purified this factor from Ehrlich cells and refer to it as GEF. CM-Sephadex chromatography of ribosomal salt wash separated two peaks of eIF-2 activity. GEF was found in association with eIF-2 in the first peak and co-purified with eIF-2 under low salt conditions. It was separated from eIF-2 in high salt buffers and further purified on hydroxylapatite and phosphocellulose. Gel electrophoresis of our purest preparations showed major bands at 85, 67, 52, 37, 27, and 21 kDa. Purified GEF increased the rate of exchange of [32P] GDP for unlabeled GDP 25-fold but did not function with phosphorylated eIF-2 (alpha subunit). The factor also stimulated markedly the rate of ternary complex formation using eIF-2 X GDP as substrate with GTP and Met-tRNAi but not using phosphorylated eIF-2 X GDP as substrate. eIF-2 is released from the 80 S initiation complex with hydrolysis of GTP. If eIF-2 X GDP is actually the complex released, then GEF is absolutely required for eIF-2 to cycle and it is therefore a new eukaryotic initiation factor. Furthermore, the inability of GEF to utilize eIF-2 (alpha P) X GDP explains how phosphorylation of eIF-2 can inhibit polypeptide chain initiation.     

Mechanisms of translational regulation by a human eIF5-mimic protein

The translation factor eIF5 is an important partner of eIF2, directly modulating its function in several critical steps. First, eIF5 binds eIF2/GTP/Met-tRNA(i)(Met) ternary complex (TC), promoting its recruitment to 40S ribosomal subunits. Secondly, its GTPase activating function promotes eIF2 dissociation for ribosomal subunit joining. Finally, eIF5 GDP dissociation inhibition (GDI) activity can antagonize eIF2 reactivation by competing with the eIF2 guanine exchange factor (GEF), eIF2B. The C-terminal domain (CTD) of eIF5, a W2-type HEAT domain, mediates its interaction with eIF2. Here, we characterize a related human protein containing MA3- and W2-type HEAT domains, previously termed BZW2 and renamed here as eIF5-mimic protein 1 (5MP1). Human 5MP1 interacts with eIF2 and eIF3 and inhibits general and gene-specific translation in mammalian systems. We further test whether 5MP1 is a mimic or competitor of the GEF catalytic subunit eIF2Bε or eIF5, using yeast as a model. Our results suggest that 5MP1 interacts with yeast eIF2 and promotes TC formation, but inhibits TC binding to the ribosome. Moreover, 5MP1 is not a GEF but a weak GDI for yeast eIF2. We propose that 5MP1 is a partial mimic and competitor of eIF5, interfering with the key steps by which eIF5 regulates eIF2 function.     

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.     

Kinetic analysis of the interaction of guanine nucleotides with eukaryotic translation initiation factor eIF5B

Eukaryotic translation initiation factor eIF5B is a ribosome-dependent GTPase that is responsible for the final step in initiation, which involves the displacement of initiation factors from the 40S ribosomal subunit in initiation complexes and its joining with the 60S subunit. Hydrolysis of eIF5B-bound GTP is not required for its function in subunit joining but is necessary for the subsequent release of eIF5B from assembled 80S ribosomes. Here we investigated the kinetics of guanine nucleotide binding to eIF5B by a fluorescent stopped-flow technique using fluorescent mant derivatives of GTP and GDP and of the GTP analogues GTPgammaS and GMPPNP. The affinity of eIF5B for mant-GTP (Kd approximately 14-18 microM) was approximately 7-fold less than for mant-GDP (Kd approximately 2.3 microM), and both guanine nucleotides dissociated rapidly from eIF5B (k-1mant-GTP approximately 22-28 s-1, k-1mant-GDP approximately 10-14 s-1). These properties of eIF5B suggest a rapid spontaneous GTP/GDP exchange on eIF5B and are therefore consistent with it having no requirement for a special guanine nucleotide exchange factor. The affinity of eIF5B for mant-GTPgammaS was about 2 times lower (Kd approximately 6.9 microM) and for mant-GMPPNP 1.5 times higher (Kd approximately 25.7 microM) than for mant-GTP, indicating that eIF5B tolerates modifications of the triphosphate moiety well.     

Localization of the Translational Guanine Nucleotide Exchange Factor eIF2B: A Common Theme for GEFs?

The eukaryotic initiation factor 2B (eIF2B) serves an essential recycling function in protein synthesis. As the guanine nucleotide exchange factor for eIF2, it recycles eIF2 from a GDP to a GTP bound form that is competent for translation initiation. Stressdependent controls target this eIF2B-recycling step allowing a re-programming of the global gene expression profile. In addition, a human disease, leukoencephalopathy with vanishing white matter (VWM), is caused by mutations in the eIF2B subunit genes. Recently, we have found that the eIF2B guanine nucleotide exchange factor resides in a specific cytoplasmic focus in the yeast, Saccharmoyces cerevisiae. eIF2B is a resident feature of this focus, whereas eIF2 shuttles to and fro. Moreover, the in vivo rate of eIF2 shuttling correlates with changes in guanine nucleotide exchange activity implicating this large cytoplasmic focus as a site of guanine nucleotide exchange. In this perspective, we discuss these findings in the wider context of the assortment of guanine nucleotide exchange factors.     

The N-terminal domain of the Caulobacter crescentus CgtA protein does not function as a guanine nucleotide exchange factor

The Caulobacter crescentus GTP binding protein CgtA is a member of the Obg/GTP1 subfamily of monomeric GTP binding proteins. In vitro, CgtA displays moderate affinity for both GDP and GTP, and rapid exchange rate constants for either nucleotide. One possible explanation for the observed rapid guanine nucleotide exchange rates is that CgtA is a bimodal protein with a C-terminal GTP binding domain and an N-terminal guanine nucleotide exchange factor (GEF) domain. In this study we demonstrate that although the N-terminus of CgtA is required for function in vivo, this domain plays no significant role in the guanine nucleotide binding, exchange or GTPase activity.     

RA-GEF, a novel Rap1A guanine nucleotide exchange factor containing a Ras/Rap1A-associating domain, is conserved between nematode and humans

A yeast two-hybrid screening for Ras-binding proteins in nematode Caenorhabditis elegans has identified a guanine nucleotide exchange factor (GEF) containing a Ras/Rap1A-associating (RA) domain, termed Ce-RA-GEF. Both Ce-RA-GEF and its human counterpart Hs-RA-GEF possessed a PSD-95/DlgA/ZO-1 (PDZ) domain and a Ras exchanger motif (REM) domain in addition to the RA and GEF domains. They also contained a region homologous to a cyclic nucleotide monophosphate-binding domain, which turned out to be incapable of binding cAMP or cGMP. Although the REM and GEF domains are conserved with other GEFs acting on Ras family small GTP-binding proteins, the RA and PDZ domains are unseen in any of them. Hs-RA-GEF exhibited not only a GTP-dependent binding activity to Rap1A at its RA domain but also an activity to stimulate GDP/GTP exchange of Rap1A both in vitro and in vivo at the segment containing its REM and GEF domains. However, it did not exhibit any binding or GEF activity toward Ras. On the other hand, Ce-RA-GEF associated with and stimulated GDP/GTP exchange of both Ras and Rap1A. These results indicate that Ce-RA-GEF and Hs-RA-GEF define a novel class of Rap1A GEF molecules, which are conserved through evolution.     

GEF-mediated GDP/GTP exchange by monomeric GTPases: A regulatory role for Mg2+?

GTPases share highly conserved guanine nucleotide-binding domains and fulfill diverse functions through a common molecular switch. An inactive GDP-bound protein is turned on by a guanine nucleotide exchange factor (GEF) that catalyzes exchange of GTP for GDP, but unfortunately little is known about the mechanism of GEF action. A common mechanism for GDP/GTP exchange can be envisioned wherein GEFs activate monomeric GTPases through transient disruption of Mg²⁺ coordination in the nucleotide-binding pocket while stabilizing a nucleotide-free (and cation-free) conformation. After guanine nucleotide exchange, Mg²⁺ coordination is restored to complete the conformational switch to the active GTP-bound state. Evidence in the literature highlighting an important regulatory role for Mg²⁺ in the mechanism of GEF-mediated GDP/GTP exchange by monomeric GTPases is summarized. BioEssays 20 :516–521, 1998. © 1998 John Wiley & Sons, Inc.     

Purification and characterization of eukaryotic initiation factor 2 and a guanine nucleotide exchange factor from rat liver

Two polypeptide chain initiation factors, eukaryotic initiation factor 2 (eIF-2) and guanine nucleotide exchange factor (GEF), were isolated from rat liver. Two forms of eIF-2 were identified, one contained three subunits (alpha, beta, and gamma), and the other contained only the alpha- and gamma-subunits. The three-subunit form was similar to eIF-2 from rabbit reticulocytes with respect to the sedimentation coefficient, Stokes radius, molecular weight of the alpha- and gamma-subunits, ability to restore protein synthesis in hemin-deficient reticulocyte lysate, and immunological cross-reactivity of the alpha-subunits using antibodies against liver eIF-2. In contrast, the beta-subunits of the liver and reticulocyte factors were distinct; they had different molecular weights, and antibodies against rat liver eIF-2 beta did not recognize the beta-subunit of the reticulocyte factor. Furthermore, the GDP dissociation constant for reticulocyte eIF-2 was more than twice that of the liver factor. GEF from rat liver reversed GDP inhibition of the ternary complex assay and catalyzed the exchange of eIF-2-bound GDP for free GDP or GTP, characteristics ascribed to the corresponding protein from rabbit reticulocytes. However, its subunit composition and molecular weight were different from those reported for reticulocyte GEF. The T1/2 for GDP exchange mediated by GEF was about 5-fold slower with two-subunit than with three-subunit eIF-2. In addition, the KD for GDP was lower for two-subunit than for three-subunit eIF-2 when GEF was present. Taken together, these data demonstrate species-associated variability in the beta-subunit of eIF-2 and suggest a crucial role for the beta-subunit in the functional interaction of eIF-2 and GEF.     

Characterisation of the Nucleotide Exchange Factor ITSN1L: Evidence for a Kinetic Discrimination of GEF-Stimulated Nucleotide Release from Cdc42

Cdc42, a member of the Ras superfamily of small guanine nucleotide binding proteins, plays an important role in regulating the actin cytoskeleton, intracellular trafficking, and cell polarity. Its activation is controlled by guanine nucleotide exchange factors (GEFs), which stimulate the dissociation of bound guanosine-5′-diphosphate (GDP) to allow guanosine-5′-triphosphate (GTP) binding. Here, we investigate the exchange factor activity of the Dbl-homology domain containing constructs of the adaptor protein Intersectin1L (ITSN1L), which is a specific GEF for Cdc42. A detailed kinetic characterisation comparing ITSN1L-mediated nucleotide exchange on Cdc42 in its GTP- versus GDP-bound state reveals a kinetic discrimination for GEF-stimulated dissociation of GTP: The maximum acceleration of the intrinsic mGDP [2′/3′-O-(N-methyl-anthraniloyl)-GDP] release from Cdc42 by ITSN1L is accelerated at least 68,000-fold, whereas the exchange of mGTP [2′/3′-O-(N-methyl-anthraniloyl)-GTP] is stimulated only up to 6000-fold at the same GEF concentration. The selectivity in nucleotide exchange kinetics for GDP over GTP is even more pronounced when a Cdc42 mutant, F28L, is used, which is characterised by fast intrinsic dissociation of nucleotides. We furthermore show that both GTP and Mg²⁺ ions are required for the interaction with effectors. We suggest a novel model for selective nucleotide exchange residing on a conformational change of Cdc42 upon binding of GTP, which enables effector binding to the Cdc42 · GTP complex but, at the same time, excludes efficient modulation by the GEF. The higher exchange activity of ITSN1L towards the GDP-bound conformation of Cdc42 could represent an evolutionary adaptation of this GEF that ensures nucleotide exchange towards the formation of the signalling-active GTP-bound form of Cdc42 and avoids dissociation of the active complex.     

Novel intermediate of Rac GTPase activation by guanine nucleotide exchange factor

The biochemical role of guanine nucleotide exchange factors (GEFs) in catalyzing small GTPase GDP-GTP exchange is thought to be twofold: stimulation of GDP dissociation and stabilization of a nucleotide-free GTPase intermediate. Here we report that TrioN, a Dbl family GEF, activates Rac1 by facilitating GTP binding to, as well as stimulating GDP dissociation from, Rac1. The TrioN-catalyzed GDP dissociation is dependent upon the structural nature and the concentration of free nucleotide, and nucleotide binding serves as the rate-limiting step of the GEF reaction. The TrioN-stimulated nucleotide exchange may undergo a novel two nucleotide-one G-protein intermediate involving two cryptic subsites on Rac1 induced by the GEF, with one subsite contributing to the recognition of the beta/gamma phosphates of the incoming GTP and another to the binding of the guanine base of the leaving GDP. We propose that the Rac GEF reaction may proceed by competitive displacement of bound GDP by GTP through a transient intermediate of GEF-[GTP-Rac-GDP].