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.     

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.     

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.     

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.     

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.     

The isolation and characterization from rabbit reticulocytes of two forms of eukaryotic initiation factor 2 having different beta-polypeptides

We have isolated from the high salt wash of rabbit reticulocyte ribosomes two forms of the polypeptide chain initiation factor 2 (eIF-2) which differ with respect to their beta-subunit, GDP content, and sensitivity to Mg2+ in ternary (eIF-2 X GTP X Met-tRNAf) and binary (eIF-2 X GDP) complex formation. The form of eIF-2 eluting first from a cation exchange (Mono S, Pharmacia) column has a beta-subunit of lower molecular weight (eIF-2(beta L] and a more acidic pI value than the form eluting at a higher salt concentration (eIF-2(beta H]. These two forms of eIF-2 beta-polypeptides are also detected in reticulocyte lysates when the proteins are resolved by two-dimensional isoelectric focusing-dodecyl sulfate polyacrylamide gel electrophoresis followed by immunoblotting. The peptide mapping of the isolated beta-subunits after limited proteolysis by papain, pancreatic protease, alpha-chymotrypsin, or Staphylococcus aureus V8 protease further demonstrates that the two forms of beta-subunits are not the product of a non-specific proteolytic action that occurred during the purification procedure, but rather reflects the existence in vivo of both forms of eIF-2. The GDP content of eIF-2(beta L) and eIF-2(beta H) is approximately 0.85 and 0.22 mol of GDP/mol of eIF-2, respectively. The KD for GDP of eIF-2(beta L) was lower (2.2 X 10(-9) M) than that of eIF-2(beta H) (6.0 X 10(-8) M). In the presence of 1 mM Mg2+, the activities of eIF-2(beta L) and eIF-2(beta H) in forming a binary and a ternary complex are inhibited 90 and 25%, respectively. The extent of Mg2+ inhibition and its reversal by the guanine nucleotide exchange factor is directly proportional to the amount of GDP bound to eIF-2. No inhibition by Mg2+ is observed when eIF-2-bound GDP is removed by alkaline phosphatase. In the presence of the guanine nucleotide exchange factor, both forms of eIF-2 are equally active in ternary complex formation, and the complex formed is quantitatively transferred to 40 S ribosomal subunits.     

Protein synthesis in rabbit reticulocytes. A study of the roles of Co-eIF-2, Co-eIF-2A80, and GDP in peptide chain initiation

The roles of Co-eIF-2, Co-eIF-2A80, and GDP in ternary complex and Met-tRNAf X 40 S initiation complex formation were studied. 1) Partially purified eukaryotic initiation factor 2 (eIF-2) (50% pure) preparations contained 0.4-0.6 pmol of bound GDP/pmol of eIF-2. eIF-2 purity was calculated from ternary complex formation in the absence of Mg2+ and in the presence of excess Co-eIF-2. 2) In the absence of Mg2+, approximately 30% of the potentially active eIF-2 molecules formed ternary complexes, and both Co-eIF-2 and Co-eIF-2A80 were equally effective in full activation of the eIF-2 molecules for ternary complex formation. 3) In the presence of Mg2+, approximately 10% of the potentially active eIF-2 molecules formed ternary complexes in the absence of ancillary factors, and the ancillary factors Co-eIF-2A80 and Co-eIF-2 raised the incorporation to 20 and 50% of the eIF-2 molecules, respectively. 4) In the absence of Mg2+, [3H]GDP in preformed eIF-2 X [3H]GDP was readily displaced by GTP during ternary complex formation. 5) In the presence of Mg2+, [3H]GDP remained tightly bound to eIF-2 and ternary complex formation was inhibited. Co-eIF-2, but not Co-eIF-2A80, was effective in promoting [3H]GDP displacement and the former was more effective in promoting ternary complex formation than the latter. 6) eIF-2 X [3H]GDP was converted to eIF-2 X [3H] GTP by incubation in the presence of nucleoside-5'-diphosphate kinase and ATP, but the eIF-2 X [3H]GTP thus formed did not bind Met-tRNAf in the presence of Mg2+ and required exogeneous addition of Co-eIF-2 and GTP for ternary complex formation and GTP displacement. 7) In the absence of Mg2+, the increased ternary complex formed in the presence of eIF-2 X [3H] GDP and Co-eIF-2A80 (with accompanying loss of [3H] GDP) was inactive in a subsequent reaction, which involves Met-tRNAf transfer to 40 S ribosomes (in the presence of Mg2+), and required trace amounts of Co-eIF-2 for such activity. Based on the above observations, we have suggested a two-step activation of eIF-2 molecules by the Co-eIF-2 protein complex for functional ternary complex formation. One of these steps involves the Co-eIF-2A component of Co-eIF-2. This activation results in stimulated Met-tRNAf binding to eIF-2 and is most apparent in the absence of Mg2+ and with aged eIF-2 molecules.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

A comparative study of the characteristics of eIF-2 and eIF-2-ancillary factor activities from yeast Saccharomyces cerevisiae and rabbit reticulocytes

The characteristics of yeast eukaryotic initiation factor 2 (eIF-2) and Co-eIF-2A have been studied and compared with those of the corresponding factors from rabbit reticulocytes. 1) Unlike eIF-2r, purified eIF-2y did not contain bound GDP. 2) Purified eIF-2y preparation contained GTPase activity and dephosphorylated GTP to GDP. 3) An anti-eIF-2r preparation which predominantly precipitated the gamma-subunit (Mr 54,000) of eIF-2r also precipitated the larger subunit (Mr 54,000) of eIF-2y. 4) Unlike eIF-2r, ternary complex formation by eIF-2y was not inhibited by Mg2+. 5) Both Co-eIF-2A20y and Co-eIF-2r significantly enhanced Met-tRNAf binding to eIF-2y and, again, Mg2+ did not have any effect on this stimulated Met-tRNAf binding to eIF-2y. 6) Both Co-eIF-2A20y and Co-eIF-2r were similarly effective in stimulating Met-tRNAf binding to eIF-2r in the absence of Mg2+. However, in the presence of Mg2+, Co-eIF-2A20y was significantly less effective than Co-eIF-2r as Co-eIF-2A20y did not promote displacement of GDP from eIF-2r X GDP. 7) eIF-2y bound [3H]GDP and this binding was significantly enhanced in the presence of Mg2+. Also, [3H]GDP in the preformed eIF-2y X [3H]GDP complex was rapidly exchanged with exogenously added unlabeled GDP in the presence of Mg2+. Co-eIF-2A20y had no effect on GDP binding to eIF-2y nor on GDP exchange reactions. 8) Reticulocyte heme-regulated protein synthesis inhibitor, which phosphorylated almost completely (in excess of 80%) the alpha-subunit (Mr 38,000) of eIF-2r, also phosphorylated similarly the smaller subunit (Mr 36,000) of eIF-2y. However, such phosphorylation had no significant effect on ternary complex formation, GDP binding, and GDP exchange reactions.     

Mechanism of stimulation of globin synthesis by adenosine-3',5'-monophosphate and guanosine 5'-triphosphate in a rabbit reticulocyte lysate system

The inhibition of globin synthesis in hemin-deficient rabbit reticulocyte lysates is due to the activation of a hemin-controlled translational inhibitor (HCI) that specifically phosphorylates eIF-2 alpha. High concentrations of cAMP (5-10 mM) and GTP (1-2 mM) stimulated the globin synthesis in hemin-deficient lysates when these compounds were added at the initial stage of incubation. The mechanism of the stimulation by cAMP and GTP was studied using hemin-deficient lysates, the N-ethylmaleimide (NEM)-treated HCI-supplemented lysates and a partially purified initiation factor, eIF-2. As the stimulation of globin synthesis by these compounds must be due to the prevention of the inhibition of globin synthesis, or due to the restoration of globin synthesis, or both, the preventive and restorative effects of these compounds were examined. As for the preventive effect, it was observed that a) the activation of HCI in the postribosomal supernatant of reticulocytes was prevented by GTP, but not by cAMP, and b) cAMP and GTP inhibited the phosphorylation of eIF-2 alpha in hemin-deficient lysates. As for the restorative effect of cAMP and GTP, it was observed that c) these compounds restored the globin synthesis and the binding of [35S]Met-tRNAf to the 40S ribosomal subunits, and promoted the dephosphorylation of eIF-2(alpha P), d) the rates of the restored synthesis of globin were lower than the control, and e) cAMP promoted the release of [3H]GDP from the eIF-2(alpha P) X [3H]GDP complex and the formation of eIF-2(alpha P) X eIF-2B complex. Finding (d) indicates that steps involved in the restorative effect of these compounds may not contribute to the stimulation of the globin synthesis in hemin-deficient lysates. The data on the preventive and restorative effects of cAMP and GTP showed that these compounds affected multiple steps. That is, cAMP inhibited the phosphorylation of eIF-2 alpha and promoted both the release of GDP from eIF-2 and the formation of eIF-2(alpha P) X eIF-2B complex, and GTP prevented both the activation of HCI and the phosphorylation of eIF-2 alpha. Though cAMP and GTP affected multiple steps, it is suggested that cAMP stimulates the globin synthesis by inhibiting the phosphorylation of eIF-2 alpha and that GTP stimulates the globin synthesis chiefly by preventing the activation of HCI in hemin-deficient lysates.     

Function of eukaryotic initiation factor 5 in the formation of an 80 S ribosomal polypeptide chain initiation complex.

Eukaryotic initiation factor 5 (eIF-5), isolated from rabbit reticulocyte lysates, is a monomeric protein of 58-62 kDa. The function of eIF-5 in the formation of an 80 S polypeptide chain initiation complex from a 40 S initiation complex has been investigated. Incubation of the isolated 40 S initiation complex (40 S.AUG.Met.tRNAf.eIF-2 GTP) with eIF-5 resulted in the rapid and quantitative hydrolysis of GTP bound to the 40 S initiation complex. The rate of this reaction was unaffected by the presence of 60 S ribosomal subunits. Analysis of eIF-5-catalyzed reaction products by gel filtration indicated that both eIF-2.GDP binary complex and Pi formed were released from the ribosomal complex whereas Met-tRNAf remained bound to 40 S ribosomes as a Met-tRNAf.40 S.AUG complex. Reactions carried out with biologically active 32P-labeled eIF-5 indicated that this protein was not associated with the 40 S.AUG.Met-tRNAf complex; similar results were obtained by immunological methods using monospecific anti-eIF-5 antibodies. The isolated 40 S.AUG.Met-RNAf complex, free of eIF-2.GDP binary complex and eIF-5, readily interacted with 60 S ribosomal subunits in the absence of exogenously added eIF-5 to form the 80 S initiation complex capable of transferring Met-tRNAf into peptide linkages. These results indicate that the sole function of eIF-5 in the initiation of protein synthesis is to mediate hydrolysis of GTP bound to the 40 S initiation complex in the absence of 60 S ribosomal subunits. This leads to formation of the intermediate 40 S.AUG.Met-tRNAf and dissociation of the eIF-2.GDP binary complex. Subsequent joining of 60 S ribosomal subunits to the intermediate 40 S.AUG.Met-tRNAf complex does not require participation of eIF-5. Thus, the formation of an 80 S ribosomal polypeptide chain initiation complex from a 40 S ribosomal initiation complex can be summarized by the following sequence of partial reactions. (40 S.AUG.Met-tRNAf.eIF-2.GTP) eIF-5----(40 S.AUG.Met-tRNAf) + (eIF-2.GDP) + Pi (1) (40 S.AUG.Met-tRNAf) + 60 S----(80 S.AUG.Met-tRNAf) (2) 80 S initiation complex.     

Protein synthesis in rabbit reticulocytes. A study of the mechanism of Co-eIF-2 action

The characteristics of component activities in Co-eIF-2 (where eIF is eukaryotic initiation factor) protein complex have been studied. (i) At limiting concentrations, Co-eIF-2 promoted rapid GDP binding to eIF-2 and also GDP displacement from eIF-2 X GDP during ternary complex formation in the presence of GTP and Mg2+ (Co-eIF-2C activity) but did not significantly stimulate ternary complex formation by eIF-2. (ii) At higher concentrations, Co-eIF-2 significantly enhanced ternary complex formation by eIF-2 and also rendered the complex stable to aurintricarboxylic acid presumably as Co-eIF-2 became physically bound to the ternary complex (Co-eIF-2A activity). (iii) Ternary complex preformed in the presence of Co-eIF-2 and without Mg2+ dissociated upon subsequent addition of Mg2+ (Co-eIF-2B activity). This dissociation reaction was presumably due to loss of interaction of the Co-eIF-2A component in Co-eIF-2 with the ternary complex (reversal of Co-eIF-2A activity) as the complex became increasingly sensitive to aurintricarboxylic acid with increasing Mg2+ concentration. In another study, purified eIF-2 was freed of bound GDP by treatment with alkaline phosphatase and the characteristics of native and GDP-free eIF-2 were compared. (i) One mM Mg2+ inhibited (60%) ternary complex formation by native eIF-2 but not by GDP-free eIF-2. Addition of exogenous GDP rendered GDP-free eIF-2 sensitive to Mg2+ indicating that Mg2+ inhibition is due to eIF-2-bound GDP. (ii) In the presence of Mg2+, Co-eIF-2 stimulated similarly ternary and Met-tRNAf X 40 S X AUG complex formation by both native and GDP-free eIF-2. Such stimulatory activity in each case was strongly inhibited by prior phosphorylation of eIF-2 alpha subunit by heme-regulated translational inhibitor. (iii) Ternary complexes preformed using either native and GDP-free eIF-2 and excess Co-eIF-2A80 in the absence of Mg2+ did not form Met-tRNAf X 40 S X AUG complex. They required trace amounts of Co-eIF-2 for such activity.     

Protein synthesis in brine shrimp embryos. Dormant and developing embryos of Artemia salina contain equivalent amounts of chain initiation factors 2.

Dormant and developing embryos of Artemia salina contain equivalent amounts of eIF-2, the eukaryotic initiation factor which forms a ternary complex with GTP and Met-tRNAf. The factor was purified from 0.5 M NH4Cl ribosomal washes by (NH4)2SO4 fractionation, followed by chromatography on heparin-Sepharose, DEAE-cellulose, hydroxyapatite and phosphocellulose. Purified preparations from dormant and developing embryos have similar specific activities and nucleotide requirements. The mobility of both proteins in dodecylsulfate gel electrophoresis is indistinguishable, and each contains three major polypeptide chains of molecular weight 52 000, 45 000 and 42 000. Both proteins are also immunologically identical, and each stimulates amino acid incorporation in a cell-free system of protein synthesis. The binding of [35S]Met-tRNAf to 40-S ribosomal subunits is catalyzed by eIF-2 isolated from dormant or developing embryos and is dependent upon GPT and AUG. Binding of [35S]Met-tRNAf to 40-S ribosomal subunits, and ternary complex formation with eIF-2, GTP, and [35S]Met-tRNAf is stimulated 2--3-fold by a factor present in the 0.5 M NH4Cl ribosomal wash and which elutes from DEAE-cellulose at 50 mM KCl. This protein does not exhibit GTP-dependent binding of [35S]Met-tRNAf. Binding of GDP and GTP was investigated with purified eIF-2 from developing embryos. The factor forms a binary complex with GDP or GTP, and eIF-2-bound [3H]GDP exchanges very slowly with free nucleotides. Our results suggest that eIF-2 does not limit resumption of embryo development following encystment, nor does it limit mRNA translation in extracts from dormant embryos.     

Photoaffinity labeling of the rabbit reticulocyte guanine nucleotide exchange factor and eukaryotic initiation factor 2 with 8-azidopurine nucleotides. Identification of GTP- and ATP-binding domains

We have covalently modified rabbit reticulocyte polypeptide chain initiation factor 2 (eIF-2) and the guanine nucleotide exchange factor (GEF) with the 8-azido analogs of GTP (8-N3GTP) and ATP (8-N3ATP). Of the five subunits of GEF, the Mr 40,000 polypeptide binds 8-[gamma-32P]N3GTP, and the Mr 55,000 and 65,000 polypeptides bind 8-[gamma-32P]N3ATP. Both 8-N3GTP and 8-N3ATP specifically label the beta-subunit of eIF-2. Covalent binding of 8-azidopurine analogs to the eukaryotic initiation factors is dependent on UV irradiation. Binding of 8-N3GTP and 8-N3ATP is specific for the guanine- and adenine-binding sites on the protein, respectively. GDP and GTP, but not ATP, inhibit the photoinsertion of 8-N3GTP to the protein. Similarly, ATP, but not GTP, inhibits the photoinsertion of 8-N3ATP. The inclusion of NADP+ in the reaction mixtures also interferes with the binding of 8-N3ATP to GEF. Mg2+ inhibits the binding of the 8-azido analogs of GTP and ATP to both eIF-2 and GEF, whereas EDTA stimulates the photoinsertion of these nucleotides. Identical results are obtained when the binding of GTP and ATP to these proteins, in the presence of Mg2+ or EDTA, is estimated by nitrocellulose membranes. In enzymatic assays, 8-N3GTP supports the activity of eIF-2 and GEF, indicating that the interaction of 8-N3GTP is catalytically relevant.     

Evidence that the primary effect of phosphorylation of eukaryotic initiation factor 2(alpha) in rabbit reticulocyte lysate is inhibition of the release of eukaryotic initiation factor-2.GDP from 60 S ribosomal subunits

The phosphorylation of eukaryotic initiation factor (eIF) 2 alpha that occurs when rabbit reticulocyte lysate is incubated in the absence of hemin or with poly(I.C) causes inhibition of polypeptide chain initiation by preventing a separate factor (termed RF) from promoting the exchange of GTP for GDP on eIF-2. When lysate was incubated in the presence of hemin and [14C] eIF-2 or [alpha-32P]GTP, we observed binding of eIF-2 and GDP or GTP to 60 S ribosomal subunits that was slightly greater than that bound to 40 S subunits and little binding to 80 S ribosomes. When incubation was in the absence of hemin or in the presence of hemin plus 0.1 microgram/ml poly(I.C), eIF-2 and GDP binding to 60 S subunits was increased 1.5- to 2-fold, that bound to 80 S ribosomes was almost as great as that bound to 60 S subunits, and that bound to 40 S subunits was unchanged. Our data indicate that about 40% of the eIF-2 that becomes bound to 60 S subunits and 80 S ribosomes in the absence of hemin or with poly(I.C) is eIF-2(alpha-P) and suggest that the eIF-2 and GDP bound is probably in the form of a binary complex. The accumulation of eIF-2.GDP on 60 S subunits occurs before binding of Met-tRNAf to 40 S subunits becomes reduced and before protein synthesis becomes inhibited. The rate of turnover of GDP (presumably eIF-2.GDP) on 60 S subunits and 80 S ribosomes in the absence of hemin is reduced to less than 10% the control rate, because the dissociation of eIF-2.GDP is inhibited. Additional RF increases the turnover of eIF-2.GDP on 60 S subunits and 80 S ribosomes to near the control rate by promoting dissociation of eIF-2.GDP but not eIF-2(alpha-P).GDP. Our findings suggest that eIF-2.GTP binding to and eIF-2.GDP release from 60 S subunits may normally occur and serve to promote subunit joining. The phosphorylation of eIF-2 alpha inhibits polypeptide chain initiation by preventing dissociation of eIF-2.GDP from either free 60 S subunits (thus inhibiting subunit joining directly) or the 60 S subunit component of an 80 S initiation complex (thereby blocking elongation and resulting in the dissociation of the 80 S complex).     

A novel role for aminoacyl-tRNA synthetases in the regulation of polypeptide chain initiation

Exposure of the temperature-sensitive leucyl-tRNA synthetase mutant of Chinese hamster ovary cells, tsH1, to the non-permissive temperature of 39.5 degrees C results in a rapid inhibition of polypeptide chain initiation. This inhibition is caused by a reduced ability of the eukaryotic initiation factor eIF-2 to participate in the formation of eIF-2.GTP.Met-tRNAf ternary complexes and thus in the formation of 43S ribosomal pre-initiation complexes. Associated with this decreased eIF-2 activity is an increased phosphorylation of the eIF-2 alpha subunit. It has previously been shown in other systems that phosphorylation of eIF-2 alpha slows the rate of recycling of eIF-2.GDP to eIF-2.GTP catalysed by the guanine nucleotide exchange factor eIF-2B. We show here that phosphorylation of eIF-2 alpha by the reticulocyte haem-controlled repressor also inhibits eIF-2B activity in cell-free extracts derived from tsH1 cells. Thus the observed increased phosphorylation of eIF-2 alpha at the non-permissive temperature in this system is consistent with impaired recycling of eIF-2 in vivo. Using a single-step temperature revertant of tsH1 cells, TR-3 (which has normal leucyl-tRNA synthetase activity at 39.5 degrees C), we demonstrate here that all inhibition of eIF-2 function reverts together with the synthetase mutation. This establishes the close link between synthetase function and eIF-2 activity. In contrast, recharging tRNALeu in vivo in tsH1 cells at 39.5 degrees C by treatment with a low concentration of cycloheximide failed to reverse the inhibition of eIF-2 function. This indicates that tRNA charging per se is not involved in the regulatory mechanism. Our data indicate a novel role for aminoacyl-tRNA synthetases in the regulation of eIF-2 function mediated through phosphorylation of the alpha subunit of this factor. However, in spite of the fact that cell-free extracts from Chinese hamster ovary cells contain protein kinase and phosphatase activities active against either exogenous or endogenous eIF-2 alpha, we have been unable to show any activation of kinase or inactivation of phosphatase following incubation of the cells at 39.5 degrees C.     

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.     

Binding of ATP to eukaryotic initiation factor 2. Differential modulation of mRNA-binding activity and GTP-dependent binding of methionyl-tRNAMetf

Eukaryotic initiation factor 2 (eIF-2) is shown to bind ATP with high affinity. Binding of ATP to eIF-2 induces loss of the ability to form a ternary complex with Met-tRNAf and GTP, while still allowing, and even stimulating, the binding of mRNA. Ternary complex formation between eIF-2, GTP, and Met-tRNAf is inhibited effectively by ATP, but not by CTP or UTP. Hydrolysis of ATP is not required for inhibition, for adenyl-5'-yl imidodiphosphate (AMP-PNP), a nonhydrolyzable analogue of ATP, is as active an inhibitor; adenosine 5'-O-(thiotriphosphate) (ATP gamma S) inhibits far more weakly. Ternary complex formation is inhibited effectively by ATP, dATP, or ADP, but not by AMP and adenosine. Hence, the gamma-phosphate of ATP and its 3'-OH group are not required for inhibition, but the beta-phosphate is indispensible. Specific complex formation between ATP and eIF-2 is shown 1) by effective retention of Met-tRNAf- and mRNA-binding activities on ATP-agarose and by the ability of free ATP, but not GTP, CTP, or UTP, to effect elution of eIF-2 from this substrate; 2) by eIF-2-dependent retention of [alpha-32P]ATP or dATP on nitrocellulose filters and its inhibition by excess ATP, but not by GTP, CTP, or UTP. Upon elution from ATP-agarose by high salt concentrations, eIF-2 recovers its ability to form a ternary complex with Met-tRNAf and GTP. ATP-induced inhibition of ternary complex formation is relieved by excess Met-tRNAf, but not by excess GTP or guanyl-5'-yl imidodiphosphate (GMP-PNP). Thus, ATP does not act by inhibiting binding of GTP to eIF-2. Instead, ATP causes Met-tRNAf in ternary complex to dissociate from eIF-2. Conversely, affinity of eIF-2 for ATP is high in the absence of GTP and Met-tRNAf (Kd less than or equal to 10(-12) M), but decreases greatly in conditions of ternary complex formation. These results support the concept that eIF-2 assumes distinct conformations for ternary complex formation and for binding of mRNA, and that these are affected differently by ATP. Interaction of ATP with an eIF-2 molecule in ternary complex with Met-tRNAf and GTP promotes displacement of Met-tRNAf from eIF-2, inducing a state favorable for binding of mRNA. ATP may thus regulate the dual binding activities of eIF-2 during initiation of translation.     

Factors from wheat germ that enhance the activity of eukaryotic initiation factor eIF-2. Isolation and characterization of Co-eIF-2 beta

A factor has been isolated from wheat germ that enhances the ability of initiation factor 2 (eIF-2) to form a ternary complex with GTP and Met-tRNAf and enhances the binding of Met-tRNAf to 40 s ribosomal subunits. This factor, designated Co-eIF2 beta, is a monomeric protein with a molecular weight of approximately 83,000. Wheat germ eIF-2 forms a stable binary complex with GDP but not with GTP. Co-eIF-2 beta enhances the formation of an eIF-2 . GDP complex, but does not enable eIF-2 to form a stable complex with GTP.     

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.