Characterization of the Mammalian Initiation Factor eIF2B Complex as a GDP Dissociation Stimulator Protein

Abstract

Initiation factor eIF2B mediates a key regulatory step in the initiation of mRNA translation, i.e. the regeneration of active eIF2·GTP complexes. It is composed of five subunits, α–ε. The largest of these (ε) displays catalytic activity in the absence of the others. The catalytic mechanism of eIF2B and the functions of the other subunits remain to be clarified. Here we show that, when present at similar concentrations to eIF2, mammalian eIF2B can mediate release of eIF2-bound GDP even in the absence of free nucleotide, indicating that it acts as a GDP dissociation stimulator protein. Consistent with this, addition of GDP to purified eIF2·eIF2B complexes causes them to dissociate. The alternative sequential mechanism would require that eIF2Bε itself bind GTP. However, we show that it is the β-subunit of eIF2B that interacts with GTP. This indicates that binding of GTP to eIF2B is not an essential element of its mechanism. eIF2B preparations that lack the α-subunit display reduced activity compared with the holocomplex. Supplementation of such preparations with recombinant eIF2Bα markedly enhances activity, indicating that eIF2Bα is required for full activity of mammalian eIF2B. Initiation factor eIF2B mediates a key regulatory step in the initiation of mRNA translation, i.e. the regeneration of active eIF2·GTP complexes. It is composed of five subunits, α–ε. The largest of these (ε) displays catalytic activity in the absence of the others. The catalytic mechanism of eIF2B and the functions of the other subunits remain to be clarified. Here we show that, when present at similar concentrations to eIF2, mammalian eIF2B can mediate release of eIF2-bound GDP even in the absence of free nucleotide, indicating that it acts as a GDP dissociation stimulator protein. Consistent with this, addition of GDP to purified eIF2·eIF2B complexes causes them to dissociate. The alternative sequential mechanism would require that eIF2Bε itself bind GTP. However, we show that it is the β-subunit of eIF2B that interacts with GTP. This indicates that binding of GTP to eIF2B is not an essential element of its mechanism. eIF2B preparations that lack the α-subunit display reduced activity compared with the holocomplex. Supplementation of such preparations with recombinant eIF2Bα markedly enhances activity, indicating that eIF2Bα is required for full activity of mammalian eIF2B. eukaryotic initiation factor guanine nucleotide dissociation stimulator guanine nucleotide exchange factor initiator methionyl-tRNA polyacrylamide gel electrophoresis Eukaryotic initiation factor (eIF)1 2B is a heteropentameric protein, which catalyzes a key step in translation initiation, i.e. the exchange of guanine nucleotides on initiation factor eIF2. eIF2 binds GTP and in this form can also interact with the initiator methionyl-tRNA (Met-tRNAi) to form the ternary complex eIF2·GTP·Met-tRNAi (1Hinnebusch A.G. Sonenberg N. Hershey J.W.B. Mathews M.B. Translational Control of Gene Expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000: 185-243Google Scholar, 2Webb B.L.J. Proud C.G. Int. J. Biochem. Cell Biol. 1998; 29: 1127-1131Crossref Scopus (77) Google Scholar). Such complexes can then bind to the 40 S ribosomal subunit to form pre-initiation complexes, bringing the Met-tRNAi into position to recognize the AUG start codon of the mRNA following ribosomal scanning. The eIF2-bound GTP molecule is then hydrolyzed to GDP and Pi in a process that requires a further factor, eIF5. The inactive eIF2·GDP complex is released from the ribosome. The rate of release of GDP from eIF2 is very slow under physiological conditions, and the exchange of bound GDP for GTP, to regenerate active eIF2·GTP, requires an additional factor, eIF2B. The activity of eIF2B plays a key role in regulating the initiation of translation and can be controlled in several ways. For example, phosphorylation of the α-subunit of eIF2 inhibits eIF2B and serves to inhibit translation under a range of stressful conditions (1Hinnebusch A.G. Sonenberg N. Hershey J.W.B. Mathews M.B. Translational Control of Gene Expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000: 185-243Google Scholar). Conversely, eIF2B is activated by many stimuli, which stimulate protein synthesis, including insulin, and this appears to involve changes in the state of phosphorylation of the largest (ε) subunit of eIF2B (3Welsh G.I. Miller C.M. Loughlin A.J. Price N.T. Proud C.G. FEBS Lett. 1998; 421: 125-130Crossref PubMed Scopus (248) Google Scholar, 4Dholakia J.N. Wahba A.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 51-54Crossref PubMed Scopus (78) Google Scholar). eIF2B has also been reported to be regulated, in vitro, by a number of allosteric modulators, including nicotinamide adenine nucleotides (5Dholakia J.N. Mueser T.C. Woodley C.L. Parkhurst L.J. Wahba A.J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 6746-6750Crossref PubMed Scopus (67) Google Scholar, 6Singh L.P. Wahba A.J. Biochem. Biophys. Res. Commun. 1998; 217: 616-623Crossref Scopus (8) Google Scholar, 7Singh L.P. Aroor A.R. Wahba A.J. Biochem. Biophys. Res. Commun. 1995; 212: 1007-1014Crossref PubMed Scopus (10) Google Scholar, 8Oldfield S. Proud C.G. Eur. J. Biochem. 1992; 208: 73-81Crossref PubMed Scopus (70) Google Scholar, 9Kimball S.R. Jefferson L.S. Biochem. Biophys. Res. Commun. 1995; 212: 1074-1081Crossref PubMed Scopus (17) Google Scholar). Studies using photoactivatable nucleotide analogs suggested that eIF2B contains binding sites for GTP and ATP, although the functions of these sites are unclear. eIF2B is an unusually complicated guanine nucleotide exchange factor (GEF), those acting on small G-proteins such as Ras, for example, being much smaller monomeric proteins. The reasons for this complexity and the roles of the five subunits are still not completely clear. A major step toward addressing these issues has been the cloning of cDNAs encoding subunits of eIF2B. This has now been achieved for several species, including yeast (see, e.g., Refs. 10Bushman J.L. Foiani M. Cigan A.M. Paddon C.J. Hinnebusch A.G. Mol. Cell. Biol. 1993; 13: 4618-4631Crossref PubMed Scopus (46) Google Scholar and 11Cigan A.M. Bushman J.L. Boal T.R. Hinnebusch A.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5350-5354Crossref PubMed Scopus (69) Google Scholar), mammals (see, e.g., Refs. 12Flowers K.M. Kimball S.R. Feldhoff R.C. Hinnebusch A.G. Jefferson L.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4274-4278Crossref PubMed Scopus (11) Google Scholar, 13Flowers K.M. Mellor H. Matts R. Kimball S.R. Jefferson L.S. Biochim. Biophys. Acta. 1996; 1317: 318-324Crossref Scopus (13) Google Scholar, 14Price N.T. Kimball S.R. Jefferson L.S. Proud C.G. Biochem. J. 1996; 318: 631-636Crossref PubMed Scopus (17) Google Scholar, 15Craddock B.L. Price N.T. Proud C.G. Biochem. J. 1995; 309: 1009-1014Crossref PubMed Scopus (11) Google Scholar, 16Price N.T. Mellor H. Craddock B.L. Flowers K.M. Kimball S.R. Wilmer T. Jefferson L.S. Proud C.G. Biochem. J. 1996; 318: 637-643Crossref PubMed Scopus (25) Google Scholar), and Drosophila(17Williams D.D. Pavitt G.D. Proud C.G. J. Biol. Chem. 2001; 276: 3733-3742Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Studies for all three sources have shown that the ε-subunit alone can catalyze nucleotide exchange on eIF2 (17Williams D.D. Pavitt G.D. Proud C.G. J. Biol. Chem. 2001; 276: 3733-3742Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 18Fabian J.R. Kimball S.R. Heinzinger N.K. Jefferson L.S. J. Biol. Chem. 1997; 272: 12359-12365Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 19Pavitt G.D. Ramaiah K.V.A. Kimball S.R. Hinnebusch A.G. Genes Dev. 1998; 12: 514-526Crossref PubMed Scopus (212) Google Scholar), although its activity is enhanced by the others (18Fabian J.R. Kimball S.R. Heinzinger N.K. Jefferson L.S. J. Biol. Chem. 1997; 272: 12359-12365Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 19Pavitt G.D. Ramaiah K.V.A. Kimball S.R. Hinnebusch A.G. Genes Dev. 1998; 12: 514-526Crossref PubMed Scopus (212) Google Scholar, 20Gomez E. Pavitt G.D. Mol. Cell. Biol. 2000; 20: 3965-3976Crossref PubMed Scopus (85) Google Scholar). eIF2Bε forms a catalytic subcomplex with eIF2Bγ, with which it shares some sequence similarity. The other three subunits also display mutual sequence similarity and form a second subcomplex, which appears to have a regulatory function (19Pavitt G.D. Ramaiah K.V.A. Kimball S.R. Hinnebusch A.G. Genes Dev. 1998; 12: 514-526Crossref PubMed Scopus (212) Google Scholar). There is some disagreement over the role of the α-subunit. This polypeptide is encoded by a non-essential gene in yeast (GCN3), although it is required for regulation of eIF2B by phosphorylation of eIF2α (21Hannig E.M. Hinnebusch A.G. Mol. Cell. Biol. 1988; 8: 4808-4820Crossref PubMed Scopus (56) Google Scholar). Similarly, studies in which mammalian eIF2B polypeptides were expressed in insect cells concluded that eIF2Bα was dispensable for activity (22Kimball S.R. Fabian J.R. Pavitt G.D. Hinnebusch A.G. Jefferson L.S. J. Biol. Chem. 1998; 273: 12841-12845Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). However, others have found that preparations of eIF2B lacking the α-subunit showed reduced activity in nucleotide exchange (23Craddock B.L. Proud C.G. Biochem. Biophys. Res. Commun. 1996; 220: 843-847Crossref PubMed Scopus (15) Google Scholar), implying that this component was required for full activity. There is also disagreement over the catalytic mechanism of eIF2B; early studies supported the so-called “ping-pong” or substituted mechanism A in A.G. R. J. Biol. Chem. 1988; Full Text PDF PubMed Google Scholar), which is the by in mRNA translation, the factor Miller J. Biol. Chem. Full Text PDF PubMed Google and by that nucleotide exchange on other H. A. 1995; PubMed Scopus Google Scholar, H. A. 1995; PubMed Scopus Google Scholar, H. J. A. 1998; PubMed Scopus Google Scholar, 2000; PubMed Scopus Google Scholar). to stimulate the release of bound guanine nucleotide and are dissociation stimulator 2000; PubMed Scopus Google studies have suggested that eIF2B the alternative sequential mechanism in Refs. J.N. Wahba A.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar and J. Pavitt G.D. Hinnebusch A.G. E.M. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). Here we that the of eIF2B the of the mechanism and that eIF2B acts as a protein. also show that contains a binding for nucleotides and that eIF2Bα is required for the full activity of mammalian eIF2B. and were from or and were from was from were from were purified as S. Proud C.G. Eur. J. Biochem. 1992; 208: 73-81Crossref PubMed Scopus (70) Google Scholar), using as the Cell The was to and the and was to exchange on and from eIF2B was by its to the release of from complexes S. Proud C.G. Eur. J. Biochem. 1992; 208: 73-81Crossref PubMed Scopus (70) Google Scholar), that for of the complexes was to some were with in the absence of as in the were using a were and of eIF2B of or of and or nucleotide as were at for to to for on were then to and were with and to to Control were at for and then on for to or on as in the eIF2Bα in was by J. of the at and and in was by Craddock of at The encoding the for subunits were and for eIF2Bα and for and into were then into and in were then and to of were on for and was with for were the by and in and at cells were and to of was using eIF2B was purified from using a of the for its from and This protein five polypeptides on polypeptides for subunits of mammalian eIF2B. This was of eIF2, as by with and by the much of with a eIF2α Such preparations are active in exchange of GDP bound to eIF2 for GTP S. Proud C.G. Eur. J. Biochem. 1992; 208: 73-81Crossref PubMed Scopus (70) Google and others J.N. Wahba A.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Pavitt G.D. Hinnebusch A.G. E.M. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google have reported that eIF2B to catalyze the release of GDP from complexes GTP was of the of this it was to show that this was also for this of which is the as shown in release of bound was when GTP was very release was in the absence of GTP. A small of release of was when the of eIF2B was and this is in have been to the catalytic mechanism of eIF2B. are in in the or substituted the binding of eIF2B to eIF2·GDP complexes the release of the GTP to bind to eIF2 to form active eIF2·GTP complexes and in the release of the eIF2B this eIF2B is acting as a eIF2, and eIF2B as the GDP is released to eIF2·eIF2B complexes. The second the of a eIF2B and GTP. The a for guanine nucleotide in to the release of bound GDP from eIF2 have been as for the sequential mechanism in J.N. Wahba A.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Pavitt G.D. Hinnebusch A.G. E.M. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). However, of the that are also with mechanism A. such is a of this mechanism as in A. eIF2B is in the by and others at catalytic in the in the of is of the mechanism shown in A that for eIF2B to it be released from the eIF2·eIF2B complex and that this requires free nucleotide would to regenerate it to function as a and to mediate further release of GDP from the complexes, which are the for this eIF2 can bind GDP or GTP, would that, under this catalytic exchange would with free GTP or GDP This is the for mammalian eIF2B and not as reported for the yeast factor A.M. Bushman J.L. Boal T.R. Hinnebusch A.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5350-5354Crossref PubMed Scopus (69) Google Scholar). Consistent with the of Cigan A.M. Bushman J.L. Boal T.R. Hinnebusch A.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5350-5354Crossref PubMed Scopus (69) Google for yeast not release of bound GDP from eIF2 not A of mechanism the not of mechanism is that of eIF2·eIF2B complexes with guanine nucleotides such complexes to dissociate. The eIF2·eIF2B complex was purified by on and then with by addition of and on this second eIF2 was from eIF2B from the also 8Oldfield S. Proud C.G. Eur. J. Biochem. 1992; 208: 73-81Crossref PubMed Scopus (70) Google Scholar and and of complexes that not been with GDP not in of the which as the eIF2·eIF2B complex not this we similar using gel exchange to eIF2 or eIF2B from eIF2·eIF2B complexes. eIF2·eIF2B complexes were to a GDP as eIF2 and eIF2B when of the eIF2·eIF2B complexes was with GDP and then to a eIF2 in eIF2B in indicating that eIF2 and eIF2B from are with mechanism not mechanism and the that the of free GDP or GTP to the release of bound to eIF2 is to the regeneration of active eIF2B. The the that eIF2B acts as a additional of mechanism A is that of eIF2B to release of GDP even in the absence of free this be the was suggested by the release of in in the of the of eIF2B. this complexes were and to gel to free eIF2B was then in similar to or the of complexes these conditions, release of GDP was was not in at a the the of release as of eIF2B were this the further toward This is the from the eIF2B can the release of of GDP similar to the of eIF2B as it into the eIF2·eIF2B complexes. The release of eIF2B from these complexes requires free GDP or GTP, the eIF2B now to Consistent with this, addition of free nucleotide, of bound GDP to the catalytic of the eIF2B requires that eIF2B contains a binding for GTP and eIF2B from mammals J.N. Wahba A.J. J. Biol. Chem. Full Text PDF PubMed Google and yeast J. Pavitt G.D. Hinnebusch A.G. E.M. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), have indicating that eIF2B can bind GTP, to a of of eIF2Bε displays nucleotide exchange activity when expressed alone (17Williams D.D. Pavitt G.D. Proud C.G. J. Biol. Chem. 2001; 276: 3733-3742Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 18Fabian J.R. Kimball S.R. Heinzinger N.K. Jefferson L.S. J. Biol. Chem. 1997; 272: 12359-12365Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 19Pavitt G.D. Ramaiah K.V.A. Kimball S.R. Hinnebusch A.G. Genes Dev. 1998; 12: 514-526Crossref PubMed Scopus (212) Google Scholar), would that, mechanism were the mechanism of of eIF2Bε a binding for GTP. However, we were to binding of to purified preparations of eIF2B complex in binding of the for eIF2 on a not we this further by using to to purified eIF2B. is free of eIF2, the of GTP binding to eIF2 to these studies using GTP the photoactivatable is in the as J.N. Wahba A.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar), of nucleotide analogs can to to subunits in a protein complex to the subunit which binds the of this is eIF2 J.N. Wahba A.J. J. Biol. Chem. Full Text PDF PubMed Google or other GTP analogs eIF2α or Biochem. Int. 1988; Google FEBS Lett. PubMed Scopus Google or as the subunit that contains the E.M. Cigan A.M. Mol. Cell. Biol. 1993; 13: PubMed Google Scholar, M. Hershey J. Biol. Chem. Full Text PDF PubMed Google Scholar). It be that the was to eIF2Bε as for mechanism were also studies using in of a polypeptide at i.e. in the position of the of J.N. Wahba A.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar), found that this polypeptide was also by However, in to that was reduced by the of as as by GTP using to of the and reported by J.N. Wahba A.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). the that by of the by a were also this was was by addition of GTP or GTP being This that and GTP bind to the sites in that GTP bound to we expressed this polypeptide in E. as a protein, purified it on and to shown in in of GTP to recombinant This that GTP interacts with this subunit and that the to this polypeptide for the complex is not a of its binding to subunit at a to in the a were in the this was the that by was to phosphorylation of by a and eIF2B is also to interact with and (5Dholakia J.N. Mueser T.C. Woodley C.L. Parkhurst L.J. Wahba A.J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 6746-6750Crossref PubMed Scopus (67) Google Scholar, 8Oldfield S. Proud C.G. Eur. J. Biochem. 1992; 208: 73-81Crossref PubMed Scopus (70) Google Scholar). the binding for these was also in we with However, this to in of of the polypeptides of although as a was not we with GTP or in the It in a in was It appears that the in is not the for binding which in the protein. The of to by or allosteric the binding sites for these reported (23Craddock B.L. Proud C.G. Biochem. Biophys. Res. Commun. 1996; 220: 843-847Crossref PubMed Scopus (15) Google Scholar), of mammalian eIF2B from a of the protein as a complex lacking the α-subunit. The is of eIF2B from cells on of eIF2B; the at showed subunits on A and the at showed The absence of eIF2Bα from the was using an for the α-subunit of eIF2B for eIF2Bα was in these a was for the factor all five subunits on the of the and preparations were the was found to display of the activity of the in the nucleotide exchange This is with for the and preparations of the factor from (23Craddock B.L. Proud C.G. Biochem. Biophys. Res. Commun. 1996; 220: 843-847Crossref PubMed Scopus (15) Google Scholar). that the absence of eIF2Bα from the was the of its activity, mammalian eIF2Bα was expressed a and This a protein on that nucleotide exchange activity on its Supplementation of eIF2B with recombinant eIF2Bα markedly its activity, to the for the protein the in activity of to the to be to lack of the α-subunit other The show that eIF2B can release of from eIF2 even in the absence of GDP or GTP. This from S. Proud C.G. Eur. J. Biochem. 1992; 208: 73-81Crossref PubMed Scopus (70) Google and (1Hinnebusch A.G. Sonenberg N. Hershey J.W.B. Mathews M.B. Translational Control of Gene Expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000: 185-243Google Scholar, J.N. Wahba A.J. J. Biol. Chem. Full Text PDF PubMed Google these were using catalytic of eIF2B compared with the have reported that of eIF2B from a not release of bound GDP from in the present show that a of eIF2B of the nucleotide bound to eIF2. The release of bound GDP at of eIF2B to eIF2 of on the rate for the eIF2·GDP eIF2B eIF2·eIF2B The mechanism that the catalytic function of eIF2B requires free nucleotide in to regenerate eIF2B from the eIF2·eIF2B complexes, which are an in this mechanism at as in this this mechanism that GDP be released from eIF2, as eIF2B the nucleotide from eIF2. with this, we show that the addition of GDP to eIF2·eIF2B complexes causes them to as from the mechanism. of these is with the other mechanism for eIF2B we also to eIF2B bound to an that be in the substituted that in the mechanism. are in with the mechanism of that this is the mechanism by which eIF2B in translation, also the substituted mechanism Miller J. Biol. Chem. Full Text PDF PubMed Google Scholar). and exchange for the small and Ras, have been shown to by an mechanism H. A. 1995; PubMed Scopus Google Scholar, H. A. 1995; PubMed Scopus Google Scholar, H. J. A. 1998; PubMed Scopus Google Scholar), and such a mechanism for the guanine nucleotide dissociation factor that acts on 2000; PubMed Scopus Google Scholar). It that the substituted mechanism is a by which such although the of the mechanism at the are to J. Biochem. Sci. Full Text Full Text PDF PubMed Scopus Google Scholar, S.R. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). The in and that eIF2B acts as a the release of GDP from eIF2 the exchange of GDP for GTP binds to the studies have that eIF2B can bind guanine which is a of the substituted mechanism. Wahba and J.N. Wahba A.J. J. Biol. Chem. Full Text PDF PubMed Google reported GTP binding to mammalian although the was Similarly, from J. Pavitt G.D. Hinnebusch A.G. E.M. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google for yeast eIF2B show a of even at a nucleotide of the dissociation this we were to binding of GTP to purified eIF2B in binding eIF2B not bind to not although this to the of of the GDP to the the or to the of the eIF2B It is that the for guanine nucleotides are very the reported by that or binding is or that the of binding by others the of a protein, although was on the shown by (1Hinnebusch A.G. Sonenberg N. Hershey J.W.B. Mathews M.B. Translational Control of Gene Expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000: 185-243Google Scholar, J.N. Wahba A.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). of GTP to to This that the protein bind guanine nucleotides and that this subunit is in the as concluded by the studies of J.N. Wahba A.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar), a GTP as of was by ATP, with this, also to this from the J.N. Wahba A.J. J. Biol. Chem. Full Text PDF PubMed Google by GTP was not by ATP, and to the and It is not this is to be that the binding in interacts with and GTP. The that the recombinant β-subunit is also by the that this subunit binds guanine and that its is not a of the binding of GTP to an subunit in the the sequence of contains its with GTP and involve a binding found that addition of not the of by GTP or ATP, indicating that the binding in is from that which nicotinamide adenine allosteric eIF2B activity. were to of eIF2B using and were also to to the binding in eIF2B for this the substituted mechanism requires that eIF2B a binding for GTP. that be such a it is to that this in and not in the catalytic This is a key eIF2B the substituted this would be present in which has been shown to catalyze nucleotide exchange on its on from three for the protein from and mammals (17Williams D.D. Pavitt G.D. Proud C.G. J. Biol. Chem. 2001; 276: 3733-3742Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 18Fabian J.R. Kimball S.R. Heinzinger N.K. Jefferson L.S. J. Biol. Chem. 1997; 272: 12359-12365Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 19Pavitt G.D. Ramaiah K.V.A. Kimball S.R. Hinnebusch A.G. Genes Dev. 1998; 12: 514-526Crossref PubMed Scopus (212) Google Scholar). also the substituted mechanism. the present that eIF2B mediates release of GDP from eIF2, the binding of GTP of these nucleotides in the this is the then eIF2B acts as a protein a nucleotide exchange factor which be to of the exchange step The physiological role of eIF2B is of to this exchange that this is a of its to the release of GDP from eIF2, with binding of GTP to the eIF2. It has been suggested that nucleotides as allosteric of eIF2B N.T. Proud C.G. PubMed Scopus Google Scholar). Kimball and Jefferson S.R. Jefferson L.S. Biochem. Biophys. Res. Commun. 1995; 212: 1074-1081Crossref PubMed Scopus (17) Google have shown that inhibits the activity of eIF2B. This is in physiological as enhanced to eIF2B inhibit suggested that the be by the of S.R. Jefferson L.S. Biochem. Biophys. Res. Commun. 1995; 212: 1074-1081Crossref PubMed Scopus (17) Google Scholar), on is by a binding in eIF2B not the of by The function of nucleotide binding to unclear. A of to eIF2B is the role of its studies that this subunit is required for the regulation of eIF2B activity by phosphorylation of eIF2α (22Kimball S.R. Fabian J.R. Pavitt G.D. Hinnebusch A.G. Jefferson L.S. J. Biol. Chem. 1998; 273: 12841-12845Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, Cigan A.M. Hinnebusch A.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: PubMed Scopus Google Scholar, R.C. Cigan A.M. Hinnebusch A.G. Cell. 1992; Full Text PDF PubMed Scopus Google Scholar). However, in yeast or of mammalian eIF2B subunits in insect cells suggested that eIF2Bα was not required for activity in or in vitro, for preparations of eIF2B that lack this subunit it was for full activity (23Craddock B.L. Proud C.G. Biochem. Biophys. Res. Commun. 1996; 220: 843-847Crossref PubMed Scopus (15) Google Scholar). The reported this for eIF2B from a cells that addition of recombinant eIF2Bα completely eIF2B activity. This the that the activity of eIF2B is to other in the protein and that, under in conditions, the mammalian factor require eIF2Bα for full activity. for the and those of studies for the yeast of by the of or the function of eIF2B in and for the studies in the of insect eIF2Bα in the complexes the subunits of eIF2B from although is at this position in the are to and for with the of initiation