Subunit Assembly and Guanine Nucleotide Exchange Activity of Eukaryotic Initiation Factor-2B Expressed in Sf9 Cells

Abstract

Eukaryotic initiation factor-2B (eIF-2B) is a guanine nucleotide exchange factor (GEF) that plays a key role in the regulation of protein synthesis. In this study, we have used the baculovirus-infected Sf9 insect cell system to express and characterize the five dissimilar subunits of rat eIF-2B. GEF activity was detected in extracts of Sf9 cells expressing the ε-subunit alone and was greatly increased when all five subunits were coexpressed. In addition, high GEF activity was observed in extracts containing a four-subunit complex lacking the α-subunit. Assembly of an eIF-2B holoprotein was confirmed by coimmunoprecipitation of all five subunits. Gel filtration chromatography revealed that recombinant eIF-2B had the same molecular mass as eIF-2B purified from rat liver and that it did indeed possess GEF activity. Phosphorylation of the substrate eIF-2 inhibited the GEF activity of the five-subunit eIF-2B; this inhibition required the eIF-2B α-subunit. The results demonstrate that eIF-2Bα functions as a regulatory subunit that is not required for GEF activity, but instead mediates the regulation of eIF-2B by substrate phosphorylation. Furthermore, eIF-2Bε is necessary and is perhaps sufficient for GEF activity in vitro. Eukaryotic initiation factor-2B (eIF-2B) is a guanine nucleotide exchange factor (GEF) that plays a key role in the regulation of protein synthesis. In this study, we have used the baculovirus-infected Sf9 insect cell system to express and characterize the five dissimilar subunits of rat eIF-2B. GEF activity was detected in extracts of Sf9 cells expressing the ε-subunit alone and was greatly increased when all five subunits were coexpressed. In addition, high GEF activity was observed in extracts containing a four-subunit complex lacking the α-subunit. Assembly of an eIF-2B holoprotein was confirmed by coimmunoprecipitation of all five subunits. Gel filtration chromatography revealed that recombinant eIF-2B had the same molecular mass as eIF-2B purified from rat liver and that it did indeed possess GEF activity. Phosphorylation of the substrate eIF-2 inhibited the GEF activity of the five-subunit eIF-2B; this inhibition required the eIF-2B α-subunit. The results demonstrate that eIF-2Bα functions as a regulatory subunit that is not required for GEF activity, but instead mediates the regulation of eIF-2B by substrate phosphorylation. Furthermore, eIF-2Bε is necessary and is perhaps sufficient for GEF activity in vitro. Protein synthesis in eukaryotic cells is regulated in response to growth factors, hormones, and changes in the external environment (reviewed in Refs. 1Kimball S.R. Vary T.C. Jefferson L.S. Annu. Rev. Physiol. 1994; 56: 321-348Crossref PubMed Scopus (182) Google Scholar and 2Redpath N.T. Proud C.G. Biochim. Biophys. Acta. 1994; 1220: 147-162Crossref PubMed Scopus (87) Google Scholar). One of the major control points for protein synthesis is the initiation of mRNA translation, which is mediated by a number of eukaryotic initiation factors (eIFs) 1The abbreviations used are: eIFs, eukaryotic initiation factors; GEF, guanine nucleotide exchange factor; PAGE, polyacrylamide gel electrophoresis; MOPS, 4-morpholinepropanesulfonic acid; Ac NPV, A. californica nuclear polyhedrosis virus; HCR, heme-controlled repressor. (recently reviewed in Ref. 3Pain V.M. Eur. J. Biochem. 1996; 236: 747-771Crossref PubMed Scopus (638) Google Scholar). Translation of mRNA begins with the binding of initiator Met-tRNAi to the 40 S ribosomal subunit and is mediated by eIF-2 as part of the eIF-2·GTP·Met-tRNAi ternary complex. During the initiation process, the GTP bound to eIF-2 is hydrolyzed, and a binary complex consisting of eIF-2 and GDP is released from the 80 S initiation complex. Since eIF-2 has a 100–400-fold higher affinity for GDP than for GTP, the guanine nucleotide exchange factor (GEF) known as eIF-2B is required to regenerate the GTP-bound form of eIF-2, which can then participate in another cycle of translation initiation. In cells subjected to a variety of stresses, e.g. heat shock, serum and amino acid deprivation, and heme deprivation in reticulocytes, protein synthesis is inhibited by a mechanism involving phosphorylation of the α-subunit of eIF-2 (reviewed in Ref. 4Clemens M.J. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory Press, Plainview, NY1996: 139-172Google Scholar). Phosphorylated eIF-2 efficiently binds to eIF-2B, but cannot undergo nucleotide exchange and therefore competitively inhibits the GEF function of eIF-2B, which is present in limiting amounts in all cell types examined (5Rhoads R.E. J. Biol. Chem. 1993; 268: 3017-3020Abstract Full Text PDF PubMed Google Scholar, 6Hershey J.W.B. J. Biol. Chem. 1989; 264: 20823-20826Abstract Full Text PDF PubMed Google Scholar, 7Dever T.E. Yang W. Astrom S. Bystrom A.S. Hinnebusch A.G. Mol. Cell. Biol. 1995; 15: 6351-6363Crossref PubMed Scopus (109) Google Scholar). In addition, eIF-2B may be regulated by other mechanisms since its activity is modulated under certain conditions in the absence of a change in phosphorylation of eIF-2α (8Welsh G.I. Proud C.G. Biochem. J. 1992; 284: 19-23Crossref PubMed Scopus (67) Google Scholar, 9Gilligan M. Welsh G.I. Flynn A. Bujalska I. Diggle T.A. Denton R.M. Proud C.G. Docherty K. J. Biol. Chem. 1996; 271: 2121-2125Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 10Kimball S.R. Jefferson L.S. Biochem. Biophys. Res. Commun. 1988; 156: 706-711Crossref PubMed Scopus (55) Google Scholar, 11Jeffrey I.W. Kelly F.J. Duncan R. Hershey J.W.B. Pain V.M. Biochimie ( Paris ). 1990; 72: 751-757Crossref PubMed Scopus (33) Google Scholar, 12Rowlands A.G. Montine K.S. Henshaw E.C. Panniers R. Eur. J. Biochem. 1988; 175: 93-99Crossref PubMed Scopus (78) Google Scholar). The ε-subunit of eIF-2B is phosphorylated in vitro by glycogen synthase kinase-3, casein kinase-1, and casein kinase-2, which may alter the GEF activity of the holoprotein (13Singh L.P. Denslow N.D. Wahba A.J. Biochem. J. 1996; 35: 3206-3212Crossref Scopus (31) Google Scholar, 14Dholakia J.N. Wahba A.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 51-54Crossref PubMed Scopus (79) Google Scholar, 15Welsh G.I. Proud C.G. Biochem. J. 1993; 294: 625-629Crossref PubMed Scopus (351) Google Scholar, 16Singh L.P. Aroor A.R. Wahba A.J. Biochemistry. 1994; 33: 9152-9157Crossref PubMed Scopus (38) Google Scholar). In addition, the activity of eIF-2B appears to be regulated by allosteric effectors,e.g. NADP+, NAD+, and ATP inhibit guanine nucleotide exchange, whereas NADPH, NADH, and polyamines activate exchange (17Dholakia 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, 18Singh L.P. Aroor A.R. Wahba A.J. Biochem. Biophys. Res. Commun. 1995; 212: 1007-1014Crossref PubMed Scopus (10) Google Scholar, 19Karinch A.M. Kimball S.R. Vary T.C. Jefferson L.S. Am. J. Physiol. 1993; 264: E101-E108Crossref PubMed Google Scholar, 20Kimball S.R. Jefferson L.S. Biochem. Biophys. Res. Commun. 1995; 212: 1074-1081Crossref PubMed Scopus (17) Google Scholar). Most GEFs, such as those stimulating GDP dissociation from the Ras superfamily of proteins, exist as single subunit proteins (21Overbeck A.F. Brtva T.R. Cox A.D. Graham S.M. Huff S.Y. Khosravi-Far R. Quilliam L.A. Solski P.A. Der C.J. Mol. Reprod. Dev. 1995; 42: 468-476Crossref PubMed Scopus (64) Google Scholar, 22Quilliam L.A. Khosravi-Far R. Huff S.Y. Der C.J. Bioessays. 1995; 17: 395-404Crossref PubMed Scopus (193) Google Scholar). In contrast, eIF-2B is a complex protein consisting of five dissimilar subunits. The cDNAs for all five subunits of eIF-2B have been cloned from both mammals (23Price 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, 24Price N.T. Kimball S.R. Jefferson L.S. Proud C.G. Biochem. J. 1996; 318: 631-636Crossref PubMed Scopus (17) Google Scholar, 25Flowers K.M. Mellor H. Kimball S.R. Jefferson L.S. Biochim. Biophys. Acta. 1995; 1264: 163-167Crossref PubMed Scopus (5) Google Scholar, 26Flowers K.M. Mellor H. Matts R.L. Kimball S.R. Jefferson L.S. Biochim. Biophys. Acta. 1996; 1307: 318-324Crossref PubMed Scopus (13) Google Scholar) and yeast (27Hinnebusch A.G. Trends Biochem. Sci. 1994; 19: 409-414Abstract Full Text PDF PubMed Scopus (157) Google Scholar). Alignment of the coding regions of mammalian and yeast subunits indicates sequence homology for the mammalian α-, β-, γ-, δ-, and ε-subunits with the yeast GCN3, GCD7, GCD1, GCD2, and GCD6 subunits, respectively. Genetic studies in yeast indicate that deletion of the α-subunit of eIF-2B (GCN3) does not affect viability and therefore is not an essential component of the GCD complex under normal growth conditions (28Hannig E.M. Hinnebusch A.G. Mol. Cell. Biol. 1988; 8: 4808-4820Crossref PubMed Scopus (56) Google Scholar). However, there is controversy over the requirement of the α-subunit for eIF-2B function since a recent report has suggested that purified rabbit eIF-2B lacking the α-subunit does not have GEF activity (29Craddock B.L. Proud C.G. Biochim. Biophys. Acta. 1996; 220: 843-847Google Scholar). The current model for translation initiation in eukaryotic cells is formulated from biochemical studies of cell-free systems derived from rabbit reticulocytes and from genetic experiments conducted in yeast. In the study reported here, we set out to determine if the baculovirus expression system could be used as a new approach to study the structure/function relationship of translation initiation factors, many of which are composed of multiple subunits with unknown functions. For example, despite the fact that the mammalian eIF-2B complex has been available in purified form for 15 years and all five yeast and mammalian eIF-2B subunits have now been cloned, relatively little is known about the function of individual subunits or the mechanism of nucleotide exchange. Here we report our studies on the expression and coexpression of rat eIF-2B subunits in Sf9 insect cells using recombinant baculoviruses and the characterization of GEF activity in extracts of cells expressing these proteins. Mouse monoclonal antibodies against the γ- and ε-subunits of eIF-2B were produced using purified protein for antigen as described previously (30Kimball S.R. Karinch A.M. Mellor H. Jefferson L.S. Biochim. Biophys. Acta. 1994; PubMed Scopus (55) Google Scholar). For a monoclonal against the of eIF-2B was used in with a rabbit or a monoclonal to the eIF-2B and the α-, β-, δ-, and ε-subunits of eIF-2B an respectively. The for the rat number used in this study was by the rat number using the rabbit B.L. N.T. Proud C.G. Biochem. J. 1995; PubMed Scopus Google Scholar) as a by of The rat eIF-2B α-, γ-, δ-, and ε-subunits were previously described (23Price 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, 24Price N.T. Kimball S.R. Jefferson L.S. Proud C.G. Biochem. J. 1996; 318: 631-636Crossref PubMed Scopus (17) Google Scholar, 25Flowers K.M. Mellor H. Kimball S.R. Jefferson L.S. Biochim. Biophys. Acta. 1995; 1264: 163-167Crossref PubMed Scopus (5) Google Scholar, 26Flowers K.M. Mellor H. Matts R.L. Kimball S.R. Jefferson L.S. Biochim. Biophys. Acta. 1996; 1307: 318-324Crossref PubMed Scopus (13) Google and are and respectively. and of rat eIF-2B subunits in Sf9 a acid were to the cDNAs the α-, β-, δ-, and ε-subunits by using the The were cloned the of the to by the The had an that was using the The used to the in a single nucleotide that did not alter the amino acid sequence of the The of all were confirmed by sequence of the and using the and which to the of the The and cDNAs were out of the and the baculovirus containing the baculovirus and the cDNAs for the and containing the were the and of The cDNAs for the γ- and were and of baculovirus the eIF-2Bε was by the from and then the the of the of the baculovirus that the alone was by the the of Sf9 insect cells were in using insect with serum and recombinant baculoviruses expressing the of eIF-2B subunits described polyhedrosis and of the recombinant were Sf9 and recombinant baculoviruses were by as described previously Press, Scholar). expressing eIF-2B subunits were by cells with the were and for protein by For expression of eIF-2B subunits, Sf9 insect cells were in The Sf9 cells were then with recombinant a of of in a of for with of and then a Sf9 insect cells were with For cells were in and then in of consisting of and were in and on for 15 was by for were by with of a monoclonal against eIF-2Bε were used to the to the were in in and containing The were then using a and with containing and with containing Protein bound to the was in of and the was for The were by and proteins in the were by as described of cell as described were by gel and were to were then with serum in for in containing and with in The rabbit and antibodies were and respectively. The were then in for with a in in and using the eIF-2B the were with a monoclonal against rat with and as described Sf9 cells were in and in of and The were by for The were for eIF-2B activity as by the exchange of bound to eIF-2 for GDP as described previously S.R. Jefferson L.S. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar). The binary complex was by containing rat liver eIF-2 S.R. Jefferson L.S. J. Biol. Chem. Full Text PDF PubMed Google and in 80 of MOPS, and serum for The was to and the binary complex was on eIF-2B activity, containing a of of cell and was to a by of binary complex. The was then for or The exchange was as a in the binding of to with For the experiments in eIF-2 was phosphorylated using the eIF-2α by of eIF-2 in of and with of purified rabbit S.R. R.M. Jefferson L.S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The was for and the was by the on of containing the and was to containing eIF-2 was for as the eIF-2 The phosphorylation of eIF-2α was by gel and eIF-2B activity, containing phosphorylated or eIF-2, a of of cell and was to a and for The complex was then to and the was of the were from the or and as described Sf9 cells were with and then in of consisting of and The extracts were by for and a gel filtration with consisting of and were and the was of containing protein were for GEF activity as described the of eIF-2B subunits in of were to of and 15 of the were by and by with and antibodies as described a consisting of the was to the of the eIF-2B α-, β-, δ-, and baculoviruses for of the eIF-2B subunits were as to express the individual subunit cDNAs under the control of the or the the number of baculoviruses to express all five subunits of eIF-2B in Sf9 single baculoviruses the and or and were using a for of the eIF-2B subunit was using a for single the of eIF-2B subunits, Sf9 cells were with baculoviruses individual eIF-2B subunits or with baculoviruses all five eIF-2B subunits. The recombinant eIF-2B proteins in Sf9 cells were by and by using and monoclonal amounts of eIF-2B α-, β-, δ-, and ε-subunit proteins were observed for Sf9 cells with baculoviruses the ε-subunit the and and the and For Sf9 cells with all coexpression of proteins for of the five eIF-2B subunits was observed by However, in these the of produced could not be with the of the other subunits a was used to than was used to the other subunits. our this is the a complex translation initiation factor has been using the baculovirus-infected Sf9 cell was to or not rat eIF-2B subunits in Sf9 cells GEF activity. The GDP dissociation is a of the GEF activity of eIF-2B in the of the of the nucleotide exchange is GDP dissociation R. A.G. Henshaw E.C. J. Biol. Chem. 1988; Full Text PDF PubMed Google Scholar). were from Sf9 cells with of subunits or the all five subunits of eIF-2B. The extracts were then for GEF activity by the of released from binary over a of GEF activity was detected in extracts of cells with baculovirus or in extracts of cells with baculoviruses the and or and of eIF-2B GEF activity was detected in from cells expressing the eIF-2B ε-subunit of the bound was released from eIF-2 over with from cells with The of GEF activity detected in extracts of cells all five eIF-2B subunits was of bound to eIF-2 was than that in extracts of cells expressing eIF-2Bε that a five-subunit eIF-2B complex was in the extracts of cells expressing the eIF-2B ε-subunit alone GEF activity with extracts of cells with we if the ε-subunit was required for the GEF activity observed in extracts of cells expressing all five subunits of eIF-2B. cells were with expressing a four-subunit complex lacking GEF activity was observed In contrast, the of GDP exchange observed in extracts of cells expressing a four-subunit complex lacking eIF-2Bα was greatly with control results demonstrate that the eIF-2B ε-subunit is required for the GEF activity of eIF-2B, whereas the eIF-2B α-subunit is The activity observed for the five-subunit eIF-2B complex in and was out of the of the and it was necessary to to determine the activity of the ε-subunit alone to the and four-subunit eIF-2B The results that of from cells expressing all five subunits of eIF-2B or the β-, γ-, δ-, and ε-subunits a of exchange to that observed for 40 of from cells expressing the ε-subunit alone the of ε-subunit in was the results indicate that the five-subunit and four-subunit and eIF-2B were than the eIF-2B ε-subunit The of GDP exchange observed in extracts of cells expressing all five subunits of eIF-2B suggested that the subunits were to form a complex. eIF-2B complex in insect we a monoclonal against the ε-subunit of rat eIF-2B previously to the five-subunit eIF-2B complex from rat liver (30Kimball S.R. Karinch A.M. Mellor H. Jefferson L.S. Biochim. Biophys. Acta. 1994; PubMed Scopus (55) Google Scholar). In these extracts of Sf9 cells expressing all five subunits of eIF-2B were using the monoclonal and the were by The proteins in the gel were by with a rabbit against the and with a monoclonal against five subunits were with the monoclonal that the subunits to form a complex in insect a control for Sf9 cells were as to express the α-, β-, γ-, and in the absence of the ε-subunit In the absence of the of the eIF-2B subunits were by the monoclonal In addition, in the absence of the monoclonal of the subunits were detected in the in Sf9 cell extracts containing all five rat eIF-2B subunits, the subunits to form a complex that can be detected by of Sf9 cells expressing and or all five rat eIF-2B subunits were on a gel filtration to the molecular mass and activity of the recombinant in GEF activity with the complex and an molecular mass to that observed for eIF-2B from rat In contrast, when extracts of cells expressing eIF-2Bε alone were GEF activity a molecular mass than observed for the protein purified from rat The that the GEF activity observed in cells expressing eIF-2Bε alone is not the of rat eIF-2Bε binding to the Sf9 cell eIF-2B α-, β-, γ-, to form an complex. the results that the eIF-2B ε-subunit alone GEF activity. In the GEF activity of eIF-2B is competitively inhibited by phosphorylation of the α-subunit of eIF-2 by the protein HCR, and Genetic and studies with mammalian eIF-2 in yeast indicate that the of eIF-2α phosphorylation are mediated in part by the α-subunit of eIF-2B T.E. A.M. Hinnebusch A.G. Proc. Natl. Acad. Sci. U. S. A. 1993; PubMed Scopus Google Scholar, T.E. A.M. Hinnebusch A.G. Cell. 1992; Full Text PDF PubMed Scopus Google Scholar). However, the studies conducted in yeast an of eIF-2B activity since the used is the of the in response to eIF-2α phosphorylation. The expression of recombinant eIF-2B subunits in Sf9 cells an for the to the of eIF-2α phosphorylation on eIF-2B activity. therefore examined the of eIF-2α phosphorylation on the activity of four-subunit and and five-subunit rat eIF-2B protein produced in Sf9 insect GEF were conducted on the cell extracts of the with eIF-2 or eIF-2 phosphorylated by in vitro. in extracts of cells expressing eIF-2B subunits and or the five-subunit complex of GDP exchange, and in both the of exchange was than in extracts of cells with The of GDP exchange both in extracts of cells expressing all five subunits and in extracts of cells with was greatly when extracts were with phosphorylated eIF-2 with with eIF-2 In contrast, the activity of the eIF-2B complex lacking the α-subunit was not inhibited by phosphorylated The of eIF-2α in the phosphorylated form the of the was the same for not that the of inhibition observed with cell extracts expressing the four-subunit eIF-2B complex and was not the of of eIF-2α the The of inhibition of the four-subunit eIF-2B complex by phosphorylated eIF-2 for the a regulatory role for the eIF-2B α-subunit using a biochemical Sf9 cells are for the of recombinant proteins the proteins express to undergo and M.J. 1992; Google Scholar). we were in the of Sf9 cells to study eIF-2B as the insect cell system has been used to recombinant for the Ras superfamily of proteins M. 1995; PubMed Scopus Google Scholar, M.J. 1995; PubMed Scopus Google Scholar). of Sf9 cells is that can be with multiple and this the coexpression of multiple In the study described here, we on expressing all five subunits of rat eIF-2B in insect cells baculovirus The of eIF-2B is complex than GEFs, which of single subunit proteins. The complex of eIF-2B that it may and that expression of the eIF-2B ε-subunit alone in GDP exchange with extracts of cells with In addition, GEF activity was to with the ε-subunit gel filtration In contrast, we have not been to GEF activity of eIF-2Bε using an not is that the conditions used the in of the we that the that eIF-2Bε alone GEF activity be with the activity of the individual subunit is insect cells with a of recombinant we were to express all five subunits of rat eIF-2B in Sf9 insect cells and to that the complex GEF activity. The of the eIF-2B ε-subunit was required for GEF activity, a role for this is with the S. B.L. Proud C.G. Eur. J. Biochem. 1994; PubMed Scopus Google Scholar) that the guanine nucleotide exchange activity of purified eIF-2B could be by monoclonal antibodies the The of an eIF-2B complex in insect cells was confirmed by coimmunoprecipitation and gel filtration In this study, we used the insect cell expression system to the role of eIF-2Bα in the regulation of eIF-2B activity. in yeast that a four-subunit eIF-2B complex lacking eIF-2Bα is since deletion of has on cell growth under normal conditions (27Hinnebusch A.G. Trends Biochem. Sci. 1994; 19: 409-414Abstract Full Text PDF PubMed Scopus (157) Google Scholar). In contrast, Craddock and Proud (29Craddock B.L. Proud C.G. Biochim. Biophys. Acta. 1996; 220: 843-847Google Scholar) reported that purified rabbit eIF-2B lacking eIF-2Bα was not and suggested that mammalian eIF-2B may therefore from yeast eIF-2B in the requirement for the α-subunit. of rat eIF-2B subunits in Sf9 we that the α-subunit of eIF-2B is not required for GEF activity. Furthermore, using gel filtration we were to that the GEF activity in Sf9 cell containing the four-subunit eIF-2B complex was to the of an four-subunit complex. is not the α-subunit was from certain purified rabbit eIF-2B reported by Craddock and Proud (29Craddock B.L. Proud C.G. Biochim. Biophys. Acta. 1996; 220: 843-847Google Scholar). One is that the absence of the α-subunit in the rabbit eIF-2B indicate in the other eIF-2B subunits that were not but that be the activity of the purified four-subunit eIF-2B complex. The α-subunit of eIF-2 is phosphorylated by protein HCR, and and this phosphorylation is an mechanism used by cells to control translation initiation in response to a variety of V.M. Eur. J. Biochem. 1996; 236: 747-771Crossref PubMed Scopus (638) Google Scholar). In addition, recent that of the eIF-2α may have a role as a in normal cells and function in the of cell growth M. S. R. Mol. Cell. Biol. 1995; 15: PubMed Scopus Google Scholar). In deletion of the eIF-2B α-subunit (GCN3) the from a or the growth from high expression of mammalian eIF-2α and T.E. A.M. Hinnebusch A.G. Proc. Natl. Acad. Sci. U. S. A. 1993; PubMed Scopus Google Scholar, M. Hinnebusch A.G. Mol. Cell. Biol. 1992; PubMed Google Scholar, K. A.G. J. 1992; PubMed Scopus Google Scholar). The genetic and expression studies in yeast that the GEF activity of eIF-2B lacking the α-subunit is to phosphorylation of Phosphorylated eIF-2 has an increased affinity for eIF-2B and as a since the complex does not undergo exchange. The inhibition of eIF-2B activity translation initiation by the of ternary In this we that the of eIF-2 phosphorylation is indeed mediated by the α-subunit of eIF-2B the GEF activity of a four-subunit eIF-2B complex lacking eIF-2Bα was to eIF-2α phosphorylation. the baculovirus-infected insect cell expression system may for the mechanism by which eIF-2B the GEF on mechanisms have been for the guanine nucleotide exchange N. Proud Biochimie ( Paris ). 1994; PubMed Scopus Google Scholar). One model is the which the of GDP and the binding of GTP to eIF-2 by eIF-2B. mechanism is of the GDP exchange and complex all of the exchange eIF-2B, and The mechanism that eIF-2B a binding for J.N. Wahba A.J. J. Biol. Chem. 1989; 264: Full Text PDF PubMed Google Scholar) reported the binding of a of GTP to the of eIF-2B and therefore suggested that eIF-2B is a The GTP binding are to with the amino acid of yeast or mammalian eIF-2B subunits since of the subunits a sequence for GTP studies using of normal and of individual eIF-2B subunits be required to in the guanine nucleotide exchange are to for