Translational Regulation of Yeast GCN4

When subjected to starvation, stress, or viral infections, mammalian cells down-regulate general protein synthesis by phosphorylating the α subunit of eukaryotic translation initiation factor 2 (eIF2) 1The abbreviations used are: eIF2, eukaryotic translation initiation factor 2; uORF, upstream open reading frame; HRI, heme-regulated inhibitor protein kinase; PKR, double-stranded RNA-dependent protein kinase; HisRS, histidyl-tRNA synthetase; EF, elongation factor.1The abbreviations used are: eIF2, eukaryotic translation initiation factor 2; uORF, upstream open reading frame; HRI, heme-regulated inhibitor protein kinase; PKR, double-stranded RNA-dependent protein kinase; HisRS, histidyl-tRNA synthetase; EF, elongation factor. (1Hershey J.W.B. Annu. Rev. Biochem. 1991; 60: 717-755Crossref PubMed Scopus (840) Google Scholar). eIF2 functions in translation initiation by delivering charged initiator tRNAMet (Met-tRNAiMet) in a ternary complex with GTP to the 40 S ribosomal subunit, forming a 43 S preinitiation complex. In the translation of most mRNAs, the 43 S complex binds near the capped 5′ end, migrates downstream, and upon reaching the first AUG codon, joins with the 60 S subunit to form an 80 S initiation complex (the scanning mechanism) (2Merrick W.C. Hershey J.W.B. Hershey J.W.B. Matthews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 31-69Google Scholar). Following AUG recognition, the GTP bound to eIF2 is hydrolyzed and eIF2 is released as an inactive eIF2·GDP binary complex. Exchange of the GDP bound to eIF2 with GTP is catalyzed by eIF2B. Phosphorylation of the α subunit of eIF2 (eIF2α) on Ser-51 prevents the recycling of eIF2 by eIF2B; in addition, the phosphorylated complex eIF2(αP)·GDP has a higher affinity than non-phosphorylated eIF2·GDP for eIF2B, such that GDP-GTP exchange on non-phosphorylated eIF2 is also impaired and ternary complex formation is blocked (2Merrick W.C. Hershey J.W.B. Hershey J.W.B. Matthews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 31-69Google Scholar). In Saccharomyces cerevisiae, eIF2α is phosphorylated when cells are deprived of an amino acid or purine, and interestingly, this leads to increased translation of a specific mRNA encoding GCN4, a transcriptional activator of at least 40 genes encoding amino acid biosynthetic enzymes (3Hinnebusch A.G. Broach J.R. Jones E.W. Pringle J.R. The Molecular and Cellular Biology of the Yeast Saccharomyces: Gene Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1992: 319-414Google Scholar).The unique induction of GCN4 translation in response to eIF2 phosphorylation is mediated by four short open reading frames (uORFs) in the leader of GCN4 mRNA located 150–360 nucleotides upstream of the authentic initiation codon. Eliminating the start codons of all four uORFs results in high level GCN4expression under both starvation and non-starvation conditions without altering the mRNA (4Mueller P.P. Hinnebusch A.G. Cell. 1986; 45: 201-207Abstract Full Text PDF PubMed Scopus (355) Google Scholar). Thus, the uORFs inhibit GCN4translation in non-starved cells by restricting the progression of scanning ribosomes through the leader to the GCN4 start codon. The first and fourth uORFs (from the 5′ end), which are sufficient for nearly wild-type regulation, have different effects onGCN4 translation. When present alone, uORF4 reducesGCN4 translation to only 1% of the level seen in the absence of all four uORFs, under both starvation and non-starvation conditions (4Mueller P.P. Hinnebusch A.G. Cell. 1986; 45: 201-207Abstract Full Text PDF PubMed Scopus (355) Google Scholar). In this situation, it appears that all ribosomes translate uORF4 and then dissociate from the mRNA. In contrast, uORF1 alone reduces GCN4 translation by only 50%, presumably because half of the ribosomes that translate uORF1 resume scanning and reinitiate at GCN4. When uORF1 is present upstream of uORF4 and amino acids are abundant, all the ribosomes that resume scanning following uORF1 translation will reinitiate at uORF4 and dissociate from the mRNA, preventing GCN4translation. Under starvation conditions, however, half of the ribosomes that resume scanning after uORF1 translation will bypass the uORF4 start site (and also those at uORFs 2–3 when present) and reinitiate at GCN4 instead (Fig. 1) (5Abastado J.P. Miller P.F. Jackson B.M. Hinnebusch A.G. Mol. Cell. Biol. 1991; 11: 486-496Crossref PubMed Scopus (165) Google Scholar). Thus, prior translation of uORF1 allows ribosomes to overcome the strong translational barrier at uORF4 by a reinitiation mechanism.We proposed that under conditions where GCN4 is repressed, the ribosomes that resume scanning following uORF1 translation are forced to reinitiate at uORF4 because they rebind the eIF2·GTP·Met-tRNAiMet ternary complex before reaching the uORF4 start codon. Under starvation conditions, phosphorylation of eIF2α reduces the concentration of ternary complexes, such that many ribosomes scan the distance between uORF1 and uORF4 without rebinding the ternary complex (6Dever T.E. Feng L. Wek R.C. Cigan A.M. Donahue T.D. Hinnebusch A.G. Cell. 1992; 68: 585-596Abstract Full Text PDF PubMed Scopus (560) Google Scholar). Lacking initiator tRNAMet, they cannot recognize the AUG codons at uORFs 2, 3, and 4 (7Cigan A.M. Feng L. Donahue T.F. Science. 1988; 242: 93-97Crossref PubMed Scopus (125) Google Scholar) and continue scanning downstream. Most of these ribosomes will bind the ternary complex while scanning between uORF4 and GCN4, enabling them to reinitiate at the GCN4start codon (6Dever T.E. Feng L. Wek R.C. Cigan A.M. Donahue T.D. Hinnebusch A.G. Cell. 1992; 68: 585-596Abstract Full Text PDF PubMed Scopus (560) Google Scholar). Thus, reducing the level of ternary complexes allows ribosomes to bypass the inhibitory uORFs 2–4 and reinitiate atGCN4 instead (6Dever T.E. Feng L. Wek R.C. Cigan A.M. Donahue T.D. Hinnebusch A.G. Cell. 1992; 68: 585-596Abstract Full Text PDF PubMed Scopus (560) Google Scholar) (Fig. 1).Genetic Evidence for the Scanning-Reinitiation Mechanism of Translating GCN4 mRNASupporting the idea that any ribosomes which translateGCN4 must have scanned past uORF4 without initiating translation (Fig. 1), it was shown that translation of auORF4-lacZ fusion decreases under conditions whereGCN4 translation is stimulated (5Abastado J.P. Miller P.F. Jackson B.M. Hinnebusch A.G. Mol. Cell. Biol. 1991; 11: 486-496Crossref PubMed Scopus (165) Google Scholar). Another strong indication that uORF4 must be skipped en route to GCN4 is that mutations in the uORF4 stop codon that make uORF4 overlap theGCN4 ORF have almost no effect on GCN4expression, indicating that the location of the uORF4 stop codon is of little consequence (5Abastado J.P. Miller P.F. Jackson B.M. Hinnebusch A.G. Mol. Cell. Biol. 1991; 11: 486-496Crossref PubMed Scopus (165) Google Scholar). In contrast, elongating uORF1 abolishesGCN4 translation (8Grant C.M. Miller P.F. Hinnebusch A.G. Mol. Cell. Biol. 1994; 14: 2616-2628Crossref PubMed Scopus (83) Google Scholar), supporting the idea that essentially all ribosomes reach the GCN4 start site by reinitiation following translation of uORF1. In addition, there is a critical requirement for nucleotides flanking the uORF1 stop codon for efficient reinitiation at GCN4. Replacing the last codon and 10 nucleotides 3′ to the uORF1 stop codon with the corresponding nucleotides from uORF4 converts uORF1 into a strong translational barrier and destroys its ability to stimulate GCN4translation when situated upstream from uORF4 (9Miller P.F. Hinnebusch A.G. Genes Dev. 1989; 3: 1217-1225Crossref PubMed Scopus (72) Google Scholar). Mutational analysis revealed that diverse AU-rich sequences at the third codon and immediately 3′ of uORF1 would promote high level reinitiation atGCN4 (10Grant C.M. Hinnebusch A.G. Mol. Cell. Biol. 1994; 14: 606-618Crossref PubMed Google Scholar). This led to the idea that base pairing between the mRNA surrounding the uORF4 stop codon and the rRNA could lengthen the time spent by the ribosome in the termination region and increase the probability of ribosome release from the mRNA.It was conceivable that following termination at uORF1, ribosomes would be shunted directly to the GCN4 start site rather than scanning the entire uORF1-GCN4 interval. This possibility is inconsistent with the fact that insertions of stem-loop structures in the vicinity of uORF4 abolish GCN4 translation. A shunting model also cannot explain the critical finding that GCN4translation gradually decreased as the uORF1–uORF4 spacing was progressively increased (5Abastado J.P. Miller P.F. Jackson B.M. Hinnebusch A.G. Mol. Cell. Biol. 1991; 11: 486-496Crossref PubMed Scopus (165) Google Scholar). According to the model in Fig. 1, under derepressing conditions, 50% of the 40 S subunits scanning from uORF1 have not re-bound the ternary complex upon reaching uORF4 and continue scanning to GCN4. When the uORF1–uORF4 interval is expanded, however, most of the 40 S subunits have bound the ternary complex and are now competent to reinitiate when they reach uORF4. Consequently, they cannot bypass uORF4 and reinitiate downstream atGCN4. A final piece of evidence inconsistent with a shunting mechanism is that the authentic uORFs were replaced with heterologous small uORFs without destroying GCN4 translational control (11Mueller P.P. Jackson B.M. Miller P.F. Hinnebusch A.G. Mol. Cell. Biol. 1988; 8: 5439-5447Crossref PubMed Scopus (23) Google Scholar, 12Williams N.P. Mueller P.P. Hinnebusch A.G. Mol. Cell. Biol. 1988; 8: 3827-3836Crossref PubMed Scopus (25) Google Scholar, 13Tzamarias D. Thireos G. EMBO J. 1988; 7: 3547-3551Crossref PubMed Scopus (18) Google Scholar). In our model, the only critical requirements for the first uORF are to be recognized efficiently by ribosomes and then to allow ribosomes to resume scanning. Presumably, both requirements can be met by heterologous uORFs, albeit with lower efficiency than occurs with authentic uORF1. Interestingly, efficient reinitiation at uORF1 depends not only on the sequence context of its stop codon but on sequences upstream of the uORF (14Grant C.M. Miller P.F. Hinnebusch A.G. Nucleic Acids Res. 1995; 23: 3980-3988Crossref PubMed Scopus (36) Google Scholar).A key feature of the model in Fig. 1 is that the reduction in ternary complex levels in starved cells is large enough to allow a fraction of reinitiating ribosomes on GCN4 mRNA to ignore uORF4 and continue scanning to GCN4 but not extensive enough to allow ribosomes to skip uORF4 if they have not translated uORF1. This distinction may exist because in conventional initiation events ribosomes bind the ternary complex before interacting with the 5′ end of the mRNA (2Merrick W.C. Hershey J.W.B. Hershey J.W.B. Matthews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 31-69Google Scholar); therefore, reducing ternary complex levels may decrease the frequency that ribosomes load at the 5′ end, but once bound to mRNA, their ability to recognize AUG codons while scanning downstream should be independent of the concentration of ternary complexes. It is also possible that reinitiating 40 S subunits are less efficient than free 40 S subunits in binding the ternary complex because they lack an initiation factor like eIF3 or eIF1A that promotes this reaction (2Merrick W.C. Hershey J.W.B. Hershey J.W.B. Matthews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 31-69Google Scholar). In any case, the reinitiation mechanism forGCN4 translation is an extremely sensitive indicator of the activity of eIF2 and associated factors that function in the formation of ternary complex or promote its binding to ribosomes.The Protein Kinase GCN2 Stimulates GCN4 Translation by Inhibiting Recycling of eIF2 by eIF2BGCN2 is a 180-kDa protein kinase that phosphorylates eIF2 and thereby induces GCN4 translation in cells starved for histidine (6Dever T.E. Feng L. Wek R.C. Cigan A.M. Donahue T.D. Hinnebusch A.G. Cell. 1992; 68: 585-596Abstract Full Text PDF PubMed Scopus (560) Google Scholar) or several other amino acids (15Wek S.A. Zhu S. Wek R.C. Mol. Cell. Biol. 1995; 15: 4497-4506Crossref PubMed Google Scholar). Substitution of Ser-51 in eIF2α with Ala (the SUI2-S51A allele) completely eliminates the increased phosphorylation in amino acid-starved cells and impairs derepression of GCN4 to the same extent as a deletion of GCN2. Immunopurified GCN2 specifically phosphorylated the α subunit of eIF2 purified from rabbit or yeast but not yeast eIF2 containing the Ala-51 substitution (6Dever T.E. Feng L. Wek R.C. Cigan A.M. Donahue T.D. Hinnebusch A.G. Cell. 1992; 68: 585-596Abstract Full Text PDF PubMed Scopus (560) Google Scholar). These results established that GCN2 stimulates GCN4 translation by phosphorylating eIF2α on Ser-51. Low level expression of the mammalian eIF2α kinases HRI and PKR in gcn2 mutants induces GCN4 translation in a manner completely dependent on Ser-51 in eIF2α (16Dever T.E. Chen J.J. Barber G.N. Cigan A.M. Feng L. Donahue T.F. London I.M. Katze M.G. Hinnebusch A.G. Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 461-462Crossref Scopus (185) Google Scholar). When expressed at high levels, PKR and HRI produce a much higher level of eIF2 phosphorylation than occurs when GCN2 is activated in amino acid-starved cells, and this severely inhibits cell growth (16Dever T.E. Chen J.J. Barber G.N. Cigan A.M. Feng L. Donahue T.F. London I.M. Katze M.G. Hinnebusch A.G. Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 461-462Crossref Scopus (185) Google Scholar, 17Chong K.L. Feng L. Schappert K. Meurs E. Donahue T.F. Friesen J.D. Hovanessian A.G. Williams B.R.G. EMBO J. 1992; 11: 1553-1562Crossref PubMed Scopus (289) Google Scholar). Mutationally activated forms of GCN2 (GCN2c kinases) also cause hyperphosphorylation of eIF2 and a general reduction in translation initiation (6Dever T.E. Feng L. Wek R.C. Cigan A.M. Donahue T.D. Hinnebusch A.G. Cell. 1992; 68: 585-596Abstract Full Text PDF PubMed Scopus (560) Google Scholar, 18Ramirez M. Wek R.C. Vazquez de Aldana C.R. Jackson B.M. Freeman B. Hinnebusch A.G. Mol. Cell. Biol. 1992; 12: 5801-5815Crossref PubMed Google Scholar, 19Wek R.C. Ramirez M. Jackson B.M. Hinnebusch A.G. Mol. Cell. Biol. 1990; 10: 2820-2831Crossref PubMed Scopus (88) Google Scholar, 20Diallinas G. Thireos G. Gene ( Amst. ). 1994; 143: 21-27Crossref PubMed Scopus (16) Google Scholar). These last findings confirm that GCN4 translation is induced at a lower level of eIF2 phosphorylation than is required for general inhibition of protein synthesis.There is strong evidence that phosphorylation of eIF2α by GCN2 down-regulates the formation of eIF2·GTP·Met-tRNAiMet ternary complexes. Mutations in the genes encoding the α, β, and γ subunits of eIF2 (encoded by SUI2 (21Cigan A.M. Pabich E.K. Feng L. Donahue T.F. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2784-2788Crossref PubMed Scopus (144) Google Scholar), SUI3(22Donahue T.F. Cigan A.M. Pabich E.K. Castilho-Valavicius B. Cell. 1988; 54: 621-632Abstract Full Text PDF PubMed Scopus (185) Google Scholar), and GCD11 (23Hannig E.M. Cigan A.M. Freeman B.A. Kinzy T.G. Mol. Cell. Biol. 1992; 13: 506-520Crossref Google Scholar), respectively) mimic the effect of eIF2 phosphorylation in derepressing GCN4 translation (Gcd− phenotype) independently of GCN2 and amino acid starvation (24Harashima S. Hinnebusch A.G. Mol. Cell. Biol. 1986; 6: 3990-3998Crossref PubMed Scopus (59) Google Scholar, 25Mueller P.P. Harashima S. Hinnebusch A.G. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 2863-2867Crossref PubMed Scopus (55) Google Scholar, 26Williams N.P. Hinnebusch A.G. Donahue T.F. Proc. Natl. Acad. Sci. U.S.A. 1989; 86: 7515-7519Crossref PubMed Scopus (60) Google Scholar, 27Castilho-Valavicius B. Yoon H. Donahue T.F. Genetics. 1990; 124: 483-495Crossref PubMed Google Scholar). These mutations produce a slow growth phenotype (Slg−) on nutrient-rich medium and thus appear to decrease eIF2 function in translation initiation. Deleting two of the fourIMT genes encoding tRNAiMetelicits the same Gcd− and Slg− phenotypes (28Dever T.E. Yang W. Astrom S. Bystrom A.S. Hinnebusch A.G. Mol. Cell. Biol. 1995; 15: 6351-6363Crossref PubMed Scopus (109) Google Scholar). Overexpression of the eIF2 complex in wild-type cells interferes with derepression of GCN4 translation and suppresses the growth inhibitory effects of GCN2 c alleles. Overexpressing eIF2 and tRNAiMettogether has a synergistic effect in suppressing the toxicity of eIF2 hyperphosphorylation (28Dever T.E. Yang W. Astrom S. Bystrom A.S. Hinnebusch A.G. Mol. Cell. Biol. 1995; 15: 6351-6363Crossref PubMed Scopus (109) Google Scholar). The β, γ, δ, and ε subunits of yeast eIF2B (encoded by GCD7 (29Bushman J.L. Asuru A.I. Matts R.L. Hinnebusch A.G. Mol. Cell. Biol. 1993; 13: 1920-1932Crossref PubMed Scopus (74) Google Scholar), GCD1 (30Hill D.E. Struhl K. Nucleic Acids Res. 1988; 16: 9253-9265Crossref PubMed Scopus (24) Google Scholar),GCD2 (31Paddon C.J. Hannig E.M. Hinnebusch A.G. Genetics. 1989; 122: 551-559Crossref PubMed Google Scholar), and GCD6 (29Bushman J.L. Asuru A.I. Matts R.L. Hinnebusch A.G. Mol. Cell. Biol. 1993; 13: 1920-1932Crossref PubMed Scopus (74) Google Scholar), respectively) were first identified by point mutations with the same Gcd− and Slg− phenotypes observed for mutations in eIF2 subunits (24Harashima S. Hinnebusch A.G. Mol. Cell. Biol. 1986; 6: 3990-3998Crossref PubMed Scopus (59) Google Scholar, 32Wolfner M. Yep D. Messenguy F. Fink G.R. J. Mol. Biol. 1975; 96: 273-290Crossref PubMed Scopus (126) Google Scholar, 33Miozzari G. Niederberger P. Huetter R. J. Bacteriol. 1978; 134: 48-59Crossref PubMed Google Scholar, 34Myers P.L. Skvirsky R.C. Greenberg M.L. Greer H. Mol. Cell. Biol. 1986; 6: 3150-3155Crossref PubMed Scopus (5) Google Scholar, 35Niederberger P. Aebi M. Huetter R. Curr. Genet. 1986; 10: 657-664Crossref PubMed Scopus (22) Google Scholar, 36Paddon C.J. Hinnebusch A.G. Genetics. 1989; 122: 543-550Crossref PubMed Google Scholar, 37Cigan A.M. Foiani M. Hannig E.M. Hinnebusch A.G. Mol. Cell. Biol. 1991; 11: 3217-3228Crossref PubMed Scopus (93) Google Scholar, 38Cigan 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). The fact that non-lethal mutations in the essential subunits of eIF2B mimic eIF2 phosphorylation in derepressingGCN4 supports the notion that eIF2 phosphorylation in yeast leads to a reduction in eIF2B function. Even stronger evidence for this conclusion comes from the fact that overexpressing the eIF2B complex in yeast overcomes the growth inhibitory effects and the derepression ofGCN4 associated with eIF2 phosphorylation (28Dever T.E. Yang W. Astrom S. Bystrom A.S. Hinnebusch A.G. Mol. Cell. Biol. 1995; 15: 6351-6363Crossref PubMed Scopus (109) Google Scholar).Mutations were obtained in the GCD2, GCD7, andGCN3 subunits of eIF2B that reverse the derepression ofGCN4 and general inhibition of translation in the presence of high level eIF2 phosphorylation. These mutations appear to make eIF2B insensitive to eIF2(αP) without decreasing the ability to catalyze nucleotide exchange on non-phosphorylated eIF2 (16Dever T.E. Chen J.J. Barber G.N. Cigan A.M. Feng L. Donahue T.F. London I.M. Katze M.G. Hinnebusch A.G. Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 461-462Crossref Scopus (185) Google Scholar, 39Hannig E.H. Williams N.P. Wek R.C. Hinnebusch A.G. Genetics. 1990; 126: 549-562Crossref PubMed Google Scholar, 40Vazquez de Aldana C.R. Hinnebusch A.G. Mol. Cell. Biol. 1994; 14: 3208-3222Crossref PubMed Scopus (51) Google Scholar, 41Pavitt G.D. Yang W. Hinnebusch A.G. Mol. Cell. Biol. 1996; 17: 1298-1313Crossref Scopus (107) Google Scholar). Interestingly, this is the only effect observed when GCN3 is deleted (16Dever T.E. Chen J.J. Barber G.N. Cigan A.M. Feng L. Donahue T.F. London I.M. Katze M.G. Hinnebusch A.G. Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 461-462Crossref Scopus (185) Google Scholar, 39Hannig E.H. Williams N.P. Wek R.C. Hinnebusch A.G. Genetics. 1990; 126: 549-562Crossref PubMed Google Scholar), indicating that GCN3 is required primarily for inhibition of eIF2B by eIF2(αP). The same conclusion was recently obtained for rat eIF2B by showing that a four-subunit complex lacking the α-subunit (homologous to GCN3), reconstituted in baculovirus-infected insect cells, was insensitive to inhibition by phosphorylated eIF2 in vitro (42Fabian 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). The GCD2 andGCD7 subunits are essential (29Bushman J.L. Asuru A.I. Matts R.L. Hinnebusch A.G. Mol. Cell. Biol. 1993; 13: 1920-1932Crossref PubMed Scopus (74) Google Scholar, 36Paddon C.J. Hinnebusch A.G. Genetics. 1989; 122: 543-550Crossref PubMed Google Scholar) and thus make important contributions to catalysis as well as regulation. GCN3, GCD7, and GCD2 show strong sequence similarities to one another throughout most of GCN3 and GCD7 and the C-terminal half of GCD2 (29Bushman J.L. Asuru A.I. Matts R.L. Hinnebusch A.G. Mol. Cell. Biol. 1993; 13: 1920-1932Crossref PubMed Scopus (74) Google Scholar, 31Paddon C.J. Hannig E.M. Hinnebusch A.G. Genetics. 1989; 122: 551-559Crossref PubMed Google Scholar). Overexpression of GCD2, GCD7, and GCN3 reduces the inhibitory effect of eIF2(αP) on general translation in vivo, and the excess amounts of these proteins form a stable subcomplex that can be co-immunoprecipitated from cell extracts. Formation of this subcomplex does not compensate for a loss of eIF2B function by mutation; thus the GCN3·GCD7·GCD2 subcomplex does not possess guanine nucleotide exchange activity but instead appears to prevent eIF2(αP) from inhibiting native eIF2B (43Yang W. Hinnebusch A.G. Mol. Cell. Biol. 1996; 16: 6603-6616Crossref PubMed Scopus (76) Google Scholar). Together these results provide strong evidence that GCN3, GCD7, and the C-terminal half of GCD2 comprise a regulatory domain in eIF2B that mediates the inhibitory effects of eIF2(αP).The majority of the regulatory mutations isolated in GCD2, GCD7, and GCN3, which decrease or abolish inhibition of eIF2B by into two of amino acids in of strong all and several mutations in two or all These results that in GCD2, GCD7, and GCN3 in the of eIF2B by eIF2(αP). possible function would be to with in eIF2α surrounding Ser-51 to increase the affinity of eIF2B for phosphorylated they could a in the complex that prevents nucleotide exchange on eIF2(αP) G.D. Yang W. Hinnebusch A.G. Mol. Cell. Biol. 1996; 17: 1298-1313Crossref Scopus (107) Google Scholar). In GCD2 regulatory nearly all of the eIF2 was phosphorylated with no effects on cell These mutations allow eIF2B to phosphorylated eIF2 as a rather than reducing the affinity for eIF2(αP) G.D. Yang W. Hinnebusch A.G. Mol. Cell. Biol. 1996; 17: 1298-1313Crossref Scopus (107) Google Scholar). the of GCD2, GCD7, and GCN3 in the of eIF2B, proposed that GCD1 and GCD6 with one another and form the for nucleotide exchange A.G. Biochem. Sci. 1994; Full Text PDF PubMed Scopus Google Scholar). This is by findings that the rat of GCD6 expressed in baculovirus-infected insect cells level exchange activity that was stimulated by the other four subunits in the same cells (42Fabian 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). The γ subunit of eIF2 (encoded is to and sequences in all proteins (23Hannig E.M. Cigan A.M. Freeman B.A. Kinzy T.G. Mol. Cell. Biol. 1992; 13: 506-520Crossref Google Scholar). mutations that eIF2 function in the domain Hannig E.M. EMBO J. 1995; 14: PubMed Scopus Google Scholar). Thus, it that GCD11 the site on eIF2 and would be to directly with the in in the cause derepression ofGCN4 translation in the absence of eIF2 phosphorylation by GCN2 and growth on medium (24Harashima S. Hinnebusch A.G. Mol. Cell. Biol. 1986; 6: 3990-3998Crossref PubMed Scopus (59) Google Scholar, 25Mueller P.P. Harashima S. Hinnebusch A.G. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 2863-2867Crossref PubMed Scopus (55) Google Scholar). These are the same phenotypes associated with mutations subunits of eIF2 or eIF2B. A reduction in observed in mutants at the is of a general in translation and a small fraction of protein was associated with and ribosomal with eIF3 activity and was shown to be to the subunit of eIF3 R. Hinnebusch A.G. Hershey J.W.B. M. Genes Dev. 1995; PubMed Scopus (59) Google Scholar) Hershey J.W.B. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). These findings that of translational reinitiation on GCN4 mRNA the subunit of on the of mammalian eIF3 (2Merrick W.C. Hershey J.W.B. Hershey J.W.B. Matthews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 31-69Google Scholar), one possibility is that mutations decrease the ability of eIF3 to stimulate formation of eIF2·GTP·Met-tRNAiMet ternary complexes or promote their binding to ribosomal of GCN2 Kinase by GCN2 was replaced by the mammalian eIF2α kinases PKR or HRI, GCN4 translation was stimulated independently of amino acid levels (16Dever T.E. Chen J.J. Barber G.N. Cigan A.M. Feng L. Donahue T.F. London I.M. Katze M.G. Hinnebusch A.G. Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 461-462Crossref Scopus (185) Google Scholar), indicating that increased phosphorylation of eIF2α in starved cells increased GCN2 not inhibition of an eIF2α is little or no increase in levels in response to amino acid starvation R.C. Ramirez M. Jackson B.M. Hinnebusch A.G. Mol. Cell. Biol. 1990; 10: 2820-2831Crossref PubMed Scopus (88) Google Scholar), showing that GCN2 not its is stimulated in starved appears to be the for GCN2 because mutations in to increased eIF2α phosphorylation by GCN2 (15Wek S.A. Zhu S. Wek R.C. Mol. Cell. Biol. 1995; 15: 4497-4506Crossref PubMed Google Scholar) with derepression ofGCN4 S. Bushman J.L. Hinnebusch A.G. H. Mueller P.P. Cell. 1992; Full Text PDF PubMed Scopus Google Scholar) and genes under its control (15Wek S.A. Zhu S. Wek R.C. Mol. Cell. Biol. 1995; 15: 4497-4506Crossref PubMed Google Scholar, F. J. J. Biochem. PubMed Scopus Google Scholar, de Aldana C.R. Wek R.C. P. A.G. Hinnebusch A.G. Mol. Cell. Biol. 1994; 14: PubMed Google Scholar) without for the amino GCN2 C-terminal to the kinase domain to the sequence of histidyl-tRNA R.C. Jackson B.M. Hinnebusch A.G. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: PubMed Scopus Google Scholar), the sequence that with the of in M. S. M. A. A. A. B. D. 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