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When starved for a single amino acid, the budding yeast Saccharomyces cerevisiae activates the eukaryotic initiation factor 2α (eIF2α) kinase GCN2 in a GCN1-dependent manner. Phosphorylated eIF2α inhibits general translation but selectively derepresses the synthesis of the transcription factor GCN4, which leads to coordinated induction of genes involved in biosynthesis of various amino acids, a phenomenon called general control response. We recently demonstrated that this response requires binding of GCN1 to the GI domain occurring at the N terminus of GCN2 (Kubota, H., Sakaki, Y., and Ito, T. (2000)J. Biol. Chem. 275, 20243–20246). Here we provide the first evidence for the involvement of GCN1-GCN2 interaction in activation of GCN2 per se. We identified a C-terminal segment of GCN1 sufficient to bind the GI domain and used a novel dual bait two-hybrid method to identify mutations rendering GCN1 incapable of interacting with GCN2. The yeast bearing such an allele,gcn1-F2291L, fails to display derepression of GCN4translation and hence general control response, as does a GI domain mutant, gcn2-Y74A, defective in association with GCN1. Furthermore, we demonstrated that phosphorylation of eIF2α is impaired in both mutants. Since GCN2 is the sole eIF2α kinase in yeast, these findings indicate a critical role of GCN1-GCN2 interaction in activation of the kinase in vivo. When starved for a single amino acid, the budding yeast Saccharomyces cerevisiae activates the eukaryotic initiation factor 2α (eIF2α) kinase GCN2 in a GCN1-dependent manner. Phosphorylated eIF2α inhibits general translation but selectively derepresses the synthesis of the transcription factor GCN4, which leads to coordinated induction of genes involved in biosynthesis of various amino acids, a phenomenon called general control response. We recently demonstrated that this response requires binding of GCN1 to the GI domain occurring at the N terminus of GCN2 (Kubota, H., Sakaki, Y., and Ito, T. (2000)J. Biol. Chem. 275, 20243–20246). Here we provide the first evidence for the involvement of GCN1-GCN2 interaction in activation of GCN2 per se. We identified a C-terminal segment of GCN1 sufficient to bind the GI domain and used a novel dual bait two-hybrid method to identify mutations rendering GCN1 incapable of interacting with GCN2. The yeast bearing such an allele,gcn1-F2291L, fails to display derepression of GCN4translation and hence general control response, as does a GI domain mutant, gcn2-Y74A, defective in association with GCN1. Furthermore, we demonstrated that phosphorylation of eIF2α is impaired in both mutants. Since GCN2 is the sole eIF2α kinase in yeast, these findings indicate a critical role of GCN1-GCN2 interaction in activation of the kinase in vivo. eukaryotic initiation factor 2 open reading frame amino acid(s) polymerase chain reaction activation domain PC motif-containing region synthetic complete synthetic dextrose 3-aminotriazole 5-fluoro-orotic acid degenerate protein kinase domain Protein synthesis in eukaryotic cells is suppressed by stress-induced phosphorylation of eukaryotic initiation factor 2α (eIF2α)1 on a serine residue at position 51 (1). The phosphorylation converts eIF2α from the substrate to an inhibitor of eIF2B, the guanine nucleotide exchange factor of eIF2; phosphorylated eIF2-GDP forms a stable complex with eIF2B to hamper recycling of eIF2-GDP to eIF2-GTP (2Merrick W.C. Microbiol. Rev. 1992; 56: 291-315Crossref PubMed Google Scholar). Scarcity of eIF2-GTP accordingly decreases the level of the ternary complex composed of eIF2, GTP, and the charged initiator tRNA, a prerequisite for translational initiation, and hence leads to general suppression of protein synthesis. Thus, eIF2α kinases play pivotal roles in this famous translational control. Mammalian cells have four eIF2α kinases, each of which is activated in response to a distinct stress. Heme-regulated inhibitor is activated by hemin deprivation (3Clemens M.J. Translational Control. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 139-172Google Scholar); double-stranded RNA-dependent kinase is activated by double-stranded RNA (3Clemens M.J. Translational Control. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 139-172Google Scholar); RNA-dependent kinase-like endoplasmic reticulum kinase is activated by unfolded proteins (4Shi Y. Vattem K.M. Sood R. An J. Liang J. Stramm L. Wek R.C. Mol. Cell. Biol. 1998; 18: 7499-7509Crossref PubMed Google Scholar, 5Harding H.P. Zhang Y. Ron D. Nature. 1999; 397: 271-274Crossref PubMed Scopus (2558) Google Scholar); and GCN2 is activated by serum or amino acid starvation (6Berlanga J.J. Santoyo J. De Haro C. Eur. J. Biochem. 1999; 265: 754-762Crossref PubMed Scopus (221) Google Scholar, 7Harding H.P. Novoa I. Zhang Y. Zeng H. Wek R. Schapira M. Ron D. Mol. Cell. 2000; 6: 1099-1108Abstract Full Text Full Text PDF PubMed Scopus (2431) Google Scholar). In contrast, the budding yeastSaccharomyces cerevisiae has the sole eIF2α kinase, GCN2, the founding member of this family. The yeast GCN2 is activated by starvation for amino acids, glucose deprivation, purine limitation, and impaired tRNA synthetase activity (8Yang R. Wek S.A. Wek R.C. Mol. Cell. Biol. 2000; 20: 2706-2717Crossref PubMed Scopus (144) Google Scholar, 9Hinnebusch A.G. Microbiol. Rev. 1988; 52: 248-273Crossref PubMed Google Scholar, 10Hinnebusch A.G. J. Biol. Chem. 1997; 272: 2161-21664Abstract Full Text Full Text PDF Scopus (434) Google Scholar). The gene for this kinase was originally identified in the studies of a response to amino acid starvation called general control of amino acid synthesis, and hence was termed GCN2 (generalcontrol nonderepressible 2). The molecular mechanism underlying general control response is currently considered as follows. When the budding yeast starves for a single particular amino acid, free tRNAs, which are not charged with amino acids, accumulate within the cells and bind to a bipartite domain composed of the histidyl tRNA synthetase-related domain and the C-terminal ribosome-binding domain of GCN2 (11Dong J. Qiu H. Garcia-Barrio M. Anderson J. Hinnebusch A.G. Mol. Cell. 2000; 6: 269-279Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar). This bipartite domain forms an inhibitory interaction with the kinase domain, which is disrupted upon binding of tRNAs (11Dong J. Qiu H. Garcia-Barrio M. Anderson J. Hinnebusch A.G. Mol. Cell. 2000; 6: 269-279Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar). In addition to uncharged tRNAs, which unmask the kinase domain, genetic evidence suggests that in vivo activation of GCN2 requires another gene, GCN1, encoding a protein bearing a region homologous to translation elongation factor 3 (12Marton M.J. Crouch D. Hinnebusch A.G. Mol. Cell. Biol. 1993; 13: 3541-3556Crossref PubMed Scopus (96) Google Scholar). GCN1 forms a stable complex with the ATP-binding cassette protein GCN20 and functions on an elongating ribosome (13Vazquez de Aldana C.R. Marton M.J. Hinnebusch A.G. EMBO J. 1995; 14: 3148-3199Crossref Scopus (125) Google Scholar, 14Marton M.J. Vazquez de Aldana C.R. Qiu H. Chalraburtty K. Hinnebusch A.G. Mol. Cell. Biol. 1997; 17: 4474-4489Crossref PubMed Google Scholar). GCN2 is activated by uncharged tRNAs in the presence of GCN1 and phosphorylates eIF2α to suppress protein synthesis via the mechanism described above. However, the mRNA encoding GCN4 is selectively translated by a unique mechanism, which depends on the four short open reading frames (ORFs) preceding the one for GCN4 (10Hinnebusch A.G. J. Biol. Chem. 1997; 272: 2161-21664Abstract Full Text Full Text PDF Scopus (434) Google Scholar). The transcription factor GCN4 induces the expression of genes involved in various amino acid synthetic pathways. In contrast to the action of uncharged tRNAs and the mechanism for derepressed translation of GCN4 mRNA, how GCN1 participates in the activation of GCN2 was poorly understood at the molecular level. Recently, we and others showed that a direct interaction between GCN1 and GCN2 is necessary for general control response, thereby providing the first insight into the underlying mechanism (15Kubota H. Sakaki Y. Ito T. J. Biol. Chem. 2000; 275: 20243-20246Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 16Garcia-Barrio M. Dong J. Ufano S. Hinnebusch A.G. EMBO J. 2000; 17: 1887-1899Crossref Google Scholar). In this study, we determine the minimal essential regions of GCN1 and GCN2 for the complex formation and demonstrate that phosphorylation of eIF2α, translational derepression of GCN4 mRNA, and general control response are impaired in the gcn1 andgcn2 mutants defective in this interaction. These results provide the first direct evidence for a crucial role of GCN1-GCN2 interaction in the activation of the eIF2α kinase. The strains used in this study are summarized in Table I.Table IGenotypes of yeast strains used in this studyStrainGenotypeSourcePJ69–4AMAT a trp1–901 leu2–3,112 ura3–52 his3Δ200 gal4Δ gal80Δ LYS2::GAL1-HIS3 GAL2-ADE2 met2::GAL7-lacZRef. 37James P. Halladay J. Craig E.A. Genetics. 1996; 144: 1425-1436Crossref PubMed Google ScholarPJ69–4AΔMAT a trp1–901 leu2–3,112 ura3–52 his3Δ200 gal4Δ gal80Δ LYS2::GAL1-HIS3 GAL2-ADE2 met2::GAL7-lacZ lig4ΔK. OtaMavχMATα trp1–901 leu2–3,112 his3Δ200 ade2Δ gal4Δ gal80Δ SPAL10::URA3 LYS2::kanMX-Lexop-HIS3 GAL1::lacZK. OtaMB758–5BMAT a ura3A. FujitaJBY1MAT a ura3 gcn2-Y74ARef. 15Kubota H. Sakaki Y. Ito T. J. Biol. Chem. 2000; 275: 20243-20246Abstract Full Text Full Text PDF PubMed Scopus (53) Google ScholarJBY2MAT a ura3 GCN2-T7::URA3Ref. 15Kubota H. Sakaki Y. Ito T. J. Biol. Chem. 2000; 275: 20243-20246Abstract Full Text Full Text PDF PubMed Scopus (53) Google ScholarJBY3MAT a ura3 gcn2-Y74A-T7::URA3Ref. 15Kubota H. Sakaki Y. Ito T. J. Biol. Chem. 2000; 275: 20243-20246Abstract Full Text Full Text PDF PubMed Scopus (53) Google ScholarJBY4MAT a ura3 GCN2-T7This studyJBY5MAT a ura3 gcn2-Y74A-T7This studyJBZ1MAT a ura3 gcn1-F2291LThis studyJBZ2MAT a ura3 GCN1-T7::URA3This studyJBZ3MAT a ura3 gcn1-F2291L-T7::URA3This study Open table in a new tab The two-hybrid vectors, pGBK and pGAD424g, were described previously (17Ito T. Nakamura R. Sumimoto H. Takeshige K. Sakaki Y. FEBS Lett. 1996; 385: 229-232Crossref PubMed Scopus (51) Google Scholar,18Nakamura R. Sumimoto H. Mizuki K. Hata K. Ago T. Kitajima S. Takeshige K. Sakaki Y. Ito T. Eur. J. Biochem. 1998; 251: 583-589Crossref PubMed Scopus (72) Google Scholar). For high efficiency transformation, the protocol of Gietz and Schiestl (19Gietz R.D. Schiestl R.H. Methods Mol. Cell. Biol. 1995; 5: 255-269Google Scholar) was adopted except for the addition of 10% dimethyl sulfoxide prior to the heat shock step (20Ito T. Tashiro K. Muta S. Ozawa R. Chiba T. Nishizawa M. Yamamoto K. Kuhara S. Sakaki Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1143-1147Crossref PubMed Scopus (667) Google Scholar). AGCN1 DNA fragment (nucleotides 6142–7146) was cloned between the segments encoding the GAL4 activation domain (AD) and the C-terminal 75-amino acid (aa) region of CDC24, which bears a PC motif and is called the PC motif-containing region (PCCR) (18Nakamura R. Sumimoto H. Mizuki K. Hata K. Ago T. Kitajima S. Takeshige K. Sakaki Y. Ito T. Eur. J. Biochem. 1998; 251: 583-589Crossref PubMed Scopus (72) Google Scholar). We subjected the GAL4 AD-GCN1-PCCR fragment to error-prone PCR in 50 μl of 1× PCR buffer containing 1 unit of Taq DNA polymerase, 200 μm dATP, 2 mm dCTP, 2 mm dGTP, 2 mm dTTP, 2 mm MgCl2, 5 pmol of each primer under the following thermal cycling: 94 °C for 3 min, followed by 30 of °C for °C for 30 and °C for 2 The of error-prone PCR were cloned into by a R. S. C. 1997; PubMed Scopus Google Scholar) as a were with bearing and The a protein between the GAL4 domain and GI domain of GCN2, and the one a protein between and the domain of which to of Ito and H. cells were synthetic complete (20Ito T. Tashiro K. Muta S. Ozawa R. Chiba T. Nishizawa M. Yamamoto K. Kuhara S. Sakaki Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1143-1147Crossref PubMed Scopus (667) Google Scholar) and with and 5-fluoro-orotic acid DNA fragment (nucleotides was cloned in a bearing the (15Kubota H. Sakaki Y. Ito T. J. Biol. Chem. 2000; 275: 20243-20246Abstract Full Text Full Text PDF PubMed Scopus (53) Google to which was with and into the yeast The were for by These were for and for the to the a ura3 For of GCN1, we first a bearing a DNA fragment encoding the of with region (nucleotides followed which is from cassette U. S. T. J. 1996; PubMed Scopus Google Scholar). the of a by an to GCN1, the was and into and to the strains a ura3 and a ura3 These strains were of or mm as described previously (15Kubota H. Sakaki Y. Ito T. J. Biol. Chem. 2000; 275: 20243-20246Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The is a which an mRNA bearing the by the region of GCN4 mRNA containing four to the translational derepression Hinnebusch A.G. Cell. Full Text PDF PubMed Scopus Google Scholar). This was into and and the were to and to or synthetic dextrose yeast amino acids, containing or the at 30 °C for 3 cells were and subjected to as described previously (18Nakamura R. Sumimoto H. Mizuki K. Hata K. Ago T. Kitajima S. Takeshige K. Sakaki Y. Ito T. Eur. J. Biochem. 1998; 251: 583-589Crossref PubMed Scopus (72) Google Scholar). The yeast and cells were to and to yeast or with or mm at 30 °C for cells were in of and by the addition of μl of buffer 2 on for min, the was by the addition of μl of were by at and with μl of Protein to cells was subjected to each of and to were by mm mm mm Phosphorylated eIF2α was by containing an that eIF2α phosphorylated at serine 51 We previously showed that the GI domain occurring at the of GCN2 to GCN1 (15Kubota H. Sakaki Y. Ito T. J. Biol. Chem. 2000; 275: 20243-20246Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). demonstrated that GCN2 with GCN1 via which the GI domain followed by an region M. Dong J. Ufano S. Hinnebusch A.G. EMBO J. 2000; 17: 1887-1899Crossref Google Scholar). the minimal essential region to with GCN1, we a of mutants for the region of GCN2 and for binding with GCN1 the yeast two-hybrid of the mutants bearing GI domain showed two-hybrid with GCN1 The yeast cells with the GCN2 protein which the GI domain, and degenerate protein kinase domain showed activity two-hybrid level of proteins and efficiency of to of binding by this the described is in with the one an in binding M. Dong J. Ufano S. Hinnebusch A.G. EMBO J. 2000; 17: 1887-1899Crossref Google Scholar). of the region not activity and we to evidence for this region to bind GCN1 In contrast, from of the GI domain the interaction We that of in the GI domain the interaction (15Kubota H. Sakaki Y. Ito T. J. Biol. Chem. 2000; 275: 20243-20246Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). These results that the GI domain as the minimal essential region for the binding to GCN1 and that the region has role in the interaction. the GI domain as bait the C-terminal of GCN1 as binding This is with the results recently by which identified the region as the sole region of GCN1 Hinnebusch A.G. EMBO J. 2000; PubMed Scopus Google Scholar). the minimal binding we this region PCR and binding to the GI domain the yeast two-hybrid were for on the and The segment of GCN1 the as as by as a domain However, we to the interaction in the for from or C-terminal the interaction 2). these we that the region amino is sufficient for binding to the GI domain of GCN2. We to gcn1 mutants defective in association with GCN2, critical for the of the GI domain and used to the role of this interaction. For this we used a However, we the minimal essential GCN1 region for GCN2 we not mutations to the protein selectively we a novel described 3 We first the that the GAL4 protein is with the of (18Nakamura R. Sumimoto H. Mizuki K. Hata K. Ago T. Kitajima S. Takeshige K. Sakaki Y. Ito T. Eur. J. Biochem. 1998; 251: 583-589Crossref PubMed Scopus (72) Google which with the domain occurring at the C-terminal of to GCN1 by error-prone were for two-hybrid interaction with the domain, which that the protein the C-terminal and hence is not within the GCN1 these incapable of interacting with GCN2 were identified the two-hybrid on gene and M. A. J. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar). this not for the of proteins but for the of with mutations to protein such display two-hybrid the In we used a dual bait two-hybrid to both We a GAL4 AD-GCN1-PCCR in These cells were with cells that GAL4 protein and that the of has genes by GAL4 and The cells by the were for to both and The to that the gene is and that an interaction is between the protein and the the is an of induction or impaired association of the protein with GCN2. the to both and we identified single amino acid and two-hybrid with the domain with that of the these single mutants to with GCN2 3 These results that the mutations the of GCN1 to with GCN2 but not the of proteins We identified each bearing amino acid and we not determine which of the is critical for is to that of these mutations in the of the GCN1 as in the single mutants the role of GCN1-GCN2 we to a yeast bearing GCN1 incapable of interacting with GCN2. For this we within a of amino acid GCN1 from various We GCN1 in this and with the at C-terminal to by in 5 of GCN1 were in thereby that the does not the protein in vivo. We these cells for to which is an inhibitor of a GCN4 and has used as an of general control response. The cells the 5 was for yeast cells bearing which GCN2 defective in interaction with GCN1 (15Kubota H. Sakaki Y. Ito T. J. Biol. Chem. 2000; 275: 20243-20246Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). these results indicate that the interaction between GCN1 and GCN2 is necessary for general control of amino acid synthesis. The described suggests that the derepression of GCN4translation is impaired in both mutants. We the translation mRNA a by the GCN4 which is for the derepression Hinnebusch A.G. Cell. Full Text PDF PubMed Scopus Google Scholar). The cells showed a induction of activity under starved or derepressed In contrast, the induction was impaired in both cells Thus, the interaction between GCN1 and GCN2 is for derepression of GCN4translation under amino we to determine the eIF2α kinase is activated in these mutants upon amino acid to translation are A.G. Microbiol. Rev. 1988; 52: 248-273Crossref PubMed Google Scholar, 10Hinnebusch A.G. J. Biol. Chem. 1997; 272: 2161-21664Abstract Full Text Full Text PDF Scopus (434) Google Scholar, J. L. R. M. K. M. Hinnebusch A.G. 1998; PubMed Scopus Google Scholar, D. P. D. 1996; PubMed Scopus Google Scholar, H. C. Anderson J. S. Hinnebusch A.G. Mol. Cell. Biol. 2000; 20: PubMed Scopus Google Scholar). The phosphorylated eIF2α in mutants and strains were under or an to eIF2α phosphorylated at phosphorylated eIF2α was under or phosphorylation of eIF2α was in the cells subjected to amino acid starvation In contrast, the induction of the phosphorylation was impaired in the cells or with of phosphorylation were Since GCN2 is the sole eIF2α kinase in the budding yeast, these results indicate that the interaction with GCN1 is necessary for the GCN2 to activated in amino GCN1 is for the activation of GCN2 in the budding yeast under amino acid or mutations of GCN1 were to phosphorylation of eIF2α, which is by the sole eIF2α kinase GCN2 (12Marton M.J. Crouch D. Hinnebusch A.G. Mol. Cell. Biol. 1993; 13: 3541-3556Crossref PubMed Scopus (96) Google Scholar, 14Marton M.J. Vazquez de Aldana C.R. Qiu H. Chalraburtty K. Hinnebusch A.G. Mol. Cell. Biol. 1997; 17: 4474-4489Crossref PubMed Google Scholar). how GCN1 activates GCN2 we and others evidence for direct association and for a general control response (15Kubota H. Sakaki Y. Ito T. J. Biol. Chem. 2000; 275: 20243-20246Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 16Garcia-Barrio M. Dong J. Ufano S. Hinnebusch A.G. EMBO J. 2000; 17: 1887-1899Crossref Google Scholar). In this study, we the minimal essential regions for GCN1-GCN2 association and for the first that the interaction is critical to the activation of GCN2 which leads to the derepression of GCN4translation and general control response. The minimal essential region of GCN2 to with GCN1 was to which we as the GI domain (15Kubota H. Sakaki Y. Ito T. J. Biol. Chem. 2000; 275: 20243-20246Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The the GI domain followed by an region were necessary for the interaction by others M. Dong J. Ufano S. Hinnebusch A.G. EMBO J. 2000; 17: 1887-1899Crossref Google Scholar). However, and in a (15Kubota H. Sakaki Y. Ito T. J. Biol. Chem. 2000; 275: 20243-20246Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) indicate that the GI domain per but not the as the for the previously (15Kubota H. Sakaki Y. Ito T. J. Biol. Chem. 2000; 275: 20243-20246Abstract Full Text Full Text PDF PubMed Scopus (53) Google the GI domain is in various proteins GCN2. of an gene that is in Y. M. K. I. T. Sakaki Y. Ito T. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar, Y. Y. K. Sakaki Y. Ito T. Biochem. 1999; PubMed Scopus Google Scholar, K. Y. T. M. M. Sakaki Y. Ito T. 2000; PubMed Scopus Google protein interacting with S. S. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google of S. C. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google yeast member of the RNA and to these GI in protein We that amino acid the minimal essential region of GCN1 to the GI domain of GCN2 2). In with this the segment of GCN1 was recently for binding to GCN2 Hinnebusch A.G. EMBO J. 2000; PubMed Scopus Google Scholar). In contrast to GCN2, the of the GI region of GCN1 We a to identify critical for the of GI For this we a unique dual bait two-hybrid which one to selectively identify mutations to defective interaction 3 Furthermore, this to mutants encoding which identified as with interaction between the domain and binding and a for of this we have identified single amino acid and each of which interaction between GCN1 and GCN2 3 that fails to with GCN2 Hinnebusch A.G. EMBO J. 2000; PubMed Scopus Google Scholar). of these mutations in the C-terminal of the In amino acid in the mutants that we identified as bearing mutations were to this These results that the C-terminal functions as the interaction with the GI The of mutants indicate that the is not involved in and a role in of the mutations in this region to and proteins and hence by as above. to the is necessary to insight into the mechanism for of the GI to the of GCN1 with of the binding for GI which to We and others showed that yeast cells defective in GCN1-GCN2 interaction display of impaired induction of which is under the of the transcription factor GCN4 (15Kubota H. Sakaki Y. Ito T. J. Biol. Chem. 2000; 275: 20243-20246Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 16Garcia-Barrio M. Dong J. Ufano S. Hinnebusch A.G. EMBO J. 2000; 17: 1887-1899Crossref Google Scholar, Hinnebusch A.G. EMBO J. 2000; PubMed Scopus Google Scholar) the derepression of GCN4 translation was impaired in these mutants However, that the derepression of GCN4 translation is not by the activation of GCN2. in of and eIF2B the of phosphorylation of eIF2α as by genes A.G. Microbiol. Rev. 1988; 52: 248-273Crossref PubMed Google Scholar, 10Hinnebusch A.G. J. Biol. Chem. 1997; 272: 2161-21664Abstract Full Text Full Text PDF Scopus (434) Google Scholar). The in (10Hinnebusch A.G. J. Biol. Chem. 1997; 272: 2161-21664Abstract Full Text Full Text PDF Scopus (434) Google Scholar) or impaired J. L. R. M. K. M. Hinnebusch A.G. 1998; PubMed Scopus Google Scholar) of initiator tRNA induces a studies demonstrated that derepression of GCN4 translation is by of D. P. D. 1996; PubMed Scopus Google encoding the RNA of 1992; 6: PubMed Scopus Google or encoding the tRNA H. C. Anderson J. S. Hinnebusch A.G. Mol. Cell. Biol. 2000; 20: PubMed Scopus Google Scholar). We the phosphorylation of eIF2α in these mutants and demonstrated the of mutants defective in GCN1-GCN2 interaction is with impaired phosphorylation of these mutants to GCN2, is the sole eIF2α kinase of the budding phosphorylation of eIF2α in these mutants that GCN1-GCN2 minimal activation of the kinase. GCN1 and GCN2 are ribosome ribosome-binding M.J. Vazquez de Aldana C.R. Qiu H. Chalraburtty K. Hinnebusch A.G. Mol. Cell. Biol. 1997; 17: 4474-4489Crossref PubMed Google Scholar, Hinnebusch A.G. EMBO J. 2000; PubMed Scopus Google Scholar, M. Wek R.C. Hinnebusch A.G. Mol. Cell. Biol. PubMed Scopus Google S. Wek R.C. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus (53) Google in such that is that the proteins a to activation of the kinase the interaction is impaired by the In this is to that of GCN2 suppress the of defective in binding to GCN2, but not that of which GCN1 the region Hinnebusch A.G. EMBO J. 2000; PubMed Scopus Google Scholar). on these we that the GI association of GCN2 to GCN1 is necessary for the activation of GCN2 kinase in vivo upon amino acid starvation and hence for derepression of GCN4 to general control response. We R. C. Wek for the of and H. Sumimoto for critical reading of the and
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