Polyribosome Binding by GCN1 Is Required for Full Activation of Eukaryotic Translation Initiation Factor 2α Kinase GCN2 during Amino Acid Starvation

The protein kinase GCN2 mediates translational control of gene expression in amino acid-starved cells by phosphorylating eukaryotic translation initiation factor 2α. In Saccharomyces cerevisiae, activation of GCN2 by uncharged tRNAs in starved cells requires its direct interaction with both the GCN1·GCN20 regulatory complex and ribosomes. GCN1 also interacts with ribosomes in cell extracts, but it was unknown whether this activity is crucial for its ability to stimulate GCN2 function in starved cells. We describe point mutations in two conserved, noncontiguous segments of GCN1 that lead to reduced polyribosome association by GCN1·GCN20 in living cells without reducing GCN1 expression or its interaction with GCN20. Mutating both segments simultaneously produced a greater reduction in polyribosome binding by GCN1·GCN20 and a stronger decrease in eukaryotic translation initiation factor 2α phosphorylation than did mutating in one segment alone. These findings provide strong evidence that ribosome binding by GCN1 is required for its role as a positive regulator of GCN2. A particular mutation in the GCN1 domain, related in sequence to translation elongation factor 3 (eEF3), decreased GCN2 activation much more than it reduced ribosome binding by GCN1. Hence, the eEF3-like domain appears to have an effector function in GCN2 activation. This conclusion supports the model that an eEF3-related activity of GCN1 influences occupancy of the ribosomal decoding site by uncharged tRNA in starved cells. The protein kinase GCN2 mediates translational control of gene expression in amino acid-starved cells by phosphorylating eukaryotic translation initiation factor 2α. In Saccharomyces cerevisiae, activation of GCN2 by uncharged tRNAs in starved cells requires its direct interaction with both the GCN1·GCN20 regulatory complex and ribosomes. GCN1 also interacts with ribosomes in cell extracts, but it was unknown whether this activity is crucial for its ability to stimulate GCN2 function in starved cells. We describe point mutations in two conserved, noncontiguous segments of GCN1 that lead to reduced polyribosome association by GCN1·GCN20 in living cells without reducing GCN1 expression or its interaction with GCN20. Mutating both segments simultaneously produced a greater reduction in polyribosome binding by GCN1·GCN20 and a stronger decrease in eukaryotic translation initiation factor 2α phosphorylation than did mutating in one segment alone. These findings provide strong evidence that ribosome binding by GCN1 is required for its role as a positive regulator of GCN2. A particular mutation in the GCN1 domain, related in sequence to translation elongation factor 3 (eEF3), decreased GCN2 activation much more than it reduced ribosome binding by GCN1. Hence, the eEF3-like domain appears to have an effector function in GCN2 activation. This conclusion supports the model that an eEF3-related activity of GCN1 influences occupancy of the ribosomal decoding site by uncharged tRNA in starved cells. Phosphorylation of the α subunit of eukaryotic translation initiation factor 2α (eIF2α) 1The abbreviations used are: eIF2α, eukaryotic translation initiation factor 2α; GAAC, general amino acid control; HisRS, histidyl-tRNA synthetase; eEF3, eukaryotic elongation factor 3; A-site, acceptor site; WCE, whole cell extract; 3AT, 3-amino-2,4-triazole; SC, synthetic complete. provides a key mechanism for down-regulating protein synthesis in response to nutrient starvation or stress in mammalian and yeast cells (reviewed in Ref. 1Hinnebusch A.G. Sonenberg N. Hershey J.W.B. Mathews M.B. Translational Control of Gene Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2000: 185-243Google Scholar). In its GTP-bound state, the heterotrimeric eIF2 complex delivers initiator methionyl-tRNA to the 40 S ribosome at an early stage of the initiation pathway. The eIF2 is ultimately released from the ribosome in an inactive GDP-bound state and must be recycled to eIF2·GTP by the guanine nucleotide exchange factor eIF2B for a new round of initiation to occur. Phosphorylation of eIF2α converts eIF2·GDP to an inhibitor of eIF2B, interfering with eIF2 recycling and decreasing the rate of translation initiation. In Saccharomyces cerevisiae, eIF2α phosphorylation also increases the translation of GCN4 mRNA, encoding a transcriptional activator of amino acid biosynthetic enzymes subject to general amino acid control (GAAC). Thus, the rate of amino acid biosynthesis is stimulated by GCN4 in parallel with the reduction in general protein synthesis as a dual mechanism for replenishing amino acids in starved yeast cells. The diminished initiator methionyl-tRNA recruitment produced by eIF2α phosphorylation allows ribosomes to overcome the inhibitory function of four upstream open reading frames present in the GCN4 mRNA leader and initiate at the GCN4 start codon instead (reviewed in Ref. 1Hinnebusch A.G. Sonenberg N. Hershey J.W.B. Mathews M.B. Translational Control of Gene Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2000: 185-243Google Scholar). In mammalian cells, expression of transcription factor ATF4 is stimulated at the translational level in response to eIF2α phosphorylation by a mechanism involving upstream open reading frames very similar to that elucidated for GCN4 mRNA in yeast (2Vattem K.M. Wek R.C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11269-11274Crossref PubMed Scopus (1135) Google Scholar, 3Lu P.D. Harding H.P. Ron D. J. Cell Biol. 2004; 167: 27-33Crossref PubMed Scopus (662) Google Scholar). Mammals contain four eIF2α kinases (PKR, PERK, HRI, and GCN2) activated in response to different kinds of stress or starvation, whereas GCN2 is the sole member of this kinase subfamily in S. cerevisiae. GCN2 is activated by uncharged tRNAs that accumulate in amino acid-starved cells and bind to a histidyl-tRNA synthetase (HisRS)-like domain located C-terminal to the kinase domain (1Hinnebusch A.G. Sonenberg N. Hershey J.W.B. Mathews M.B. Translational Control of Gene Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2000: 185-243Google Scholar). We showed previously that GCN2 activation in starved cells additionally requires interaction of the GCN2 N-terminal domain with the GCN1·GCN20 regulatory complex through a C-terminal segment of GCN1 (residues 2052–2428 within area D shown in Fig. 1A) (4Garcia-Barrio M. Dong J. Ufano S. Hinnebusch A.G. EMBO J. 2000; 19: 1887-1899Crossref PubMed Google Scholar, 5Sattlegger E. Hinnebusch A.G. EMBO J. 2000; 19: 6622-6633Crossref PubMed Scopus (91) Google Scholar). GCN1 is a large, 296-kDa protein containing a central domain with strong sequence similarity to the N-terminal portion of fungal translation elongation factor 3 (eEF3) (6Marton M.J. Crouch D. Hinnebusch A.G. Mol. Cell. Biol. 1993; 13: 3541-3556Crossref PubMed Scopus (93) Google Scholar). eEF3 promotes release of deacylated tRNAs from the ribosomal exit site and thereby stimulates delivery of charged tRNAs to the acceptor site (A-site) by eEF1A·GTP (7Triana-Alonso F.J. Chakraburtty K. Nierhaus K.H. J. Biol. Chem. 1995; 270: 20473-20478Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). The eEF3-like domain in GCN1 contains the binding domain for the N-terminal portion of GCN20 (8Marton M.J. Vazquez de Aldana C.R. Qiu H. Chakraburtty K. Hinnebusch A.G. Mol. Cell. Biol. 1997; 17: 4474-4489Crossref PubMed Google Scholar), whereas the remainder of GCN20 is related to the C terminus of eEF3, including the two ATP-binding cassettes (9Vazquez de Aldana C.R. Marton M.J. Hinnebusch A.G. EMBO J. 1995; 14: 3184-3199Crossref PubMed Scopus (124) Google Scholar). Thus, formation of the GCN1·GCN20 complex juxtaposes the domains in these two proteins that are related to different segments of eEF3 (Fig. 1). GCN1 associates with elongating ribosomes (polyribosomes) in cell extracts, and this interaction is stimulated by ATP in a manner dependent on the ATP-binding cassettes in GCN20 (8Marton M.J. Vazquez de Aldana C.R. Qiu H. Chakraburtty K. Hinnebusch A.G. Mol. Cell. Biol. 1997; 17: 4474-4489Crossref PubMed Google Scholar). GCN2 also has ribosome binding activity that appears to be critical for its function in vivo (10Ramirez M. Wek R.C. Hinnebusch A.G. Mol. Cell. Biol. 1991; 11: 3027-3036Crossref PubMed Scopus (121) Google Scholar, 11Zhu S. Wek R.C. J. Biol. Chem. 1998; 273: 1808-1814Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). GCN1 and GCN2 both bind to polyribosomes in cell extracts independent of the respective domains required for their interaction with one another (5Sattlegger E. Hinnebusch A.G. EMBO J. 2000; 19: 6622-6633Crossref PubMed Scopus (91) Google Scholar, 10Ramirez M. Wek R.C. Hinnebusch A.G. Mol. Cell. Biol. 1991; 11: 3027-3036Crossref PubMed Scopus (121) Google Scholar, 11Zhu S. Wek R.C. J. Biol. Chem. 1998; 273: 1808-1814Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Likewise, complex formation between GCN1 and GCN2 can occur in the absence of their ribosome binding domains (4Garcia-Barrio M. Dong J. Ufano S. Hinnebusch A.G. EMBO J. 2000; 19: 1887-1899Crossref PubMed Google Scholar, 5Sattlegger E. Hinnebusch A.G. EMBO J. 2000; 19: 6622-6633Crossref PubMed Scopus (91) Google Scholar). Hence, it appears that GCN1 and GCN2 independently contact one another and the 80 S ribosome to promote assembly of a GCN1·GCN20·GCN2 regulatory complex on polyribosomes. As gcn1Δ mutants are more resistant than wild type to the drug paromomycin (5Sattlegger E. Hinnebusch A.G. EMBO J. 2000; 19: 6622-6633Crossref PubMed Scopus (91) Google Scholar), which binds to the A-site of small ribosomal subunits in bacteria (12Schroeder R. Waldsich C. Wank H. EMBO J. 2000; 19: 1-9Crossref PubMed Scopus (209) Google Scholar), GCN1 binding to the ribosome apparently alters A-site function. Together, these findings led us to propose that GCN1·GCN20 functions on translating ribosomes to promote binding of uncharged tRNA to the A-site or in the transfer of uncharged tRNA from the A-site to the HisRS-like domain in GCN2 for kinase activation (5Sattlegger E. Hinnebusch A.G. EMBO J. 2000; 19: 6622-6633Crossref PubMed Scopus (91) Google Scholar, 8Marton M.J. Vazquez de Aldana C.R. Qiu H. Chakraburtty K. Hinnebusch A.G. Mol. Cell. Biol. 1997; 17: 4474-4489Crossref PubMed Google Scholar). This proposed mechanism resembles that demonstrated in Escherichia coli for activation of RelA protein by uncharged tRNA bound to the A-site, which mediates the stringent response to amino acid starvation in bacteria (13Cashel M. Rudd K.E. Neidhardt F.C. Ingraham J.L. Magasanik B. Low K.B. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. American Society for Microbiology, Washington, D. C.1987: 1410-1438Google Scholar, 14Goldman E. Jakubowski H. Mol. Microbiol. 1990; 4: 2035-2040Crossref PubMed Scopus (45) Google Scholar). We showed previously that deletions of regions A, B, or C in GCN1, encompassing the N-terminal 77% of the protein (see Fig. 1A), destroys GCN1 regulatory function and impairs ATP-dependent polyribosome binding by GCN1 in cell extracts (5Sattlegger E. Hinnebusch A.G. EMBO J. 2000; 19: 6622-6633Crossref PubMed Scopus (91) Google Scholar). This result suggested that GCN1 has multiple points of contact with the ribosome. Although the deletions of regions A, B, or C did not destabilize the remainder of GCN1 protein or impair its interaction with GCN2, it is possible that these large deletions destroy another function of GCN1 other than ribosome binding. Hence, it was uncertain whether stable ribosome association by the GCN1·GCN20 complex is crucial for its function in activating GCN2. To validate this important aspect of our model, we set out to isolate point mutations in GCN1 that would impair polyribosome binding by GCN1 without affecting GCN1 expression or complex formation with GCN2 or GCN20. We also wanted to determine the effects of these mutations on activation of GCN2 and the GAAC response. From analysis of such mutations in conserved residues found in regions B and C, we have obtained strong evidence that GCN1-ribosome interaction is essential for GCN2 activation. Our results further demonstrate that specific residues in the eEF3-related domain of GCN1 function in signal transduction beyond their role in promoting ribosome binding by the GCN1·GCN20 complex. Yeast Strains and Plasmids—The yeast strain used in this study was gcn1Δ strain H2556 (MATα ura3–52 trp1–63 leu2–3, leu2–112 GAL2+) constructed previously. 2C. R. Vazquez de Aldana, and A. G. Hinnebusch, unpublished observations. Plasmids used in this study are listed in Table I, and details of their construction are provided below. Vectors used were pRS316 and pRS425 (15Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar).Table IPlasmids used in this studyPlasmidRelevant gene under its own promotorRelevant featuresVectorSourcep2367GCN1-mycaCoding sequences for a single c-myc epitope appended to the 3′ end of the open reading frameURA3, CEN6/ARSH4pRS316Ref. 8Marton M.J. Vazquez de Aldana C.R. Qiu H. Chakraburtty K. Hinnebusch A.G. Mol. Cell. Biol. 1997; 17: 4474-4489Crossref PubMed Google ScholarpES176–4gcn1-M1A-mycaCoding sequences for a single c-myc epitope appended to the 3′ end of the open reading frameURA3, CEN6/ARSH4pRS316This studypES182–8gcn1-M7A-mycaCoding sequences for a single c-myc epitope appended to the 3′ end of the open reading frameURA3, CEN6/ARSH4pRS316This studypES257–12-2gcn1-M7D-mycaCoding sequences for a single c-myc epitope appended to the 3′ end of the open reading frameURA3, CEN6/ARSH4pRS316This studypES263–34-1gcn1-M1A/M7A-mycaCoding sequences for a single c-myc epitope appended to the 3′ end of the open reading frameURA3, CEN6/ARSH4pRS316This studyp1747GCN20LEU2, 2μpRS425Ref. 8Marton M.J. Vazquez de Aldana C.R. Qiu H. Chakraburtty K. Hinnebusch A.G. Mol. Cell. Biol. 1997; 17: 4474-4489Crossref PubMed Google Scholara Coding sequences for a single c-myc epitope appended to the 3′ end of the open reading frame Open table in a new tab For construction of the low copy plasmid pES176-4 containing gcn1-M1A, two were as the and and the and (see Table In a the two were and The was with plasmid with and In this the sequences of were used in this to the reading site in in to GCN1 the mutations are to GCN1 the mutations are a site are and site is in in and to GCN1 to to GCN1 to GCN1 of to GCN1 the mutations are to GCN1 to the mutations are a site are and site is in in and to GCN1 to the mutations are to GCN1 to the mutations are a site are and site is in in to GCN1 to the reading the mutations are a site are and site is in Open table in a new tab For the construction of the low copy plasmid containing two were as and and and In a the two were and The was the plasmid The low copy plasmid containing was constructed to plasmid and instead of and For the construction of the low copy plasmid containing the of was by the from of Cell from vivo association of proteins with polyribosomes was as previously with a K.H. B. A. Hinnebusch A.G. EMBO J. 2004; PubMed Scopus Google Scholar). yeast cells were to in of to an of in were a containing of and of were and on for with were for 3 at with of inhibitor without and of and and in of were with a of to the of cells by for with on Cell was in a for at and of were on a by for at at the were whereas for the model were by and to or to the were by the GCN20 (9Vazquez de Aldana C.R. Marton M.J. Hinnebusch A.G. EMBO J. 1995; 14: 3184-3199Crossref PubMed Scopus (124) Google eIF2α Wek R.C. Hinnebusch A.G. Cell. Full Text PDF PubMed Scopus Google eIF2α on J. D. A. A. D. D. J. Cell Biol. PubMed Scopus Google from or c-myc were to to of c-myc or to protein A of in B of GCN1 for in showed previously that ribosome binding by GCN1 was by deletions of A, B, or C, which amino acids or of the protein (Fig. 1A) (5Sattlegger E. Hinnebusch A.G. EMBO J. 2000; 19: 6622-6633Crossref PubMed Scopus (91) Google Scholar). This suggested that GCN1 has multiple ribosome contact In an to specific amino acids in ribosome we sequence of GCN1 proteins from different eukaryotic and a that is in amino acids and is to an (Fig. To the of this conserved for ribosome binding by S. GCN1, we the amino acids in this conserved with uncharged residues or charged residues and the GCN1 proteins for ribosome binding in yeast GCN1. the GCN1 proteins from on low copy under the GCN1 were with to GCN1 to ribosomes in vivo to whole cell extracts The were by through and the of GCN1, and the S ribosomal subunit protein the were by In the GCN1 GCN1 a The of the protein and the polyribosomes were between and more than 40 S subunits were in (Fig. GCN20 showed a similar the with the that these two proteins bind to ribosomes as a complex (8Marton M.J. Vazquez de Aldana C.R. Qiu H. Chakraburtty K. Hinnebusch A.G. Mol. Cell. Biol. 1997; 17: 4474-4489Crossref PubMed Google Scholar). We showed previously that the of GCN1 and GCN20 with polyribosomes can be to 80 S by a of the with which converts the polyribosomes to 80 S that GCN1 and GCN20 are with the polyribosomes (8Marton M.J. Vazquez de Aldana C.R. Qiu H. Chakraburtty K. Hinnebusch A.G. Mol. Cell. Biol. 1997; 17: 4474-4489Crossref PubMed Google Scholar). To determine the effects of the and on polyribosome binding by GCN1 and we the in and for for the in the and the as a of the for GCN1 (Fig. B and GCN1 proteins showed a similar in our with of the polyribosomes and GCN1 and GCN20 in and From the results of two we found that the and mutations reduced polyribosome binding by GCN1 to and of wild and similar effects on polyribosome binding by GCN20 (Fig. These results that the residues in the conserved in area B of GCN1 are required for ribosome binding by the GCN1·GCN20 complex in between GCN1 and eEF3 in C with in B to binding by GCN1 in has to the terminus of eEF3, and the eEF3 terminus acids interacts with S in H. K. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar), that the eEF3-like is in ribosome binding. In a we to amino acids in the eEF3-like that are essential for GCN1 function. of amino acids conserved the eEF3-like regions in GCN1 proteins were by in GCN1, and gcn1Δ the were for on the amino acid starvation, and to GCN2 can overcome starvation and on containing In this not we found that one of our mutations GCN1 in activating GCN2. This mutation in a of conserved residues that in also conserved in eEF3 Fig. In this we whether these amino acids are in ribosome binding. We found that the mutation led to of in polyribosome association of GCN1 and GCN20 (Fig. B and the mutations the of GCN1 and GCN20 produced by the with the GCN1·GCN20 binding to polyribosomes of wild than did the single mutants of wild (Fig. These findings that the and residues function in promoting ribosome binding by GCN1. of GCN1 from in GCN1 or GCN20 out the that the reduced ribosome binding of GCN1·GCN20 produced by the and mutations results from decreased expression of the proteins by analysis of from the and (Fig. we evidence that ribosome binding by GCN1 is by its interaction with GCN20 (5Sattlegger E. Hinnebusch A.G. EMBO J. 2000; 19: 6622-6633Crossref PubMed Scopus (91) Google Scholar, 8Marton M.J. Vazquez de Aldana C.R. Qiu H. Chakraburtty K. Hinnebusch A.G. Mol. Cell. Biol. 1997; 17: 4474-4489Crossref PubMed Google it was important to determine whether the and mutations impair the interaction in This is for the mutation it alters residues in to shown previously to be required for interaction of GCN1 with GCN20. analysis of association in we found that of the GCN1 proteins were with GCN20 to the as for GCN1 (Fig. A and evidence that the GCN1 mutations not the GCN1·GCN20 complex is that not the expression of GCN20 (Fig. We showed previously that GCN1 with interaction and the level of GCN20 of GCN20 is more to of the GCN1·GCN20 complex (8Marton M.J. Vazquez de Aldana C.R. Qiu H. Chakraburtty K. Hinnebusch A.G. Mol. Cell. Biol. 1997; 17: 4474-4489Crossref PubMed Google Scholar). Thus, the that the and not GCN20 further supports that interaction is by these GCN1 We a in vivo to that mutating the residues not impair The of interaction by the mutation impairs the GAAC response to starvation (8Marton M.J. Vazquez de Aldana C.R. Qiu H. Chakraburtty K. Hinnebusch A.G. Mol. Cell. Biol. 1997; 17: 4474-4489Crossref PubMed Google Scholar). A in activation of GCN2 kinase function by with to GCN4 and amino acid biosynthetic under its cell on containing the inhibitor of we found that the of the but not that of the gcn1Δ is by GCN20 (Fig. and This of can be by a of complex formation between GCN20 and the by at of GCN20. the of the strain was not by GCN20 our conclusion that the residues not GCN20 binding GCN1 in with in GCN2 and the GAAC have proposed that the GCN1·GCN20 complex must bind to the ribosome to activation of GCN2 by uncharged tRNAs bound to the ribosomal A-site of elongating ribosomes (5Sattlegger E. Hinnebusch A.G. EMBO J. 2000; 19: 6622-6633Crossref PubMed Scopus (91) Google Scholar). this model is the mutations in the and which GCN1 binding to decrease phosphorylation of eIF2α by GCN2 and impair the GAAC response. To this we the of eIF2α in of the and by analysis specific for eIF2α on We also the of eIF2α that the protein of its phosphorylation (Fig. The of eIF2α on were for the of eIF2α and to the for the GCN1 strain (Fig. In we the of the (Fig. The and mutations in GCN1 produced in eIF2α phosphorylation of and in cells starved for by (Fig. The of these with the of by these two mutants (Fig. and also with the in polyribosome binding by GCN1·GCN20 (Fig. These findings the that polyribosome binding by GCN1 is critical for its ability to GCN2 and the GAAC response. The mutation a stronger than or on eIF2α phosphorylation and to (Fig. B and it with to the in polyribosome binding by GCN1·GCN20 (Fig. This that the mutation has a on GCN2 the ability of GCN1 to GCN2 activation by uncharged tRNA in to reducing ribosome association by the mutations impair ribosome binding by the and mutations produced a more reduction in eIF2α phosphorylation than did single of eIF2α on (Fig. A and This reduction is with the in GCN1 polyribosome association produced by these two mutations (Fig. The mutation did not greater to than did the single the is from the for this (Fig. In this we in conserved residues located in two segments of GCN1 that lead to in polyribosome association by the GCN1·GCN20 complex in the residues in the segment located in area B with residues led to a greater reduction in polyribosome binding by GCN1·GCN20 than did at the residues and it produced a greater in both eIF2α phosphorylation by GCN2 and the GAAC response. In in the segment located in area C with in area B led to in polyribosome binding by in eIF2α and in the GAAC response. of these mutations produced in the of GCN1 or did impair GCN1·GCN20 or complex formation (Fig. and not Together, these findings provide strong for the that binding of GCN1·GCN20 to polyribosomes is required for activation of GCN2 and the of GCN4 translation in amino acid-starved cells. The that independent to between GCN1 and the ribosome an interaction of GCN1 with of the on both 40 S and S that to stable association of the GCN1·GCN20 complex with the 80 S ribosome. and mutations led to similar in GCN1·GCN20 association with but produced a much greater in eIF2α phosphorylation than did which that the residues by in area C an important role in signal transduction in to ribosome binding by these residues are located within a of similarity between GCN1 and the N-terminal domain of In of evidence that eEF3 stimulates release of deacylated tRNA from the exit site and the binding of tRNA to the A-site (7Triana-Alonso F.J. Chakraburtty K. Nierhaus K.H. J. Biol. Chem. 1995; 270: 20473-20478Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), it is to propose that interaction of the eEF3-related in GCN1 with the ribosome a in the This be in binding of uncharged tRNA to the A-site, or it stimulate the transfer of uncharged tRNA from the A-site to the HisRS-like domain in GCN2 for kinase activation. we found that binding of GCN1 and GCN20 to polyribosomes in was dependent on the of ATP to the In the we GCN1·GCN20 binding to polyribosomes in ATP that were from cells with the cells. we this to the association of translation initiation with ribosomal subunits through us to translation initiation complex assembly in living yeast cells K.H. B. A. Hinnebusch A.G. EMBO J. 2004; PubMed Scopus Google Scholar). Our of polyribosome association of GCN1·GCN20 in from cells allows us to state that these proteins are with translating ribosomes in vivo in amino cells. In we can be that the and mutations polyribosome association by GCN1·GCN20 in vivo by similar the much stronger of on GCN2 activation a in signal transduction ribosome association of the regulatory complex. The of of living cells be an for specific residues in the eEF3-related domain to critical functions in ribosome binding GCN2 activation. We and for on the of the of Gene and for and and for in of the