Title: The GTP-binding Release Factor eRF3 as a Key Mediator Coupling Translation Termination to mRNA Decay
Abstract: GTP is essential for eukaryotic translation termination, where the release factor 3 (eRF3) complexed with eRF1 is involved as the guanine nucleotide-binding protein. In addition, eRF3 regulates the termination-coupled events, eRF3 interacts with poly(A)-binding protein (Pab1) and the surveillance factor Upf1 to mediate normal and nonsense-mediated mRNA decay. However, the roles of GTP binding to eRF3 in these processes remain largely unknown. Here, we showed in yeast that GTP is essentially required for the association of eRF3 with eRF1, but not with Pab1 and Upf1. A mutation in the GTP-binding motifs of eRF3 impairs the eRF1-binding ability without altering the Pab1- or Upf1-binding activity. Interestingly, the mutation causes not only a defect in translation termination but also delay of normal and nonsense-mediated mRNA decay, suggesting that GTP/eRF3-dependent termination exerts its influence on the subsequent mRNA degradation. The termination reaction itself is not sufficient, but eRF3 is essential for triggering mRNA decay. Thus, eRF3 is a key mediator that transduces termination signal to mRNA decay. GTP is essential for eukaryotic translation termination, where the release factor 3 (eRF3) complexed with eRF1 is involved as the guanine nucleotide-binding protein. In addition, eRF3 regulates the termination-coupled events, eRF3 interacts with poly(A)-binding protein (Pab1) and the surveillance factor Upf1 to mediate normal and nonsense-mediated mRNA decay. However, the roles of GTP binding to eRF3 in these processes remain largely unknown. Here, we showed in yeast that GTP is essentially required for the association of eRF3 with eRF1, but not with Pab1 and Upf1. A mutation in the GTP-binding motifs of eRF3 impairs the eRF1-binding ability without altering the Pab1- or Upf1-binding activity. Interestingly, the mutation causes not only a defect in translation termination but also delay of normal and nonsense-mediated mRNA decay, suggesting that GTP/eRF3-dependent termination exerts its influence on the subsequent mRNA degradation. The termination reaction itself is not sufficient, but eRF3 is essential for triggering mRNA decay. Thus, eRF3 is a key mediator that transduces termination signal to mRNA decay. In eukaryotes, the process of translation is divided into at least three steps: initiation, elongation, and termination, and all the three steps are in common regulated by GTP-binding proteins (1Hershey J.W.B. Merrick W.C. Sonenberg N. Hershey J.W.B. Mathews M.B. Translational Control of Gene Expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000: 33-88Google Scholar, 2Merrick W.C. Nyborg J. Sonenberg N. Hershey J.W.B. Mathews M.B. Translational Control of Gene Expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000: 89-125Google Scholar, 3Welch E.M. Wang W. Peltz S.W. Sonenberg N. Hershey J.W.B. Mathews M.B. Translational Control of Gene Expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000: 467-485Google Scholar). The structure of the GTP-binding proteins functioning at each step is well conserved from yeast to mammals, and these proteins are fundamental to living cells (4Bourne H.R. Sanders D.A. McCormick F. Nature. 1990; 348: 125-132Crossref PubMed Scopus (1836) Google Scholar). In the initiation and elongation steps eIF2 1The abbreviations used are: eIF, eukaryotic initiation factor; eRF, eukaryotic release factor; PABP, polyadenylate-binding protein; GST, glutathione S-transferase; NMD, nonsense-mediated mRNA decay; HA, hemagglutinin; GTPγS, guanosine 5′-3-O-(thio)triphosphate; CL, CAT and luciferase reporters; CSL, CAT and luciferase reporters with a stop codon between them; EF, elongation factor; GDPNP, 5′-guanylylimidodiphosphate.1The abbreviations used are: eIF, eukaryotic initiation factor; eRF, eukaryotic release factor; PABP, polyadenylate-binding protein; GST, glutathione S-transferase; NMD, nonsense-mediated mRNA decay; HA, hemagglutinin; GTPγS, guanosine 5′-3-O-(thio)triphosphate; CL, CAT and luciferase reporters; CSL, CAT and luciferase reporters with a stop codon between them; EF, elongation factor; GDPNP, 5′-guanylylimidodiphosphate. and eEF1A, respectively, bring aminoacyl tRNAs to the A site of the ribosome (1Hershey J.W.B. Merrick W.C. Sonenberg N. Hershey J.W.B. Mathews M.B. Translational Control of Gene Expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000: 33-88Google Scholar, 2Merrick W.C. Nyborg J. Sonenberg N. Hershey J.W.B. Mathews M.B. Translational Control of Gene Expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000: 89-125Google Scholar). While in the termination step, eRF3 was identified as the GTP-binding protein (5Kisselev L.L. Buckingham R.H. Trends Biochem. Sci. 2000; 25: 561-566Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 6Bertram G. Innes S. Minella O. Richardson J. Stansfield I. Microbiology. 2001; 147: 255-269Crossref PubMed Scopus (112) Google Scholar, 7Inge-Vechtomov S. Zhouravleva G. Philippe M. Biol. Cell. 2003; 95: 195-209Crossref PubMed Scopus (100) Google Scholar, 8Kisselev L. Ehrenberg M. Frolova L. EMBO J. 2003; 22: 175-182Crossref PubMed Scopus (199) Google Scholar). Translation termination is regulated by a heterodimeric release factor consisting of eRF1 and eRF3 (9Frolova L. Le Goff X. Rasmussen H.H. Cheperegin S. Drugeon G. Kress M. Arman I. Haenni A.L. Celis J.E. Philippe M. Jastesen J. Kisselev L. Nature. 1994; 372: 701-703Crossref PubMed Scopus (374) Google Scholar, 10Zhouravleva G. Frolova L. Le Goff X. Le Guellec R. Inge-Vechtomov S. Kisselev L. Philippe M. EMBO J. 1995; 14: 4065-4072Crossref PubMed Scopus (518) Google Scholar, 11Stansfield I. Jones K.M. Kushnirov V.V. Dagkesamanskaya A.R. Poznyakovski A.I. Paushkin S.V. Nierras C.R. Cox B.S. Ter-Avanesyan M.D. Tuite M.F. EMBO J. 1995; 14: 4365-4373Crossref PubMed Scopus (426) Google Scholar). eRF1 directly recognizes all three termination codons to release completed polypeptide chain from the ribosome (9Frolova L. Le Goff X. Rasmussen H.H. Cheperegin S. Drugeon G. Kress M. Arman I. Haenni A.L. Celis J.E. Philippe M. Jastesen J. Kisselev L. Nature. 1994; 372: 701-703Crossref PubMed Scopus (374) Google Scholar, 12Drugeon G. Jean-Jean O. Frolova L. Le Goff X. Philippe M. Kisselev L. Haenni A.L. Nucleic Acids Res. 1997; 25: 2254-2258Crossref PubMed Scopus (62) Google Scholar), and eRF3 stimulates the termination reaction in a GTP-dependent manner (10Zhouravleva G. Frolova L. Le Goff X. Le Guellec R. Inge-Vechtomov S. Kisselev L. Philippe M. EMBO J. 1995; 14: 4065-4072Crossref PubMed Scopus (518) Google Scholar, 13Frolova L. Le Goff X. Zhouravleva G. Davydova E. Philippe M. Kisselev L. RNA (N. Y.). 1996; 2: 334-341PubMed Google Scholar). In the yeast Saccharomyces cerevisiae, eRF1 and eRF3 are encoded by the essential genes SUP45 and SUP35 (9Frolova L. Le Goff X. Rasmussen H.H. Cheperegin S. Drugeon G. Kress M. Arman I. Haenni A.L. Celis J.E. Philippe M. Jastesen J. Kisselev L. Nature. 1994; 372: 701-703Crossref PubMed Scopus (374) Google Scholar, 11Stansfield I. Jones K.M. Kushnirov V.V. Dagkesamanskaya A.R. Poznyakovski A.I. Paushkin S.V. Nierras C.R. Cox B.S. Ter-Avanesyan M.D. Tuite M.F. EMBO J. 1995; 14: 4365-4373Crossref PubMed Scopus (426) Google Scholar). The eRF3 genes are conserved from yeast to mammals. The mammalian eRF3 gene, GSPT, was first identified by the ability to complement temperature-sensitive growth arrest phenotype of sup35/gst1-1 mutation in S. cerevisiae, which is defective in G1 to S phase transition (14Hoshino S. Miyazawa H. Enomoto T. Hanaoka F. Kikuchi Y. Kikuchi A. Ui M. EMBO J. 1989; 8: 3807-3814Crossref PubMed Scopus (107) Google Scholar). Later, two subtypes of GSPT genes, GSPT1 and GSPT2, were identified (15Hoshino S. Imai M. Mizutani M. Kikuchi Y. Hanaoka F. Ui M. Katada T. J. Biol. Chem. 1998; 273: 22254-22259Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Structural analyses revealed that eRF3 consists of two domains, the unique N-terminal region (N-domain) and the C-terminal region (C-domain) that contains eEF1A-like GTP-binding motifs. The C-domain of eRF3 is well conserved among several species, and eRF3 associates with eRF1 through the C-domain (15Hoshino S. Imai M. Mizutani M. Kikuchi Y. Hanaoka F. Ui M. Katada T. J. Biol. Chem. 1998; 273: 22254-22259Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 16Ito K. Ebihara K. Nakamura Y. RNA (N. Y.). 1998; 4: 958-972Crossref PubMed Scopus (101) Google Scholar, 17Merkulova T.I. Frolova L.Y. Lazar M. Camonis J. Kisselev L.L. FEBS Lett. 1999; 443: 41-47Crossref PubMed Scopus (125) Google Scholar, 18Ebihara K. Nakamura Y. RNA (N. Y.). 1999; 5: 739-750Crossref PubMed Scopus (75) Google Scholar). Moreover, the C-domain of eRF3 is required and sufficient for the termination reaction (10Zhouravleva G. Frolova L. Le Goff X. Le Guellec R. Inge-Vechtomov S. Kisselev L. Philippe M. EMBO J. 1995; 14: 4065-4072Crossref PubMed Scopus (518) Google Scholar). In addition to translation termination, eRF3 functions in translation termination-coupled events. We previously showed that eRF3 interacts with poly(A)-binding protein (PABP) through its N-domain (19Hoshino S. Imai M. Kobayashi T. Uchida N. Katada T. J. Biol. Chem. 1999; 274: 16677-16680Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). The interaction is evolutionarily conserved from yeast to mammals (19Hoshino S. Imai M. Kobayashi T. Uchida N. Katada T. J. Biol. Chem. 1999; 274: 16677-16680Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 20Kozlov G. Trempe J.F. Khaleghpour K. Kahvejian A. Ekiel I. Gehring K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4409-4413Crossref PubMed Scopus (171) Google Scholar, 21Cosson B. Berkova N. Couturier A. Chabelskaya S. Philippe M. Zhouravleva G. Biol. Cell. 2002; 94: 205-216Crossref PubMed Scopus (50) Google Scholar, 22Cosson B. Couturier A. Chabelskaya S. Kiktev D. Inge-Vechtomov S. Philippe M. Zhouravleva G. Mol. Cell. Biol. 2002; 22: 3301-3315Crossref PubMed Scopus (124) Google Scholar, 23Uchida N. Hoshino S. Imataka H. Sonenberg N. Katada T. J. Biol. Chem. 2002; 277: 50286-50292Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 24Hosoda N. Kobayashi T. Uchida N. Funakoshi Y. Kikuchi Y. Hoshino S. Katada T. J. Biol. Chem. 2003; 278: 38287-38291Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). PABP binds to the 3′-poly(A) tail of mRNA and plays important roles in translation initiation and mRNA decay (25Jacobson A. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 451-480Google Scholar, 26Sachs A.B. Sarnow P. Hentze M.W. Cell. 1997; 89: 831-838Abstract Full Text Full Text PDF PubMed Scopus (588) Google Scholar, 27Schwartz D.C. Parker R. Sonenberg N. Hershey J.W.B. Mathews M.B. Translational Control of Gene Expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000: 807-825Google Scholar). Recently, we have reported in yeast that eRF3 regulates the initiation of normal mRNA decay at poly(A) tail-shortening step through the interaction with PABP in a manner coupled to translation termination (24Hosoda N. Kobayashi T. Uchida N. Funakoshi Y. Kikuchi Y. Hoshino S. Katada T. J. Biol. Chem. 2003; 278: 38287-38291Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). We also showed in mammals that eRF3 forms a complex with the initiation factor eIF4G through the interaction with PABP and contribute to the cap- and poly(A)-dependent translation suggesting that eRF3 mediates efficient recycling of ribosome to stimulate the next translation initiation in a manner coupled to translation termination (23Uchida N. Hoshino S. Imataka H. Sonenberg N. Katada T. J. Biol. Chem. 2002; 277: 50286-50292Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). On the other hand, Upf1, which is a key component of the surveillance complex that recognizes and degrades aberrant mRNAs containing premature termination codons, was identified as a binding partner of eRF3 in yeast and humans (28Czaplinski K. Ruiz-Echevarria M.J. Paushkin S.V. Han X. Weng Y. Perlick H.A. Dietz H.C. Ter-Avanesyan M.D. Peltz S.W. Genes Dev. 1998; 12: 1665-1677Crossref PubMed Scopus (298) Google Scholar). In nonsense-containing mRNA, translation termination is thought to occur by the termination complex eRF1-eRF3 at the premature termination codon. The eRF1-eRF3 associates with Upf1-Upf2-Upf3 to form a "surveillance complex" and triggers rapid mRNA decay via nonsense-mediated mRNA decay (NMD) pathway (28Czaplinski K. Ruiz-Echevarria M.J. Paushkin S.V. Han X. Weng Y. Perlick H.A. Dietz H.C. Ter-Avanesyan M.D. Peltz S.W. Genes Dev. 1998; 12: 1665-1677Crossref PubMed Scopus (298) Google Scholar, 29Wang W. Czaplinski K. Rao Y. Peltz S.W. EMBO J. 2001; 20: 880-890Crossref PubMed Scopus (170) Google Scholar). These findings allowed us to speculate that eRF3 functions as a molecular switch in the process that couples translation termination to mRNA decay and/or re-initiation, where GTP-binding to eRF3 plays regulatory roles. In this study, we present the first evidence that GTP is essential for the association between eRF3 and eRF1 as well as for the termination reaction. Although the binding of eRF3 to Pab1 and Upf1 is independent of the guanine nucleotides and not affected by a mutation in the GTP-binding motifs of eRF3, the mutation causes a defect in both normal and nonsense-mediated mRNA decay. Furthermore, eRF3 plays an indispensable role in the termination-coupled mRNA decay, and the termination reaction itself is not sufficient for triggering the mRNA degradation. These results provide a model for the mechanism whereby translation termination-coupled mRNA decay is regulated by the GTP-binding protein eRF3. Yeast Strains and Growth Conditions—The yeast strains used in this study are listed in Table I. The yeast cells were grown in standard culture media and transformed with DNA by the lithium acetate method. Deletion of the SUP35 gene was performed as described previously (24Hosoda N. Kobayashi T. Uchida N. Funakoshi Y. Kikuchi Y. Hoshino S. Katada T. J. Biol. Chem. 2003; 278: 38287-38291Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Epitope tagging of SUP35, SUP45, PAB1, and UPF1 was performed by the one-step method described by Knop et al. (30Knop M. Siegers K. Pereira G. Zachariae W. Winsor B. Nasmyth K. Schiebel E. Yeast. 1999; 15: 963-972Crossref PubMed Scopus (812) Google Scholar). Transformants were checked by PCRs for the correct integration. All epitope-tagged proteins expressed in this study were fully functional. The sequences of the oligonucleotides used for the epitope tagging are as follows: SUP35, GTA CCA CAA TAG CAA TTG GTA AAA TTG TTA AAA TTG CCG AGC GTA CGC TGC AGG TCG AC and GGT ATT ATT GTG TTT GCA TTT ACT TAT GTT TGC AAG AAA TAT CGA TGA ATT CGA GCT CG; SUP45, GAA TAT TAT GAC GAA GAT GAA GGA TCC GAC TAT GAT TTC ATT CGT ACG CTG CAG GTC GAC and AAT TCT TTT TGA TTC GAT TTT TTT CTC CCC CTT TTA TTT ATA TCG ATG AAT TCG AGC TCG; PAB1, GAG TCT TTC AAA AAG GAG CAA GAA CAA CAA ACT GAG CAA GCT CGT ACG CTG CAG GTC GAC and ATA AGT TTG TTG AGT AGG GAA GTA GGT GAT TAC ATA GAG CAA TCG AGC TCG; and UPF1, CAA AAG CAT GAA TTG TCA AAA GAC TTC AGC AAT TTG GGA ATA CGT ACG CTG CAG GTC GAC and CAA GCC AAG TTT AAC ATT TTA TTT TAA CAG GGT TCA CCG AAA TCG ATG AAT TCG AGC TCG.Table IYeast strains used in this studyNameDerivative and genotypeyTK3MBS pab1-9MycyTK12MBS sup35-9Myc, sup45-proA, pab1-3HAyTK13MBS sup35-9Myc, sup45-proA, upf1-3HAyTK19MBS pEAU-Flag-SUP45yTK41YK21-02 pEAU-Flag-SUP45yTK42YK21-02 pEAU-FlagyTK48YK21-02 pEAU-Flag-SUP35CyTK54YK21-02 YCplac22-Flag-SUP35/WTyTK55YK21-02 YCplac22-Flag-SUP35/N406IyTK58MBS sup45-proA, sup35::CgHIS3, YCplac22-Flag-SUP35/WT, pEAU-Flag-SUP45yTK59MBS sup45-proA, sup35::CgHIS3, YCplac22-Flag-SUP35/WT, pEAU-FlagyTK60MBS sup45-proA, sup35::CgHIS3, YCplac22-Flag-SUP35/Y420S, pEAU-Flag-SUP45yTK61MBS sup45-proA, sup35::CgHIS3, YCplac22-Flag-SUP35/Y420S, pEAU-FlagyTK65MBS pEAU-Flag-PAB1yTK66MBS pURAGAL1-Flag-UPF1 Open table in a new tab Plasmid Construction—pEAU was derived from YEplac195 by insertion of the yeast TRP5 terminator and ADC1 promoter. To obtain pEAU-Flag-SUP45, the PCR fragment of SUP45 was inserted into the region between XbaI and KpnI sites of pEAU. The XbaI site and FLAG epitope sequences were added to the 5′-primer, and the KpnI site was added to the 3′-primer. Inserting the XbaI–BamHI fragment of PAB1 was amplified by PCR into pEAU-Flag to generate pEAU-Flag-Pab1. The XbaI–BglII fragment of pEAU was ligated with the synthetic FLAG adaptor to make pEAU-Flag. For pURAGAL1-Flag-Upf1, the UPF1 fragment amplified by PCR was subcloned into the region between SalI site of pURAGAL1-Flag (31Araki Y. Takahashi S. Kobayashi T. Kajiho H. Hoshino S. Katada T. EMBO J. 2001; 20: 4684-4693Crossref PubMed Scopus (165) Google Scholar). To produce full-length eRF3 and its deletion mutants fused with N terminally GST and C terminally His6 epitope, the EcoRI–XhoI fragment of SUP35 amplified by PCR was inserted into the region between EcoRI and SalI sites of pGPH6 (23Uchida N. Hoshino S. Imataka H. Sonenberg N. Katada T. J. Biol. Chem. 2002; 277: 50286-50292Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). The construction of YCplac22-Flag-SUP35 was carried out as follows. The EcoRI–XhoI fragment of the SUP35 gene including the promoter region was excised from pYK807 (32Kikuchi Y. Shimatake H. Kikuchi A. EMBO J. 1988; 7: 1175-1182Crossref PubMed Scopus (103) Google Scholar) and subcloned into the region between EcoRI and SalI sites of YCplac22. FLAG sequence was inserted into just before the first ATG by a one-day mutagenesis method (33Imai Y. Matsushima Y. Sugimura T. Terada M. Nucleic Acids Res. 1991; 19: 2785Crossref PubMed Scopus (314) Google Scholar). To obtain pGEM-SUP35 N406I or D409N, the BamHI–SalI fragment of the SUP35 gene from pGPH6-SUP35 was inserted into pGEM-T Easy vector (Promega), and N406I and D409N mutants were constructed by the one-day mutagenesis method (33Imai Y. Matsushima Y. Sugimura T. Terada M. Nucleic Acids Res. 1991; 19: 2785Crossref PubMed Scopus (314) Google Scholar). The BamHI–SalI fragment of the N406I or D409N mutant was subcloned into the region between BamHI and SalI sites of pGPH6-SUP35. To generate YCplac22-Flag-SUP35 N406I, the StuI–NcoI fragment of the mutant was inserted into the region between the StuI and NcoI sites of YCplac22-Flag-SUP35. To construct pURAGAL1-CL and CSL, CAT and luciferase genes were amplified with PCR from pCAT-control (Promega) and pGL2 (Promega), respectively. The EcoRI site was added to 5′-primer, and the XhoI site was added to 3′-primer for the CAT gene. The XhoI site was added to 5′-primer, and the SalI site was added to the 3′-primer for luciferase gene. Amplified DNA was subcloned into pURAGAL1. For CSL, a stop codon was inserted into 3′-primer for the CAT gene. Preparation of Yeast Lysate and Immunoprecipitation Assay—Logarithmically growing yeast cells (1 × 109) in standard yeast extract/peptone medium supplemented with glucose and adenine (YPDA) were resuspended in 500 μl of a lysis buffer (50 mm Tris-HCl, pH 7.5, 50 mm NaCl, 5 mm EDTA, 1 mm dithiothreitol, 0.1% Triton X-100, 10% glycerol, and protease inhibitors). The cells were mixed with glass beads (1 g) and disrupted by 12 cycles of vortexing for 30 s followed by incubating on ice for 1 min. After centrifugation at 15,000 × g for 20 min, the clear supernatant was used as the yeast lysate. The lysate was incubated at 4 °C for 30 min with protein G-Sepharose (Amersham Biosciences) and centrifuged at 3000 rpm for 10 s. The supernatant was mixed with protein G-Sepharose and an anti-Myc monoclonal antibody (9E10, Sigma) and further incubated at 4 °C for 2 h. The Sepharose resin was pelleted and washed three times with the ice-cold lysis buffer containing 10 mm MgCl2 instead of EDTA. When necessary, the indicated nucleotides were also added and further incubated at 30 °C for 1 h. After centrifugation, proteins retained in the resin were eluted with an SDS-PAGE sample buffer by boiling for 5 min. The eluted proteins were separated by SDS-PAGE and immunoblotted with anti-Myc (9E10) and anti-HA (12CA5) antibodies. eRF1 conjugated with protein A was detected with an anti-GST polyclonal antibody (Sigma). Production of Recombinant Proteins—Various forms of eRF3 proteins were induced by the addition of 0.1 mm isopropyl-1-thio-β-d-galactopyranoside at 20 °C for 12 h in Escherichia coli JM109 containing the pGH6-SUP35. The cells were resuspended in buffer A consisting of 50 mm Tris-HCl, pH 7.5, 50 mm NaCl, 0.5 mm EDTA, 0.1% Triton X-100, 2% glycerol, and protease inhibitors, and incubated with 1 mg/ml lysozyme at 4 °C for 30 min. The mixture was sonicated for 5 min on ice and centrifuged at 100,000 × g for 60 min. The expressed proteins were purified from the clear supernatant using glutathione-Sepharose 4B (Amersham Biosciences) and/or nickel-nitrilotriacetic acid-agarose (Qiagen) according to the manufacturer's instructions. Flag-eRF1, Flag-Pab1, and Flag-Upf1 were expressed in yeast cells and purified using Anti-FLAG M2-agarose affinity gel (Sigma). In Vitro Binding Assay—GST- and His-tagged eRF3 proteins purified from E. coli were mixed with the yeast lysate and incubated with glutathione-Sepharose 4B at 4 °C for 2 h. The resin was pelleted and washed three times with an ice-cold buffer A containing 0.5 mm dithiothreitol. When necessary, the indicated nucleotides and Mg2+ were added and further incubated at 30 °C for 30 min. After centrifugation and washing, proteins retained in the resin were eluted with the SDS-PAGE sample buffer by boiling for 5 min. The eluted proteins were separated by SDS-PAGE and immunoblotted with anti-GST, anti-FLAG (M2), and anti-Myc (9E10) antibodies. The binding experiments in supplemental Fig. S1 were performed in the presence of 10 μg/ml of RNase A. Read-through Assay—Yeast cells in the selective medium containing 2% galactose (2 ml) were grown at 26 °C to A600 = ∼0.6, and further incubated at 37 °C 2 h. The cells were harvested and resuspended in 100 μl of the lysis buffer lacking Triton X-100. The cells were mixed with glass beads (0.1 g) and disrupted by 12 cycles of vortexing for 30 s followed by incubating on ice for 1 min. After centrifugation at 15,000 × g for 20 min, the 5 μl of clear supernatant was mixed with 45 μl of Bright-Glo luciferase assay regent (Promega), and luciferase activity was measured using a multiplate reader (Wallac). RNA Analysis—RNA isolation and Northern blot hybridization were performed as described previously (24Hosoda N. Kobayashi T. Uchida N. Funakoshi Y. Kikuchi Y. Hoshino S. Katada T. J. Biol. Chem. 2003; 278: 38287-38291Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Radiolabeled probes for PGK1 and CYH2 mRNAs were prepared by random priming. SCR1 RNA was detected by using an oligonucleotide probe (o77), TCT AGC CGC GAG GAA GGA. All experiments were performed at least three times with different samples of the yeast strains, and the results were fully reproducible. Hence, most of the data shown are representative of several independent experiments. Association of eRF3/Sup35 with eRF1/Sup45 Requires GTP—Previous in vitro binding studies have revealed that eRF3 is capable of associating with eRF1 in several species (10Zhouravleva G. Frolova L. Le Goff X. Le Guellec R. Inge-Vechtomov S. Kisselev L. Philippe M. EMBO J. 1995; 14: 4065-4072Crossref PubMed Scopus (518) Google Scholar, 11Stansfield I. Jones K.M. Kushnirov V.V. Dagkesamanskaya A.R. Poznyakovski A.I. Paushkin S.V. Nierras C.R. Cox B.S. Ter-Avanesyan M.D. Tuite M.F. EMBO J. 1995; 14: 4365-4373Crossref PubMed Scopus (426) Google Scholar, 15Hoshino S. Imai M. Mizutani M. Kikuchi Y. Hanaoka F. Ui M. Katada T. J. Biol. Chem. 1998; 273: 22254-22259Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 16Ito K. Ebihara K. Nakamura Y. RNA (N. Y.). 1998; 4: 958-972Crossref PubMed Scopus (101) Google Scholar, 17Merkulova T.I. Frolova L.Y. Lazar M. Camonis J. Kisselev L.L. FEBS Lett. 1999; 443: 41-47Crossref PubMed Scopus (125) Google Scholar, 18Ebihara K. Nakamura Y. RNA (N. Y.). 1999; 5: 739-750Crossref PubMed Scopus (75) Google Scholar, 34Eurwilaichitr L. Graves F.M. Stansfield I. Tuite M.F. Mol. Microbiol. 1999; 32: 485-496Crossref PubMed Scopus (79) Google Scholar). eRF1 structurally mimics the stem of an aminoacyl-tRNA (35Song H. Mugnier P. Das A.K. Webb H.M. Evans D.R. Tuite M.F. Hemmings B.A. Barford D. Cell. 2000; 100: 311-321Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar), whereas eRF3 is a GTP-binding protein related to the translation elongation factor eEF1A (13Frolova L. Le Goff X. Zhouravleva G. Davydova E. Philippe M. Kisselev L. RNA (N. Y.). 1996; 2: 334-341PubMed Google Scholar). eEF1A binds to aminoacyl-tRNA in the GTP-bound form, whereas it dissociates from the RNA in the GDP-bound form (2Merrick W.C. Nyborg J. Sonenberg N. Hershey J.W.B. Mathews M.B. Translational Control of Gene Expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000: 89-125Google Scholar). Moreover, Mg2+ exerts its influence on the conformation of many types of GTP-binding proteins. It is, therefore, very likely that the association between eRF3 and eRF1 is also regulated by GTP and Mg2+. In this study, we first investigated whether such GTP and Mg2+-dependent association is observed in the cell lysate of S. cerevisiae. For the analysis, we constructed a yeast strain (yTK12), in which the chromosomal copies of SUP35 (encoding yeast eRF3) and SUP45 (yeast eRF1) were tagged with epitopes, nine Myc and protein A, respectively. These proteins were physiologically produced under the control of their own promoters to prevent the artificial effect of overproduction. The yeast cells that had been extracted under various conditions were immunoprecipitated with an anti-Myc antibody, and associated eRF1 was detected with an anti-protein A antibody. As shown in Fig. 1A, eRF3 co-immunoprecipitated with eRF1 (lane 1). When a non-hydrolyzable GTP analog, GTPγS, was added to the extraction buffer containing 10 mm Mg2+, there was a marked increase in the immunoprecipitated amount of eRF1 (Fig. 1A, lane 3). In sharp contrast, GDP failed to enhance the association of eRF1 (Fig. 1A, lane 2). Interestingly, a considerable amount of eRF1 was immunoprecipitated even in the absence of GTP (Fig. 1A, lane 4) or the presence of GDP (lane 5) if Mg2+ was excluded from the extraction buffer. Thus, guanine nucleoside triphosphates appeared to be required for the association between eRF3 and eRF1 at the physiological concentrations of Mg2+ but not in the absence of the divalent cation. We further investigated the properties of eRF3-eRF1 association in this assay system. The immunoprecipitated eRF3-eRF1 complex was washed and incubated with various nucleotides in the presence of 10 mm Mg2+ (Fig. 1B). eRF1 still associated with eRF3 after the incubation if the reaction mixture contained GTP or GTPγS. However, other nucleotides, such as ATP, ADP, CTP, UTP, and ITP, failed to protect the eRF1 dissociation (data partly not shown). These results suggest that eRF1 specifically associates with the GTP-bound form of eRF3 but dissociates from the GDP-bound or free form. The effects of various concentrations of guanine nucleotides and Mg2+ were also investigated (Fig. 1C). The half-maximum eRF1 binding to eRF3 required 10–100 μm GTP (Fig. 1C, left), and the concentration is apparently higher than the dissociation constant (Kd) of eEF1A for GTP (36Saha S.K. Chakraburtty K. J. Biol. Chem. 1986; 261: 12599-12603Abstract Full Text PDF PubMed Google Scholar). Moreover, GTPγS-supported association between eRF3 and eRF1 was maximally observed at physiological concentrations (0.1–1 mm) of Mg2+ (Fig. 1C, right). A GTP-binding Motif on the C-domain of eRF3/Sup35 Is Responsible for the Association to eRF1/Sup45—To confirm that GTP binding to eRF3 is required for the eRF1 association, we produced several eRF3 mutants that had been tagged with GST/His in E. coli. The mutant proteins were purified, mixed with extract from a yeast expressing FLAG-tagged eRF1, and subjected to pull-down assay with glutathione-Sepharose resin. In accordance with previous studies (15Hoshino S. Imai M. Mizutani M. Kikuchi Y. Hanaoka F. Ui M. Katada T. J. Biol. Chem. 1998; 273: 22254-22259Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 16Ito K. Ebihara K. Nakamura Y. RNA (N. Y.). 1998; 4: 958-972Crossref PubMed Scopus (101) Google Scholar, 17Merkulova T.I. Frolova L.Y. Lazar M. Camonis J. Kisselev L.L. FEBS Lett. 1999; 443: 41-47Crossref PubMed Scopus (125) Google Scholar, 18Ebihara K. Nakamura Y. RNA (N. Y.). 1999; 5: 739-750Crossref PubMed Scopus (75) Google Scholar), the C-domain (the amino acid sequence of 254–685) of eRF3 but not its N-domain (1–253) associated with eRF1 (Fig. 2A). The GTP-dependent association between eRF3 and eRF1 was also observed in this assay system (Fig. 2B). eRF3 mutants, of which a GTP-binding motif (NKXD) on the C-domain was replaced by Ile (N406I) or Asn (D409N), were subjected to the in vitro binding assay. The mutations in this motif of various GTP-binding proteins are demonstrated to result in reduced affinity for GTP (37Walter M. Clark S.G. Levinson A.D. Science. 1986; 233: 649-652Crossref PubMed Scopus (85) Google Scholar, 38Hwang Y.W. Miller D.L. J. Biol. Chem. 1987; 262: 13081-13085Abstract Full Text PDF PubMed Google Scholar). As shown in Fig. 2C, eRF1 association was marke