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Translation Initiation Factor eIF4G-1 Binds to eIF3 through the eIF3e Subunit

2006; Elsevier BV; Volume: 281; Issue: 32 Linguagem: Inglês

10.1074/jbc.m605418200

ISSN

1083-351X

Autores

Aaron K. LeFebvre, Nadejda L. Korneeva, Marjan Trutschl, Urška Cvek, Roy Duzan, C.A. Bradley, John W.B. Hershey, Robert E. Rhoads,

Tópico(s)

PI3K/AKT/mTOR signaling in cancer

Resumo

eIF3 in mammals is the largest translation initiation factor (∼800 kDa) and is composed of 13 nonidentical subunits designated eIF3a-m. The role of mammalian eIF3 in assembly of the 48 S complex occurs through high affinity binding to eIF4G. Interactions of eIF4G with eIF4E, eIF4A, eIF3, poly(A)-binding protein, and Mnk1/2 have been mapped to discrete domains on eIF4G, and conversely, the eIF4G-binding sites on all but one of these ligands have been determined. The only eIF4G ligand for which this has not been determined is eIF3. In this study, we have sought to identify the mammalian eIF3 subunit(s) that directly interact(s) with eIF4G. Established procedures for detecting protein-protein interactions gave ambiguous results. However, binding of partially proteolyzed HeLa eIF3 to the eIF3-binding domain of human eIF4G-1, followed by high throughput analysis of mass spectrometric data with a novel peptide matching algorithm, identified a single subunit, eIF3e (p48/Int-6). In addition, recombinant FLAG-eIF3e specifically competed with HeLa eIF3 for binding to eIF4G in vitro. Adding FLAG-eIF3e to a cell-free translation system (i) inhibited protein synthesis, (ii) caused a shift of mRNA from heavy to light polysomes, (iii) inhibited cap-dependent translation more severely than translation dependent on the HCV or CSFV internal ribosome entry sites, which do not require eIF4G, and (iv) caused a dramatic loss of eIF4G and eIF2α from complexes sedimenting at ∼40 S. These data suggest a specific, direct, and functional interaction of eIF3e with eIF4G during the process of cap-dependent translation initiation, although they do not rule out participation of other eIF3 subunits. eIF3 in mammals is the largest translation initiation factor (∼800 kDa) and is composed of 13 nonidentical subunits designated eIF3a-m. The role of mammalian eIF3 in assembly of the 48 S complex occurs through high affinity binding to eIF4G. Interactions of eIF4G with eIF4E, eIF4A, eIF3, poly(A)-binding protein, and Mnk1/2 have been mapped to discrete domains on eIF4G, and conversely, the eIF4G-binding sites on all but one of these ligands have been determined. The only eIF4G ligand for which this has not been determined is eIF3. In this study, we have sought to identify the mammalian eIF3 subunit(s) that directly interact(s) with eIF4G. Established procedures for detecting protein-protein interactions gave ambiguous results. However, binding of partially proteolyzed HeLa eIF3 to the eIF3-binding domain of human eIF4G-1, followed by high throughput analysis of mass spectrometric data with a novel peptide matching algorithm, identified a single subunit, eIF3e (p48/Int-6). In addition, recombinant FLAG-eIF3e specifically competed with HeLa eIF3 for binding to eIF4G in vitro. Adding FLAG-eIF3e to a cell-free translation system (i) inhibited protein synthesis, (ii) caused a shift of mRNA from heavy to light polysomes, (iii) inhibited cap-dependent translation more severely than translation dependent on the HCV or CSFV internal ribosome entry sites, which do not require eIF4G, and (iv) caused a dramatic loss of eIF4G and eIF2α from complexes sedimenting at ∼40 S. These data suggest a specific, direct, and functional interaction of eIF3e with eIF4G during the process of cap-dependent translation initiation, although they do not rule out participation of other eIF3 subunits. Eukaryotic translation initiation involves numerous initiation factors (eIFs) 2The abbreviations used are: eIF, eukaryotic initiation factor; BSA, bovine serum albumin; CSFV, classical swine fever virus; HCV, hepatitis C virus; IRES, internal ribosome entry site; MALDI-TOF-MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; Mnk, mitogen-activated protein kinase-interacting serine/threonine kinase; PABP, poly(A)-binding protein; RRL, rabbit reticulocyte lysate; DTT, dithiothreitol; SASD, sulfosuccinimidyl-2-[p-azidosalicylamido]ethyl-1,3′-dithiopropionate; MMTV, mouse mammary tumor virus. that participate in recruitment of initiator tRNA and mRNA to the 40 S ribosomal subunit, recognition of the initiator AUG codon, and joining of the 40 S and 60 S ribosomal subunits, culminating in formation of the first peptide bond (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). The factors required for recruitment of mRNA include eIF3, eIF4A, eIF4B, eIF4E, eIF4G, eIF4H, and PABP. eIF4E and PABP bind the 5′ cap and 3′ poly(A) tract of mRNA, respectively, whereas eIF4A unwinds 5′-terminal secondary structure in an ATP-dependent process that also involves the RNA-binding proteins eIF4B and eIF4H. eIF4G forms specific complexes with eIF4E, eIF4A, and PABP, thereby linking the processes of cap recognition, poly(A) binding, and secondary structure melting. eIF4G in turn is recruited to the 40 S ribosomal subunit via binding to the multisubunit complex eIF3. eIF3 in mammals is the largest initiation factor (∼800 kDa) and includes 13 nonidentical polypeptides designated eIF3a-m 3A unified nomenclature system for eIF3 subunits assigns the same letter to homologous subunits across all eukaryotic organisms (2Browning K.S. Gallie D.R. Hershey J.W. Hinnebusch A.G. Maitra U. Merrick W.C. Norbury C. Trends. Biochem. Sci. 2001; 26: 284Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The subunits are named in order of highest to lowest molecular mass (e.g., eIF3a is 170 kDa, eIF3b is 116 kDa, etc.), although subunits discovered after establishment of this nomenclature system do not follow this rule (e.g. eIF3l is 69 kDa (3Morris-Desbois C. Rety S. Ferro M. Garin J. Jalinot P. J. Biol. Chem. 2001; 276: 45988-45995Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) and eIF3m is 42 kDa (64Unbehaun A. Borukhov S.I. Hellen C.U. Pestova T.V. Genes Dev. 2004; 18: 3078-3093Crossref PubMed Scopus (176) Google Scholar)). (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, 2Browning K.S. Gallie D.R. Hershey J.W. Hinnebusch A.G. Maitra U. Merrick W.C. Norbury C. Trends. Biochem. Sci. 2001; 26: 284Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 3Morris-Desbois C. Rety S. Ferro M. Garin J. Jalinot P. J. Biol. Chem. 2001; 276: 45988-45995Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 4Zhou C. Arslan F. Wee S. Krishnan S. Ivanov A.R. Oliva A. Leatherwood J. Wolf D.A. BMC Biol. 2005; 3: 14Crossref PubMed Scopus (111) Google Scholar). In contrast, eIF3 in Saccharomyces cerevisiae contains only six subunits (eIF3a, -b, -c, -g, -i, and -j) (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, 5Hinnebusch A.G. Sonenberg N. Hershey J.W.B. Mathews M.B. Translational Control of Gene Expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000: 185-244Google Scholar). Five of these (eIF3a, -b, -c, -g, and -i) are conserved in all eukaryotes and are considered to be the "conserved core" of eIF3 (2Browning K.S. Gallie D.R. Hershey J.W. Hinnebusch A.G. Maitra U. Merrick W.C. Norbury C. Trends. Biochem. Sci. 2001; 26: 284Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 6Phan L. Zhang X. Asano K. Anderson J. Vornlocher H.-P. Greenberg J.R. Qin J. Hinnebusch A.G. Mol. Cell. Biol. 1998; 18: 4935-4946Crossref PubMed Scopus (158) Google Scholar). Most of the eight mammalian "non-core" subunits are found in other higher eukaryotes, Schizosaccharomyces pombe lacking only eIF3j, -k, and -l; Triticum aesitivum lacking eIF3j, -l, and -m; Arabidopsis thaliana lacking eIF3j and -m; and Drosophila melanogaster and Caenorhabditis elegans lacking eIF3m (4Zhou C. Arslan F. Wee S. Krishnan S. Ivanov A.R. Oliva A. Leatherwood J. Wolf D.A. BMC Biol. 2005; 3: 14Crossref PubMed Scopus (111) Google Scholar, 7Rhoads R.E. Dinkova T.D. Korneeva N.L. WormBook, The C. elegans Research Community, Wormbook. 2005; doi/10.1895/wormbook.1.7.1Google Scholar, 8Burks E.A. Bezerra P.P. Le H. Gallie D.R. Browning K.S. J. Biol. Chem. 2001; 276: 2122-2131Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 9Asano K. Vornlocher H.P. Richter-Cook N.J. Merrick W.C. Hinnebusch A.G. Hershey J.W. J. Biol. Chem. 1997; 272: 27042-27052Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). In addition to subunit composition, subunit-subunit interactions have been extensively characterized for both mammalian (10Fraser C.S. Lee J.Y. Mayeur G.L. Bushell M. Doudna J.A. Hershey J.W. J. Biol. Chem. 2004; 279: 8946-8956Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 11Mayeur G.L. Fraser C.S. Peiretti F. Block K.L. Hershey J.W. Eur. J. Biochem. 2003; 270: 4133-4139Crossref PubMed Scopus (49) Google Scholar, 12Shalev A. Valasek L. Pise-Masison C.A. Radonovich M. Phan L. Clayton J. He H. Brady J.N. Hinnebusch A.G. Asano K. J. Biol. Chem. 2001; 276: 34948-34957Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) and S. cerevisiae eIF3 (13Nielsen K.H. Valasek L. Sykes C. Jivotovskaya A. Hinnebusch A.G. Mol. Cell. Biol. 2006; 26: 2984-2998Crossref PubMed Scopus (52) Google Scholar). Although the subunit compositions of S. cerevisiae and mammalian eIF3 are different, the functions of the core subunits appear to be the same, as suggested by the observation that the S. cerevisiae conserved core can substitute for mammalian eIF3 in an assay for Met-puromycin synthesis containing all other eIFs of mammalian origin (6Phan L. Zhang X. Asano K. Anderson J. Vornlocher H.-P. Greenberg J.R. Qin J. Hinnebusch A.G. Mol. Cell. Biol. 1998; 18: 4935-4946Crossref PubMed Scopus (158) Google Scholar). eIF3 is required for both 43 S and 48 S initiation complex formation and is also involved in dissociation of 80 S complexes and anti-association of 40 S and 60 S subunits (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). The 43 S complex consists of eIF3, eIF2·GTP·Met-tRNAi, eIF5, eIF1, eIF1A, and the 40 S ribosomal subunit (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). In S. cerevisiae, a multifactor complex has been identified consisting of eIF3, eIF2·GTP·Met-tRNAi, eIF5, and eIF1 (14Asano K. Clayton J. Shalev A. Hinnebusch A.G. Genes Dev. 2000; 14: 2534-2546Crossref PubMed Scopus (229) Google Scholar). eIF3 stabilizes this complex through direct interactions with eIF1, eIF2, eIF5, and the 40 S ribosomal subunit (15Valasek L. Nielsen K.H. Zhang F. Fekete C.A. Hinnebusch A.G. Mol. Cell. Biol. 2004; 24: 9437-9455Crossref PubMed Scopus (128) Google Scholar). Most of these interactions have been mapped to individual eIF3 subunits. For example, eIF3a and eIF3c bind to eIF1 (14Asano K. Clayton J. Shalev A. Hinnebusch A.G. Genes Dev. 2000; 14: 2534-2546Crossref PubMed Scopus (229) Google Scholar, 16Valasek L. Nielsen K.H. Hinnebusch A.G. EMBO J. 2002; 21: 5886-5898Crossref PubMed Scopus (113) Google Scholar), eIF3c binds to eIF5 (14Asano K. Clayton J. Shalev A. Hinnebusch A.G. Genes Dev. 2000; 14: 2534-2546Crossref PubMed Scopus (229) Google Scholar), and eIF3a binds to eIF2β (16Valasek L. Nielsen K.H. Hinnebusch A.G. EMBO J. 2002; 21: 5886-5898Crossref PubMed Scopus (113) Google Scholar). eIF3 also binds eIF1A (17Olsen D.S. Savner E.M. Mathew A. Zhang F. Krishnamoorthy T. Phan L. Hinnebusch A.G. EMBO J. 2003; 22: 193-204Crossref PubMed Scopus (110) Google Scholar), although the subunit that mediates this interaction has not been reported. In mammals, eIF3 binds eIF1 (through eIF3c) (18Fletcher C.M. Pestova T.V. Hellen C.U. Wagner G. EMBO J. 1999; 18: 2631-2637Crossref PubMed Scopus (109) Google Scholar) and eIF5 (subunit unknown) (19Bandyopadhyay A. Maitra U. Nucleic Acids Res. 1999; 27: 1331-1337Crossref PubMed Scopus (31) Google Scholar). eIF3 contacts the 40 S ribosomal subunit through eIF3b and -j in mammals (10Fraser C.S. Lee J.Y. Mayeur G.L. Bushell M. Doudna J.A. Hershey J.W. J. Biol. Chem. 2004; 279: 8946-8956Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) and S. cerevisiae (13Nielsen K.H. Valasek L. Sykes C. Jivotovskaya A. Hinnebusch A.G. Mol. Cell. Biol. 2006; 26: 2984-2998Crossref PubMed Scopus (52) Google Scholar). Finally, eIF3a in mammals (20Methot N. Song M.S. Sonenberg N. Mol. Cell. Biol. 1996; 16: 5328-5334Crossref PubMed Scopus (157) Google Scholar) and eIF3g in plants (21Park H.S. Browning K.S. Hohn T. Ryabova L.A. EMBO J. 2004; 23: 1381-1391Crossref PubMed Scopus (39) Google Scholar) and S. cerevisiae (22Vornlocher H.P. Hanachi P. Ribeiro S. Hershey J.W. J. Biol. Chem. 1999; 274: 16802-16812Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) interacts with eIF4B. Mammalian eIF3 participates in assembly of the 48 S complex through high affinity binding to eIF4G (23Korneeva N.L. Lamphear B.J. Hennigan F.L.C. Rhoads R.E. J. Biol. Chem. 2000; 275: 41369-41376Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). This may not be the case for S. cerevisiae, where a requirement for eIF4G in mRNA recruitment to the 43 S complex has not been demonstrated (24Jivotovskaya A.V. Valasek L. Hinnebusch A.G. Nielsen K.H. Mol. Cell. Biol. 2006; 26: 1355-1372Crossref PubMed Scopus (100) Google Scholar), correlating with the lack of an observed eIF3-eIF4G interaction in this organism. Binding of eIF4G to eIF3 in mammals is regulated by insulin via association of mTOR with eIF3, specifically eIF3f (25Holz M.K. Ballif B.A. Gygi S.P. Blenis J. Cell. 2005; 123: 569-580Abstract Full Text Full Text PDF PubMed Scopus (921) Google Scholar, 26Harris T.E. Chi A. Shabanowitz J. Hunt D.F. Rhoads R.E. Lawrence Jr., J.C. EMBO J. 2006; 25: 1659-1668Crossref PubMed Scopus (101) Google Scholar). Interactions with eIF3, eIF4E, eIF4A, PABP, and Mnk1/2 have been mapped to separate sites on eIF4G (27Lamphear B.J. Kirchweger R. Skern T. Rhoads R.E. J. Biol. Chem. 1995; 270: 21975-21983Abstract Full Text Full Text PDF PubMed Scopus (473) Google Scholar, 28Tarun S.Z. Sachs A.B. EMBO J. 1996; 15: 7168-7177Crossref PubMed Scopus (584) Google Scholar, 29Imataka H. Gradi A. Sonenberg N. EMBO J. 1998; 17: 7480-7489Crossref PubMed Scopus (473) Google Scholar, 30Pyronnet S. Imataka H. Gingras A.-C. Fukunaga R. Hunter T. Sonenberg N. EMBO J. 1999; 18: 270-279Crossref PubMed Scopus (538) Google Scholar, 31Imataka H. Sonenberg N. Mol. Cell. Biol. 1997; 17: 6940-6947Crossref PubMed Scopus (241) Google Scholar, 32Waskiewicz A.J. Johnson J.C. Penn B. Mahalingam M. Kimball S.R. Cooper J.A. Mol. Cell. Biol. 1999; 19: 1871-1880Crossref PubMed Scopus (404) Google Scholar). The minimum portion of eIF4G that retains full affinity for eIF3 is located between amino acids 1015 and 1118 (23Korneeva N.L. Lamphear B.J. Hennigan F.L.C. Rhoads R.E. J. Biol. Chem. 2000; 275: 41369-41376Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). The eIF4G-binding sites on all but one of these ligands have also been determined, viz. eIF4E (30Pyronnet S. Imataka H. Gingras A.-C. Fukunaga R. Hunter T. Sonenberg N. EMBO J. 1999; 18: 270-279Crossref PubMed Scopus (538) Google Scholar, 33Marcotrigiano J. Gingras A.-C. Sonenberg N. Burley S.K. Mol. Cell. 1999; 3: 707-716Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar, 34Ptushkina M. von der Haar T. Karimm M.M. Hughers J.M. McCarthy J.E.G. EMBO J. 1999; 18: 4068-4075Crossref PubMed Scopus (106) Google Scholar), eIF4A (35Korneeva N.L. First E.A. Benoit C.A. Rhoads R.E. J. Biol. Chem. 2005; 280: 1872-1881Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), Mnk1 (32Waskiewicz A.J. Johnson J.C. Penn B. Mahalingam M. Kimball S.R. Cooper J.A. Mol. Cell. Biol. 1999; 19: 1871-1880Crossref PubMed Scopus (404) Google Scholar), Mnk2 (36Scheper G.C. Parra J.L. Wilson M. Van Kollenburg B. Vertegaal A.C. Han Z.G. Proud C.G. Mol. Cell. Biol. 2003; 23: 5692-5705Crossref PubMed Scopus (90) Google Scholar), and PABP (29Imataka H. Gradi A. Sonenberg N. EMBO J. 1998; 17: 7480-7489Crossref PubMed Scopus (473) Google Scholar, 37Kessler S.H. Sachs A.B. Mol. Cell. Biol. 1998; 18: 51-57Crossref PubMed Scopus (127) Google Scholar, 38Otero L.J. Ashe M.P. Sachs A.B. EMBO J. 1999; 18: 3153-3163Crossref PubMed Scopus (110) Google Scholar). The only eIF4G ligand for which this has not been determined is eIF3. However, the recently solved cryo-EM structure of an eIF4G·eIF3·40 S complex suggests a small surface contact between eIF4G and eIF3 (39Siridechadilok B. Fraser C.S. Hall R.J. Doudna J.A. Nogales E. Science. 2005; 310: 1513-1515Crossref PubMed Scopus (226) Google Scholar), likely composed of one or a few eIF3 subunits. The interaction between eIF3 and eIF4G is critical because it is essential for bringing mRNA and all of the proteins bound to eIF4G to the 43 S initiation complex. In this study, we have sought to identify the mammalian eIF3 subunit(s) that directly interact with eIF4G. Binding of peptides from partially proteolyzed eIF3 suggests one subunit, eIF3e, is more involved than the other 12. Adding FLAG-tagged eIF3e to a cell-free translation system decreases the overall rate of protein synthesis and polysome accumulation. FLAG-eIF3e inhibits eIF4G-dependent translation significantly more than eIF4G-independent (IRES-driven) translation. Finally, FLAG-eIF3e causes a dramatic reduction in the amount of eIF4G and eIF2 associated with complexes sedimenting at ∼40 S. These functional activities would be expected for an eIF3 subunit involved in eIF4G binding. Materials—An ECL+ Western blotting development kit and Low Range Rainbow™ molecular weight markers were obtained from Amersham Biosciences. Sf9 cells and Sf-900 II SFM media were obtained from Invitrogen. [35S]Met was obtained from MP Biomedicals (Irvine, CA). S-protein-agarose and S-protein-coupled alkaline phosphatase was purchased from Novagen (Madison, WI). Beetle luciferin, mass spectrometry grade trypsin gold, RQ1 RNase-free DNase, and all DNA restriction enzymes were purchased from Promega. Ni2+-nitrilotriacetic acid-agarose was obtained from Qiagen (Chatsworth, CA). RNase A, EZView Red anti-FLAG M2 affinity gel, mouse monoclonal anti-FLAG M2 antibody, and FLAG peptide were from Sigma. pXL.HCV(40-373).NS′ and pXL.CSFV(1-442).NS′ were described previously (40Reynolds J.E. Kaminski A. Kettinen H.J. Grace K. Clarke B.E. Carroll A.R. Rowlands D.J. Jackson R.J. EMBO J. 1995; 14: 6010-6020Crossref PubMed Scopus (309) Google Scholar, 41Pestova T.V. Shatsky I.N. Fletcher S.P. Jackson R.J. Hellen C.U. Genes Dev. 1998; 12: 67-83Crossref PubMed Scopus (631) Google Scholar) and were kindly provided by Tatyana Pestova (State University of New York, Brooklyn). Protein Expression and Purification—The plasmids encoding recombinant His6- and S-peptide-tagged eIF4G(1015-1118), 4There are both multiple genes for eIF4G (47Yan R. Rychlik W. Etchison D. Rhoads R.E. J. Biol. Chem. 1992; 267: 23226-23231Abstract Full Text PDF PubMed Google Scholar, 86Gradi A. Imataka H. Svitkin Y.V. Rom E. Raught B. Morino S. Sonenberg N. Mol. Cell. Biol. 1998; 18: 334-342Crossref PubMed Scopus (249) Google Scholar) and multiple protein isoforms encoded by a single gene (87Byrd M.P. Zamora M. Lloyd R.E. Mol. Cell. Biol. 2002; 22: 4499-4511Crossref PubMed Scopus (72) Google Scholar, 88Bradley C.A. Padovan J.C. Thompson T.L. Benoit C.A. Chait B.T. Rhoads R.E. J. Biol. Chem. 2002; 277: 12559-12571Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The recombinant proteins used in this work are all derived from human eIF4G-1, and the amino acid numbers all refer to the longest form, eIF4G-1f (1600 amino acids) (88Bradley C.A. Padovan J.C. Thompson T.L. Benoit C.A. Chait B.T. Rhoads R.E. J. Biol. Chem. 2002; 277: 12559-12571Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The nomenclature system recommended by an ad hoc committee appointed by the IUBMB is used for eIF4G (89Clark B.F.C. Grunberg-Manago M. Gupta N.K. Hershey J.W.B. Hinnebusch A.G. Jackson R.J. Maitra U. Mathews M.B. Merrick W.C. Rhoads R.E. Sonenberg N. Spremulli L.L. Trachsel H. Voorma H.O. Biochimie (Paris). 1996; 78: 1119-1122Crossref PubMed Scopus (41) Google Scholar). Forms of eIF4G-1 containing amino acids 653-1118, 1015-1118, and 1118-1600 plus N-terminal His6 and S-peptide tags are referred to as S-eIF4G(653-1118), S-eIF4G(1015-1118), and S-eIF4G(1118-1600), respectively. eIF4G(653-1118), and eIF4G(1118-1600) and purification of S-eIF4G(653-1118), and S-eIF4G(1118-1600) have been described previously (42Korneeva N.L. Lamphear B.J. Hennigan F.L.C. Merrick W.C. Rhoads R.E. J. Biol. Chem. 2001; 276: 2872-2879Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). S-eIF4G(1015-1118) was expressed in Escherichia coli strain BL21(DE3)pLysS (Novagen) and purified by Ni2+-nitrilotriacetic acid-agarose chromatography. One-liter cultures were grown at 37 °C to A600 0.4-0.6, and expression was induced with 1 mm isopropyl β-d-thiogalactoside for 3.5 h. Cell pellets were harvested and stored at −80 °C. Thawed cell pellets were resuspended in lysis buffer containing Complete Mini EDTA-free Protease Inhibitor tablets (Roche Applied Science) and disrupted by sonication. Cleared extracts were incubated in batch with Ni2+-nitrilotriacetic acid-agarose (1 ml packed) with rotation for 2 h at 4°C. Bound material was washed five times batchwise with 20 ml of Buffer A (20 mm Tris-HCl, 200 mm KCl, 10% glycerol, 20 mm imidazole, pH 7.6) and twice with 20 ml of Buffer B (same as Buffer A except 500 mm KCl), after which the resin was equilibrated in 10 ml of Buffer C (same as Buffer A except 100 mm KCl) and loaded into a gravity flow column for elution. S-eIF4G(1015-1118) was eluted with 5 ml of Buffer D (same as Buffer C except 300 mm imidazole), and 1-ml fractions were collected. Fractions containing purified S-eIF4G(1015-1118) were passed over Econo-Pac© 10 DG columns (Bio-Rad) to exchange Buffer D with Buffer E (20 mm HEPES-KOH, 100 mm KCl, 10% glycerol, 1 mm EDTA, 2 mm DTT, pH 7.4). Recombinant baculoviruses for expression of FLAG-tagged eIF3 subunits, inserted into the FLAG-FastBac donor vector, were prepared as described previously (10Fraser C.S. Lee J.Y. Mayeur G.L. Bushell M. Doudna J.A. Hershey J.W. J. Biol. Chem. 2004; 279: 8946-8956Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 11Mayeur G.L. Fraser C.S. Peiretti F. Block K.L. Hershey J.W. Eur. J. Biochem. 2003; 270: 4133-4139Crossref PubMed Scopus (49) Google Scholar). Sf9 cells from frozen stocks were grown in 250-ml sterile flasks at 27 °C and 135 rpm in Sf-900 II SFM media in a New Brunswick Scientific Innova® 44 orbital shaker. For expression of recombinant proteins, cells were expanded to ∼2 × 106 cells/ml in 50 ml of Sf-900 II SFM media prior to infection with recombinant baculoviruses expressing FLAG-eIF3e, FLAG-eIF3i, or FLAG-eIF3j. Cells were harvested after 72 h by centrifugation at 3000 rpm for 5 min followed by a single wash with 1× phosphate-buffered saline. A single cell pellet from each 50-ml culture was stored at −80 °C. Each thawed cell pellet was incubated on ice for 15 min, with occasional mixing, in 25 ml of Buffer F (20 mm Tris-HCl, 120 mm KCl, 10% glycerol, 5 mm β-mercaptoethanol, 1% Triton X-100, pH 7.5) containing one Complete Mini EDTA-free Protease Inhibitor tablet. Uncleared lysates for two pellets representing the same eIF3 subunit were then mixed and centrifuged at 10,000 × g for 20 min. Cleared lysates were incubated batchwise with EZView Red Anti-FLAG M2 affinity gel (100 μl packed), equilibrated in Buffer F, for 2 h at 4°C. The resin was washed three times in batch with 1 ml of Buffer F. FLAG-tagged proteins were eluted with three sequential 100-μl aliquots of Buffer G (20 mm Tris-HCl, 70 mm KCl, 10% glycerol, 1 mm DTT, 2 mm magnesium acetate, 200 μg/ml FLAG peptide, pH 7.5) at 4 °C for 45 min each. Recombinant protein purity was assessed by SDS-PAGE followed by Coomassie Blue staining and anti-FLAG Western blotting. eIF3 from RRL (23Korneeva N.L. Lamphear B.J. Hennigan F.L.C. Rhoads R.E. J. Biol. Chem. 2000; 275: 41369-41376Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar) and HeLa cells (10Fraser C.S. Lee J.Y. Mayeur G.L. Bushell M. Doudna J.A. Hershey J.W. J. Biol. Chem. 2004; 279: 8946-8956Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) was purified as described previously. The concentrations of purified proteins were determined with the protein assay kit (Bio-Rad) with BSA as standard. In Vitro Transcription—Unlabeled capped mRNAs were synthesized in vitro as described previously (43Jemielity J. Fowler T. Zuberek J. Stepinski J. Lewdorowicz M. Niedzwiecka A. Stolarski R. Darzynkiewicz E. Rhoads R.E. RNA (N. Y.). 2003; 9: 1108-1122Crossref PubMed Scopus (213) Google Scholar) except that the final volume of reactions was 100 μl; 1.5 μg of template DNA was used; and the final concentration of m7GpppG was 1.0 mm. The templates used were EcoRI-linearized pXL.HCV(40-373).NS′, EcoRI-linearized pXL.CSFV(1-442).NS′, HpaI-linearized pluc-A+ (43Jemielity J. Fowler T. Zuberek J. Stepinski J. Lewdorowicz M. Niedzwiecka A. Stolarski R. Darzynkiewicz E. Rhoads R.E. RNA (N. Y.). 2003; 9: 1108-1122Crossref PubMed Scopus (213) Google Scholar), and HpaI-linearized pluc-A60 (44Grudzien E. Kalek M. Jemielity J. Darzynkiewicz E. Rhoads R.E. J. Biol. Chem. 2006; 281: 1857-1867Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). The mRNAs generated from these four templates are referred to as m7G-CycB2-HCV-NS′, m7G-CycB2-CSFV-NS′, m7G-Luc-A31, and m7G-Luc-A60, respectively. 32P-Labeled nucleotides were included when the pluc-A60 template was used to make 32P-labeled mRNA for measurement of polysomal distribution. Following incubation for 45 min at 37 °C, fresh T7 RNA polymerase was added, and the reaction was continued for an additional 45 min at 37 °C. DNA was digested by addition of 3.0 units of RQ1 RNase-Free DNase followed by incubation for 10 min at 65 °C. mRNA was then purified using an RNeasy mini kit (Qiagen). mRNA concentration and purity were assessed by absorbance at 260 and 280 nm. In Vitro Translation—All in vitro translation reactions were performed at 30 °C with a micrococcal nuclease-treated RRL system (45Cai A. Jankowska-Anyszka M. Centers A. Chlebicka L. Stepinski J. Stolarski R. Darzynkiewicz E. Rhoads R.E. Biochemistry. 1999; 38: 8538-8547Crossref PubMed Scopus (115) Google Scholar). For measurement of translational inhibition utilizing the luciferase assay, m7G-Luc-A60 and nonradioactive amino acids were used. For measurement of translational inhibition utilizing m7G-CycB2-HCV-NS′ or m7G-CycB2-CSFV-NS′ bicistronic mRNAs, nonradioactive Met was omitted and [35S]Met was included at 0.5 μCi/μl. For time course measurements, reactions contained m7G-Luc-A31 and [35S]Met at 0.2 μCi/μl. When inhibition of protein synthesis was measured, FLAG-tagged eIF3 subunits and nonradiolabeled mRNA were added simultaneously. When polysomal distribution of mRNA was determined, reaction mixtures containing nonradiolabeled amino acids were preincubated with FLAG-tagged eIF3 subunits for 15 min at 30 °C prior to addition of 32P-labeled m7G-Luc-A60. In all reaction mixtures, the volume of added Buffer G (except lacking FLAG peptide) was kept constant. For measurement of luciferase synthesis in the presence of FLAG-eIF3e, luciferase assays were performed using beetle luciferin following the manufacturer's protocol (Promega). For inhibition of bicistronic mRNA translation, 2 μl of each reaction were analyzed by SDS-PAGE on 15% gels followed by Coomassie Blue staining. Newly synthesized protein was detected by exposure of each dried gel to a PhosphorScreen for 2 h and scanning with a STORM 860 PhosphorImager (Amersham Biosciences). Cyclin B2 (∼50 kDa) and NS′ (∼20 kDa) were quantitated with ImageQuant software, version 5.0 (Amersham Biosciences). Curves were fit, as if for competitive inhibition of an enzymatic reaction, with Kaleidagraph software (version 3.5; Synergy Software, Reading, PA) using the equation y = m1/(1 + M0/m2), where y = synthesis of cyclin B2, NS′, or luciferase in the presence of a FLAG-tagged eIF3 subunit; m1 = cyclin B2, NS′, or luciferase synthesized in the absence of the eIF3 subunit; M0 = concentration of the eIF3 subunit in μm, and m2 = the inhibition constant (KI). For time course measurements of inhibition of m7G-Luc-A31 mRNA translation, 10-μl aliquots of each reaction were removed at the indicated times and diluted 1:10 in 10 μg/ml BSA, 10 mm Met, and 10 μm cycloheximide on ice. Aliquots of 5 μl were spotted on Whatman 540 filter disks, and total trichloroacetic acid-precipitatable 35S radioactivity was measured by scintillation spectrometry. Ultracentrifugal Analysis of Initiation Complexes and Polysomes—For polysomal distribution of radiolabeled mRNA, 100-μl translation reaction mixtures were layered immediately after incubation on a 10-ml 10-35% linear sucrose density gradient in Buffer K (50 mm Tris-HCl, 50 mm KCl, 10 mm MgCl2,1 mm DTT, 50 μg/ml cycloheximide, pH 7.5) and centrifuged at 38,000 rpm in a Beckman SW41Ti rotor at 4 °C for 3 h. Fractions of 250 μl were collected using an ISCO gradient fractionator with continuous monitoring at 260 nm. The presence of 32P-labeled mRNA in each frac

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