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• W1994421775 abstract "Eukaryotic initiation factor 1 (eIF1) is a low molecular weight factor critical for stringent AUG selection in eukaryotic translation. It is recruited to the 43 S complex in the multifactor complex (MFC) with eIF2, eIF3, and eIF5 via multiple interactions with the MFC constituents. Here we show that FLAG epitope tagging of eIF1 at either terminus abolishes its in vitro interactions with eIF5 and eIF2β but not that with eIF3c. Nevertheless, both forms of FLAG-eIF1 fail to bind eIF3 and are incorporated into the 43 S complex inefficiently in vivo. C-terminal FLAG tagging of eIF1 is lethal; overexpression of C-terminal FLAG-eIF1 severely impedes 43 S complex formation and derepresses GCN4 translation due to limiting of (eIF2·GTP·Met-tRNAiMet)ternary complex binding to the ribosome. Furthermore, N-terminal FLAG-eIF1 overexpression reduces eIF2 binding to the ribosome and moderately derepresses GCN4 translation. Our results provide the first in vivo evidence that eIF1 plays an important role in promoting 43 S complex formation as a core of factor interactions. We propose that the coordinated recruitment of eIF1 to the 40 S ribosome in the MFC is critical for the production of functional 40 S preinitiation complex. Eukaryotic initiation factor 1 (eIF1) is a low molecular weight factor critical for stringent AUG selection in eukaryotic translation. It is recruited to the 43 S complex in the multifactor complex (MFC) with eIF2, eIF3, and eIF5 via multiple interactions with the MFC constituents. Here we show that FLAG epitope tagging of eIF1 at either terminus abolishes its in vitro interactions with eIF5 and eIF2β but not that with eIF3c. Nevertheless, both forms of FLAG-eIF1 fail to bind eIF3 and are incorporated into the 43 S complex inefficiently in vivo. C-terminal FLAG tagging of eIF1 is lethal; overexpression of C-terminal FLAG-eIF1 severely impedes 43 S complex formation and derepresses GCN4 translation due to limiting of (eIF2·GTP·Met-tRNAiMet)ternary complex binding to the ribosome. Furthermore, N-terminal FLAG-eIF1 overexpression reduces eIF2 binding to the ribosome and moderately derepresses GCN4 translation. Our results provide the first in vivo evidence that eIF1 plays an important role in promoting 43 S complex formation as a core of factor interactions. We propose that the coordinated recruitment of eIF1 to the 40 S ribosome in the MFC is critical for the production of functional 40 S preinitiation complex. In eukaryotic translation initiation, the small ribosomal subunit (40 S ribosome) binds the eukaryotic initiation factor 2 (eIF2) 1The abbreviations used are: eIF, eukaryotic initiation factor; r-eIF1, recombinant form of eIF1; TC, ternary complex; CTD, carboxyl-terminal domain; NTD, amino-terminal domain; MFC, multifactor complex; WCE, whole cell extract(s); K-box, lysine-rich box; AA-box, acidic and aromatic amino acid box; GST, glutathione S-transferase; ORF, open reading frame; uORF, upstream open reading frame; HA, hemagglutinin; 3-AT, 3-aminotriazole; FOA, 5-fluoroorotic acid. ·GTP· (Met-tRNAiMet)ternary complex (TC) to form the 43 S preinitiation complex. Subsequent joining of mRNA carried by eIF4F produces the 48 S preinitiation complex. The eIF3 stimulates recruitment of (Met-tRNAiMet)and mRNA to the 40 S ribosome by binding to eIF2 and the eIF4G subunit of eIF4F, either directly or indirectly (for a review, see Ref. 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, Cold Spring Harbor, NY2000: 33-88Google Scholar). The 48 S complex searches for the first AUG codon in the mRNA with the help of low molecular weight factors, eIF1 and eIF1A, and the helicase and its cofactor, eIF4A and eIF4B, respectively. Correct base pairing between the (Met-tRNAiMet)anticodon and the AUG codon triggers the hydrolysis of GTP bound to eIF2; this reaction is dependent on the GTPase-activating function of the eIF5 N-terminal domain (NTD). This GTP hydrolysis then leads to dissociation of preassembled eIFs and formation of an initiation complex containing the AUG anticodon base pair in the ribosomal P site. The second GTP-binding protein eIF5B then stimulates joining of the 40 S initiation complex with the 60 S subunit to form the 80 S initiation complex. The elongation of the polypeptide chain starts from the methionine linked to the 80 S initiation complex. eIFs are highly conserved from yeast to mammals. Mammalian eIFs were purified from a high salt wash fraction (e.g. in a 500 mm KCl buffer) of ribosome-associated proteins and characterized in crude mammalian cell extracts or partially or fully reconstituted initiation systems (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, Cold Spring Harbor, NY2000: 33-88Google Scholar, 2Pestova T.V. Borukhov S.I. Hellen C.U.T. Nature. 1998; 394: 854-859Google Scholar, 3Pestova T.V. Lomakin I.B. Lee J.H. Choi S.K. Dever T.E. Hellen C.U.T. Nature. 2000; 403: 332-335Google Scholar). Despite the progress in elucidating the function of individual eIFs, the precise order of and the key element(s) critical for their assembly in vivo remain to be elucidated. Using yeast Saccharomyces cerevisiae as a model organism, it was proposed that the C-terminal domain (CTD) of eIF5 plays a critical role in the assembly and integrity of the functional 43 and 48 S complexes (4Asano K. Clayton J. Shalev A. Hinnebusch A.G. Genes Dev. 2000; 14: 2534-2546Google Scholar, 5Asano K. Shalev A. Phan L. Nielsen K. Clayton J. Valasek L. Donahue T.F. Hinnebusch A.G. EMBO J. 2001; 20: 2326-2337Google Scholar). eIF5-CTD interacts concurrently with the β subunit of eIF2 and the c subunit of eIF3 via a conserved motif called aromatic/acidic boxes (AA-boxes) 1 and 2 (6Asano K. Krishnamoorthy T. Phan L. Pavitt G.D. Hinnebusch A.G. EMBO J. 1999; 18: 1673-1688Google Scholar). Lysine-rich segments (K-boxes) in eIF2β-NTD are responsible for its binding to the eIF5-CTD (6Asano K. Krishnamoorthy T. Phan L. Pavitt G.D. Hinnebusch A.G. EMBO J. 1999; 18: 1673-1688Google Scholar). These interactions plus interactions between eIF3c and eIF1 (7Asano K. Phan L. Anderson J. Hinnebusch A.G. J. Biol. Chem. 1998; 273: 18573-18585Google Scholar), between eIF3a and eIF1, and between eIF2β and eIF3a (8Valásek L. Nielsen K.H. Hinnebusch A.G. EMBO J. 2002; 21: 5886-5898Google Scholar) were proposed to mediate formation of the multifactor complex (MFC) containing eIF1, eIF2, eIF3, eIF5, and (Met-tRNAiMet)(4Asano K. Clayton J. Shalev A. Hinnebusch A.G. Genes Dev. 2000; 14: 2534-2546Google Scholar). Accumulating evidence supports the model that the constituents of the MFC bind to the 40 S ribosome as a preformed unit to form the 43 S complex (5Asano K. Shalev A. Phan L. Nielsen K. Clayton J. Valasek L. Donahue T.F. Hinnebusch A.G. EMBO J. 2001; 20: 2326-2337Google Scholar, 9Valásek L. Phan L. Schoenfeld L.W. Valásková V. Hinnebusch A.G. EMBO J. 2001; 20: 891-904Google Scholar, 10Phan L. Schoenfeld L.W. Valasek L. Nielsen K. Hinnebusch A.G. EMBO J. 2001; 20: 2954-2965Google Scholar). Disruption of MFC by an AA-box 2 mutation in eIF5 leads to a defect in (Met-tRNAiMet)and mRNA binding to the 40 S ribosome in vitro. Evidence also suggests that this mutation impedes a step subsequent to the 43 S complex formation in vivo (5Asano K. Shalev A. Phan L. Nielsen K. Clayton J. Valasek L. Donahue T.F. Hinnebusch A.G. EMBO J. 2001; 20: 2326-2337Google Scholar). Among the components of the MFC, eIF1 is a small factor (12 kDa) whose importance has just begun to be appreciated. Genetic and biochemical studies indicate that eIF1 is required for discrimination of the 48 S complex against near cognate codon pairing with the (tRNAiMet)anticodon, to ensure initiation from AUG only (2Pestova T.V. Borukhov S.I. Hellen C.U.T. Nature. 1998; 394: 854-859Google Scholar, 11Yoon H.J. Donahue T.F. Mol. Cell. Biol. 1992; 12: 248-260Google Scholar, 12Pestova T.V. Kolupaeva V.G. Gene Dev. 2002; 16: 2906-2922Google Scholar). Besides its role in the 48 S complex, in vitro assays using the 40 S ribosome, (Met-tRNAiMet), and a limited set of eIFs indicate that eIF1 stimulates eIF2 TC binding to the 40 S ribosome both in yeast (13Algire M.A. Maag D. Savio P. Acker M.G. Tarun S.Z.J. Sachs A.B. Asano K. Nielsen K.H. Olsen D.S. Phan L. Hinnebusch A.G. Lorsch J.R. RNA. 2002; 8: 382-397Google Scholar) and mammals (14Majumdar R. Bandyopadhyay A. Maitra U. J. Biol. Chem. 2003; 278: 6580-6587Google Scholar). In this paper, we investigated mutual interactions of eIF1 with other components in the MFC and found that eIF1 binds to the other two components of MFC, eIF2β and eIF5, in addition to eIF3c, as was previously shown (7Asano K. Phan L. Anderson J. Hinnebusch A.G. J. Biol. Chem. 1998; 273: 18573-18585Google Scholar). These newly identified eIF1 interactions, but not that with eIF3c, are significantly reduced by tagging of eIF1 with the highly charged FLAG peptide at either terminus. The FLAG-tagged forms of eIF1 reduce binding to eIF3 and are recruited to the 40 S ribosome inefficiently. C-terminal FLAG-eIF1 produces a recessive lethal phenotype, and its overexpression severely impedes 43 S complex formation. N-terminal FLAG-eIF1 overexpression also compromises 43 S complex formation and reduces eIF2 binding to the 40 S ribosome. Together these results provide firm evidence that eIF1 plays an important role in promoting 43 S complex formation in vivo. Plasmids and Yeast Strains—Plasmids and yeast strains used in this study are listed in Tables I and II, respectively. The 1.3-kb SphI fragment of p1128 (11Yoon H.J. Donahue T.F. Mol. Cell. Biol. 1992; 12: 248-260Google Scholar) containing the chromosomal SUI1 locus was cloned into YCplac111 and YEplac181 (15Gietz R.D. Sugino A. Gene (Amst.). 1988; 74: 527-534Google Scholar), yielding YCpL-SUI1 and YEpL-SUI1, respectively. The 1.6-kb PstI-SphI fragment of p4231 carrying pGPD-FL-SUI1-tPGK was cloned into YCplac111 to generate YCpL-GPDFL-SUI1 encoding the N-terminal FLAG-eIF1 (designated FL-eIF1) under the GPD promoter.Table IPlasmids employed in this studyPlasmidDescriptionSourcepGEX vectorsExpression vectors for GST fusionsAmersham BiosciencespGEX-TIF5eIF5 cloned in pGEXRef. 6Asano K. Krishnamoorthy T. Phan L. Pavitt G.D. Hinnebusch A.G. EMBO J. 1999; 18: 1673-1688Google ScholarpGEX-TIF5-B6eIF5-(241–405) cloned in pGEXRef. 6Asano K. Krishnamoorthy T. Phan L. Pavitt G.D. Hinnebusch A.G. EMBO J. 1999; 18: 1673-1688Google ScholarpGEX-TIF5-B6-12ApGEX-TIF5-B6 carrying tif5-12ARef. 5Asano K. Shalev A. Phan L. Nielsen K. Clayton J. Valasek L. Donahue T.F. Hinnebusch A.G. EMBO J. 2001; 20: 2326-2337Google ScholarpGEX-TIF5-B6-7ApGEX-TIF5-B6 carrying tif5-7ARef. 5Asano K. Shalev A. Phan L. Nielsen K. Clayton J. Valasek L. Donahue T.F. Hinnebusch A.G. EMBO J. 2001; 20: 2326-2337Google ScholarpGEX-SUI3eIF2β cloned in pGEXThis studypGEX-SUI3-3KpGEX-SUI3 altering all 3 K-boxes to alaninesThis studypGEX-SUI3ΔSeIF2β-(1–140) cloned in pGEXThis studypGEX-NIP1-NeIF3c-(1–156) cloned in pGEXRef. 4Asano K. Clayton J. Shalev A. Hinnebusch A.G. Genes Dev. 2000; 14: 2534-2546Google ScholarpHis-NIP1-NHis6-tagged eIF3c-(1–156) under a T7 promoterRef. 4Asano K. Clayton J. Shalev A. Hinnebusch A.G. Genes Dev. 2000; 14: 2534-2546Google ScholarpHis-SUI3ΔSHis6-tagged eIF2β-(1–140) under a T7 promoterRef. 5Asano K. Shalev A. Phan L. Nielsen K. Clayton J. Valasek L. Donahue T.F. Hinnebusch A.G. EMBO J. 2001; 20: 2326-2337Google ScholarpHis-TIF5-B6His6-tagged eIF5-(241–405) under a T7 promoterRef. 4Asano K. Clayton J. Shalev A. Hinnebusch A.G. Genes Dev. 2000; 14: 2534-2546Google ScholarpET-FL-SUI1FL-eIF1 cloned under a T7 promoterThis studypET-SUI1-FLeIF1-FL cloned under a T7 promoterThis studypT7-7Cloning vector with T7 promoterRef. 30Tabor S. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4767-4771Google ScholarpT7-SUI1eIF1 cloned in pT7-7Ref. 7Asano K. Phan L. Anderson J. Hinnebusch A.G. J. Biol. Chem. 1998; 273: 18573-18585Google ScholarpT7-SUI1-FLeIF1-FL cloned in pT7-7This studyYCpL-SUI1Single copy SUI1 LEU2 plasmidThis studyYEpL-SUI1High copy SUI1 LEU2 plasmidThis studyYCpL-SUI1-FLSingle copy SUI1-FL LEU2 plasmidThis studyYCpL-FL-SUI1Single copy FL-SUI1 LEU2 plasmidThis studyYCpL-FL-SUI1*Single copy FL-SUI1* LEU2 plasmidThis studyYEpL-SUI1-FLHigh copy SUI1-FL LEU2 plasmidThis studyYEpL-FL-SUI1*High copy FL-SUI1* LEU2 plasmidThis studyp4231High copy pGPD-FL-SUI1-tPGK TRP1 plasmidRef. 31Cui Y. Dinman J.D. Kinzy T.G. Peltz S.W. Mol. Cell. Biol. 1998; 18: 1506-1516Google ScholarYCpL-GPDFL-SUI1Single copy pGPD-FL-SUI1-tPGK LEU2 plasmidThis studyp1780-IMTHigh copy SUI2 SUI3 GCD11 IMT URA3 plasmidRef. 6Asano K. Krishnamoorthy T. Phan L. Pavitt G.D. Hinnebusch A.G. EMBO J. 1999; 18: 1673-1688Google Scholarp180 and derivativesSingle copy GCN4::lacZ URA3 plasmidsRef. 20Mueller P.P. Hinnebusch A.G. Cell. 1986; 45: 201-207Google Scholar Open table in a new tab Table IIYeast S. cerevisiae strains employed in this studyStrainDescriptionSourceY217MATα leu2 lys11 ura3–52 trp1Δ mof2(sui1)::hisG p[SUI1 URA3]Ref. 31Cui Y. Dinman J.D. Kinzy T.G. Peltz S.W. Mol. Cell. Biol. 1998; 18: 1506-1516Google ScholarY218Y217 with p[SUI1 TRP1] replacing p[SUI1 URA3]Ref. 31Cui Y. Dinman J.D. Kinzy T.G. Peltz S.W. Mol. Cell. Biol. 1998; 18: 1506-1516Google ScholarKAY146Y217 with YCpL-SUI1 (SUI1 LEU2) replacing p[SUI1 URA3]This studyKAY156Y217 with YCpL-FL-SUI1 (FL-SUI1 LEU2) replacing p[SUI1 URA3]This studyKAY178Y217 with YCpL-FL-SUI1* (FL-SUI1* LEU2) replacing p[SUI1 URA3]This studyKAY142Y217 with YCpL-GPDFL-SUI1 (GPDFL-SUI1 LEU2) replacing p[SUI1 URA3]This studyKAY6MATa ura3–52 leu2–3,–112 trp1-Δ63 tif34Δ gcn2Δ p[TIF34 URA3]Ref. 7Asano K. Phan L. Anderson J. Hinnebusch A.G. J. Biol. Chem. 1998; 273: 18573-18585Google ScholarKAY107MATa ura3–52 leu2–3,–112 trp1-Δ63 tif34Δ gcn2Δ tif5Δ p[TIF34-HA TRP1] p[TIF5 URA3]This studyKAY37MATa ura3–52 leu2–3,–112 trp1-Δ63 tif5Δ gcn2Δ p[TIF5-FL TRP1]Ref. 6Asano K. Krishnamoorthy T. Phan L. Pavitt G.D. Hinnebusch A.G. EMBO J. 1999; 18: 1673-1688Google Scholar Open table in a new tab To introduce a NcoI site at the 5′-end of the SUI1 ORF, a SUI1 5′-untranslated region DNA was amplified with oligo-1 (5′-CCC GAGCTC GAA TCT ATT CTG GAC ATC CTG-3′) and oligo-2 (5′-GCG GGATCCATGGGA TTT GCT TCA GCT ATA TTA ATA TAT TC-3′). Restriction enzyme sites are underlined. The SacI-BamHI fragment of this DNA fragment was replaced with the SacI-BamHI segment of YCpL-SUI1. The resulting plasmid, YCpSUI1ΔNco, has a unique NcoI site following the 5′-untranslated region and lacks the 5′-half of SUI1 ORF up to the unique BamHI site. The 0.21-kb NcoI-BamHI fragment of p4231 containing the FL-SUI1 ORF 5′-half was subcloned into YCpL-SUI1ΔNco to generate YCpL-FL-SUI1 encoding FL-eIF1 under the natural promoter. The 0.21-kb NcoI-BamHI fragment from PCR with oligo-3 5′-CCC CCATGG ACT ACA AGG ACG ACG ATG ACA AGCTGT CCA TTG AGA ATC TGA AAT C-3′ (to replace the second ATG of FL-eIF1 ORF with CTG; underlined) and oligo-4 5′-CTC CCC CAT TTC TGGATCCTT GAC-3′ (covering the unique BamHI site in SUI1 ORF) was cloned into YCpL-SUI1ΔNco to generate YCpL-FL-SUI1* encoding another version of FLAG-eIF1 designated FL-eIF1*. To introduce an NdeI site at the 5′-end of the SUI1 ORF, YCpL-SUI1ΔNde was prepared similarly to YCpL-SUI1ΔNco except using oligo-5 (5′-CGC GGATCC GACATATGA TTT GCT TCA GCT ATA TTA AT-3′) (with a NdeI site) in place of oligo-2. To make a plasmid encoding C-terminal FLAG-eIF1 (eIF-FL), the NdeI-SalI fragment of SUI1-FL ORF from PCR with oligo-6 (5′-GGC CAT ATG TCC ATT GAG AAT CTG AAA TC-3′) and oligo-7 (5′-CGC GTCGAC TTA TTT GTC ATC GTC GTC CTT GTA GTC AAA CCC ATG AAT TTT AAT GTT C-3′) and the SalI-HindIII fragment of the SUI1 3′-untranslated region from PCR with oligo-8 (5′-GGC GTCGAC GTT CAA GGC TTA CGC CG-3′) and oligo-9 (5′-CAT AAGCTT GGG ATT CCA TGA TTT-3′) were cloned together into pET23a to generate pET-SUI1-FL. Since this plasmid did not produce 35S-eIF1-FL in T7/TNT system (Promega), we replaced the 0.5-kb AflIII-HindIII fragment of pT7-SUI1 with that of pET-SUI1-FL to generate pT7-SUI1-FL that was used for 35S-eIF1-FL synthesis in the T7/TNT system. The NdeI-SphI fragment of pET-SUI1-FL was cloned into the same sites of YCpL-SUI1ΔNde to generate YCpL-SUI1-FL. The 1.5-kb SacI-SphI fragment of YCpL-FL-SUI1* or YCpL-SUI1-FL was cloned into YEplac181 to generate YEpL-FL-SUI1* or YEpL-SUI1-FL, respectively. Yeast strains KAY146, KAY156, KAY178, and KAY142 were constructed by plasmid shuffling (16Boeke J.D. LaCroute F. Fink G.R. Mol. Gen. Genet. 1984; 197: 345-346Google Scholar) using Y217 and the corresponding SUI1 LEU2 plasmids listed in Table II. Biochemical Assays—GST pull-down assays with 35S-labeled proteins, synthesized in a rabbit reticulocyte lysate, were conducted as described previously (7Asano K. Phan L. Anderson J. Hinnebusch A.G. J. Biol. Chem. 1998; 273: 18573-18585Google Scholar). The amounts of bound 35S-labeled proteins were quantitated with STORM or TYPHOON PhosphorImagers (Amersham Biosciences). Polyhistidine-tagged eIF5-CTD-(241–405), eIF3c-(1–156), and eIF2β-(1–140) fragments designated His-eIF5-B6, His-NIP1-N, and His-eIF2β-N were expressed and purified from BL21(DE3) carrying pHis-TIF5-B6, pHis-NIP1-N, and pHis-SUI3ΔS, respectively, as described (5Asano K. Shalev A. Phan L. Nielsen K. Clayton J. Valasek L. Donahue T.F. Hinnebusch A.G. EMBO J. 2001; 20: 2326-2337Google Scholar). FLAG-eIF1 constructs designated FL-eIF1 and eIF1-FL were purified from BL21 (DE3) carrying pET-FL-SUI1 and pET-SUI1-FL, respectively, as described (17Asano K. Lon P. Krishnamoorthy T. Pavitt G.D.E., G. Hannig E.M. Nika J. Donahue T.F. Huang H.-K. Hinnebusch A.G. Methods Enzymol. 2002; 351: 221-247Google Scholar). Peptide sequencing of FL-eIF1 indicated that the majority of its N terminus was blocked, probably by a formyl group attached to the first methionine, whereas a subpopulation of the protein had an unblocked N terminus with the sequence MDYK, confirming its first four amino acids. 2G. Radke, T. Iwamoto, and J. Tomich, unpublished observations. FLAG-tagged eIF2 or HA-tagged eIF3 was affinity-purified from strain KAY42 (gcd6-7A p1780-FL) (6Asano K. Krishnamoorthy T. Phan L. Pavitt G.D. Hinnebusch A.G. EMBO J. 1999; 18: 1673-1688Google Scholar) or H2557 transformant carrying pLPY-PRT1His-TIF34HA-TIF35FLAG and pLPY-NIP1-TIF32 (10Phan L. Schoenfeld L.W. Valasek L. Nielsen K. Hinnebusch A.G. EMBO J. 2001; 20: 2954-2965Google Scholar), attached to anti-FLAG (Sigma) or anti-HA (made by preadsorbing protein A-Sepharose beads (Amersham Biosciences) with anti-HA antibodies (Babco) as described (7Asano K. Phan L. Anderson J. Hinnebusch A.G. J. Biol. Chem. 1998; 273: 18573-18585Google Scholar)) affinity resin, respectively, and used for binding assays with recombinant forms of eIF1. Co-immunoprecipitation was done essentially as described (6Asano K. Krishnamoorthy T. Phan L. Pavitt G.D. Hinnebusch A.G. EMBO J. 1999; 18: 1673-1688Google Scholar) with the following modification. Whole cell extracts (WCE) were prepared in buffer A, but immune complex binding and washing were done in buffer A supplemented with 0.1% Triton X-100. Polysome analysis was conducted as described previously (4Asano K. Clayton J. Shalev A. Hinnebusch A.G. Genes Dev. 2000; 14: 2534-2546Google Scholar, 5Asano K. Shalev A. Phan L. Nielsen K. Clayton J. Valasek L. Donahue T.F. Hinnebusch A.G. EMBO J. 2001; 20: 2326-2337Google Scholar). To quantitate the amount of factors in the precipitated fractions, we used WCE prepared from a wild type strain (KAY146) as a standard, based on the following information. Using purified FL-eIF1 as a reference, we determined that 40 μg of WCE contains 3.6 pmol of eIF1 (see Fig. 3A). Because the intracellular molar ratio of eIF1, eIF2, eIF3, and eIF5 is 1:0.77:0.65:0.82 in our WCE, 3H. He, C. R. Singh, and K. Asano, unpublished data. the same amount of WCE was calculated to contain 2.8, 1.9, and 3.0 pmol of eIF2, eIF3, and eIF5, respectively. The amounts of precipitated eIFs were determined in reference to these values. We determined individual eIF levels by anti-FLAG immunoblotting of WCE prepared from strains encoding FLAG-eIF1, -eIF2β, -eIF3c, or -eIF5 as its sole source. 3H. He, C. R. Singh, and K. Asano, unpublished data. The values obtained are in better agreement with data from Ref. 18von der Haar T. McCarthy J.E.G. Mol. Microbiol. 2002; 46: 531-544Google Scholar than the data from Ref. 19Ghaemmaghami S. Huh W.K. Bower K. Howson R.W. Belle A. Dephoure N. O'Shea E.K. Weissman J.S. Nature. 2003; 425: 737-741Google Scholar. GCN4 translational control in KAY142 (GPDFL-SUI1) was tested by assaying β-galactosidase from its transformant carrying p180 (GCN4::lacZ) or its derivative p226 or p227 (20Mueller P.P. Hinnebusch A.G. Cell. 1986; 45: 201-207Google Scholar). The results of these experiments were described in Ref. 21, He, H. (2003) Genetic and Biochemical Characterization of Factor Interactions Involving Eukaryotic Translation Initiation Factors eIF1 and eIF5. Master's thesis, Kansas State University, Manhattan, KSGoogle Scholar. All of the biochemical assays were done at least three times, and a typical result is shown. Yeast eIF1 Interacts with eIF5-CTD via AA-boxes and eIF2β-NTD via K-boxes—It was previously reported that a GST fusion form of eIF5-CTD (165 amino acids), known as GST-eIF5-B6, can interact with 35S-labeled eIF1, synthesized in a rabbit reticulocyte lysate (4Asano K. Clayton J. Shalev A. Hinnebusch A.G. Genes Dev. 2000; 14: 2534-2546Google Scholar) (also see Fig. 1A, lanes 2 and 3). Because eIF1 stimulates eIF2 TC binding to the ribosome (13Algire M.A. Maag D. Savio P. Acker M.G. Tarun S.Z.J. Sachs A.B. Asano K. Nielsen K.H. Olsen D.S. Phan L. Hinnebusch A.G. Lorsch J.R. RNA. 2002; 8: 382-397Google Scholar, 14Majumdar R. Bandyopadhyay A. Maitra U. J. Biol. Chem. 2003; 278: 6580-6587Google Scholar, 22Thomas A. Spaan W. van Steeg H. Voorma H.O. Benne R. FEBS Lett. 1980; 116: 67-71Google Scholar), we also examined whether individual eIF2 subunits can bind eIF1 and found that GST-eIF2β and its C-terminal deletion GST-eIF2β-N (covering amino acids 1–140) specifically bound 35S-eIF1 (Fig. 1B, lanes 3 and 4). To further examine interactions between eIF1 and eIF5 or eIF2β, the bacterial extract containing a recombinant form of eIF1 (r-eIF1) was incubated with GST-eIF5-B6, GST-eIF2β-N, GST-eIF3c-N, or GST alone. GST-eIF3c-N contains the N-terminal 156 amino acids of eIF3c, sufficient for eIF1 binding (4Asano K. Clayton J. Shalev A. Hinnebusch A.G. Genes Dev. 2000; 14: 2534-2546Google Scholar), and was used as a positive control. GST fusion proteins bound to r-eIF1 were one-step purified with glutathione and analyzed by SDS-PAGE. As shown in Fig. 1C, top panel, Coomassie staining of the eluted proteins indicates that GST-eIF3c-N (lane 6), but not GST alone (lane 4), specifically bound to a nearly stoichiometric amount of r-eIF1, whereas no protein from mock-induced extracts bound to GST-eIF3c-N (lane 5). Under these conditions, GST-eIF5-B6 and GST-eIF2β-N bound recombinant eIF1, albeit weakly as determined by Western blotting (Fig. 1C, bottom panel, lanes 9 and 12; note that 5 times less eluted fraction was analyzed in lanes 5–7 than in lanes 8–13). Therefore, r-eIF1 binds GST-eIF5-B6 and GST-eIF2β-N. The treatment of the GST fusion complexes with RNase A prior to the elution step did not reduce these interactions (Fig. 1C, lanes 7, 10, and 13), ruling out the possibility that r-eIF1 was tethered to the proteins via RNA fortuitously bound to r-eIF1 or the GST fusion protein. As mentioned above, the new interactions of eIF1 with eIF2β and eIF5 are weaker than the previously known interaction between eIF1 and eIF3c (Fig. 1C). However, it is these weaker interactions that contribute to cooperative formation of eIF1·eIF2β·eIF5 subcomplex in the MFC, as shown in Fig. 1, E and F. Consistent with this model, FLAG epitope tagging of eIF1 compromises these interactions, leading to inefficient incorporation of FLAG-eIF1 into the MFC (see Fig. 2C). Both the novel interactions of eIF1 depend on the AA-boxes of eIF5 and K-boxes of eIF2β, because the interaction between GST-eIF5-B6 and 35S-eIF1 was abolished by the AA-box 1 mutation, tif5-12A, or the AA-box 2 mutation, tif5-7A (Fig. 1A, lanes 3–5), and altering all of the lysines in the K-boxes to alanines reduced GST-eIF2β binding to eIF1 by 4-fold (Fig. 1B, lane 5). We also found that these interactions were salt-sensitive, since they were not detected when a buffer containing 150 mm Na+ was used at the washing step (7Asano K. Phan L. Anderson J. Hinnebusch A.G. J. Biol. Chem. 1998; 273: 18573-18585Google Scholar) instead of a buffer containing 75 mm KCl as employed in Fig. 1 (data not shown). We confirmed, however, that 35S-eIF1 interacts with eIF5-B6 and eIF2β-N in the same binding buffer (7Asano K. Phan L. Anderson J. Hinnebusch A.G. J. Biol. Chem. 1998; 273: 18573-18585Google Scholar) with 100 mm KCl; this salt concentration has been used in a variety of translation initiation assays (23Hinnebusch 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). The salt sensitivity of these interactions explains our previous failure to identify these interactions. To confirm that eIF1 can bind directly to the trimeric eIF2 complex, we affinity-purified FLAG-tagged eIF2 from yeast and allowed it to bind r-eIF1 expressed from bacteria. The Coomassie-stained gel of the purified eIF2 is shown in Fig. 1D, lane 2. The eIF2·r-eIF1 complex was precipitated with anti-FLAG affinity resin and then analyzed by Western blotting. As shown in Fig. 1D, second panel, r-eIF1 bound specifically to FLAG-eIF2 as detected by anti-eIF1 (lanes 4–9). To examine whether this interaction is mediated by eIF3 or eIF5 associated with FLAG-eIF2, we analyzed the complex with antibodies against eIF2α, eIF3g, and eIF5. As shown in Fig. 1D, bottom two panels, we found little eIF3 and eIF5 in the precipitated FLAG-eIF2 fractions (lanes 6 and 9), whereas the amounts of eIF1 and eIF2α in the FLAG-eIF2·r-eIF1 fraction was judged to be nearly stoichiometric (lane 6), when compared with their amounts in yeast WCE (lane 3). These results indicate that eIF1 can bind native eIF2 as well as its β subunit. eIF2β-NTD Interacts Simultaneously with eIF1 and eIF5-CTD—Having observed separate interactions of eIF1 with eIF2β-NTD and eIF5-CTD, we pondered whether these interactions occur simultaneously. If so, the formation of a trimeric eIF1·eIF2·eIF5 complex as a part of MFC would contribute to stable eIF2 TC binding to the 40 S ribosome. Thus, we tested whether the interaction between GST-eIF5-B6 and eIF1 can be enhanced by the addition of eIF2β-NTD by a bridging mechanism. As a control, we used the eIF3c-NTD segment, since it is known to bridge the same interaction between GST-eIF5-CTD and eIF1 (4Asano K. Clayton J. Shalev A. Hinnebusch A.G. Genes Dev. 2000; 14: 2534-2546Google Scholar). As shown in Fig. 1E, bottom panel, the eIF2β-N segment increases 35S-eIF1 binding to GST-eIF5-B6 by forming a bridge, as efficiently as the eIF3c-N segment does (top panel). The addition of equivalent amounts of bovine serum albumin did not increase GST-eIF5-B6/35S-eIF1 interaction (data not shown), indicating that the increased interaction in Fig. 1E is not due to increasing the efficiency of the pull downs by nonspecific mechanisms. Thus, eIF2β-NTD binds simultaneously to eIF1 and eIF5-CTD. Likewise, Fig. 1F shows that the His-eIF5-B6 segment can bridge GST-eIF2β-N and 35S-eIF1, thereby enhancing this interaction (compare lanes 2–4), indicating that eIF5-CTD binds simultaneously to eIF1 and eIF2β-NTD. Together, these results support the idea that a trimeric eIF1·eIF2·eIF5 complex can be formed as a part of MFC for stimulation of TC binding to the ribosome. FLAG Tagging of eIF1 Impairs the Interaction with eIF5-CTD, eIF2β-NTD, and the Native eIF3 Complex in Vitro—To analyze the functional significance of a protein interaction in vivo, it is important to obtain a mutation that specifically reduces it. During the course of our study, we noted that some of the interactions involving eIF1 were compromised by its epitope tagging at either terminus with the bulky highly charged FLAG peptide (DYKDDDDK). As shown in Fig. 2A, middle and bottom panels, C-terminally and N-terminally FLAG-tagged eIF1, designated eIF1-FL and FL-eIF1, respectively, had reduced interactions with GST-eIF2β-N and GST-eIF5-B6 (lanes 3 and 6) but not with GST-eIF3c-N (lane 4). In addition, we found that both forms of FLAG-eIF1 had reduced interactions with the C-terminal HEAT domain of eIF4G2 (lane 5) (24He H. von der Haar T. Singh R.C. Ii M. Li B. McCarthy J.E.G. Hinnebusch A.G. Asano K. Mol. Cell. Biol. 2003; 23: 5441-5445Google Scholar) as well as with the C-terminal domain of eIF3a (GST-TIF32Δ1 (8Valásek L. Nielsen K.H. Hinnebusch A.G. EMBO J. 2002; 21: 5886-5898Google Scholar); data not shown). To examine the interaction of FLAG-eIF1 derivatives with native eIF3, we affinity-purified native HA epitope-tagged eIF3, attached it to anti-HA affinity resin, and allowed it to bind recombinant forms of eIF1, FL-eIF1, and eIF1-FL in vitro. As shown in Fig. 2B, lane 2, the purified HA-eIF3 contains stoichiometric amounts of a, b, and g subunits of eIF3, a substoichiometric amount of eIF5, and no detectable eIF1. This eIF3 complex bound untagged eIF1 very efficiently but bound FLAG-eIF1s less efficiently. Thus, FLAG tagging of eIF1 reduces the interaction with e" @default.
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• W1994421775 cites W1969737313 @default.
• W1994421775 cites W1976327985 @default.
• W1994421775 cites W1993691117 @default.
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• W1994421775 cites W2072933194 @default.
• W1994421775 cites W2077545733 @default.
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• W1994421775 cites W2110598934 @default.
• W1994421775 cites W2116415909 @default.
• W1994421775 cites W2117163223 @default.
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