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• W2077985310 abstract "Members of the p56 family of mammalian proteins are strongly induced in virus-infected cells and in cells treated with interferons or double-stranded RNA. Previously, we have reported that human p56 inhibits initiation of translation by binding to the “e” subunit of eukaryotic initiation factor 3 (eIF3) and subsequently interfering with the eIF3/eIF2·GTP·Met-tRNAi (ternary complex) interaction. Here we report that mouse p56 also interferes with eIF3 functions and inhibits translation. However, the murine protein binds to the “c” subunit, not the “e” subunit, of eIF3. Consequently, it has only a marginal effect on eIF3·ternary complex interaction. Instead, the major inhibitory effect of mouse p56 is manifested at a different step of translation initiation, namely the binding of eIF4F to the 40 S ribosomal subunit·eIF3·ternary complex. Thus, mouse and human p56 proteins block different functions of eIF3 by binding to its different subunits. Members of the p56 family of mammalian proteins are strongly induced in virus-infected cells and in cells treated with interferons or double-stranded RNA. Previously, we have reported that human p56 inhibits initiation of translation by binding to the “e” subunit of eukaryotic initiation factor 3 (eIF3) and subsequently interfering with the eIF3/eIF2·GTP·Met-tRNAi (ternary complex) interaction. Here we report that mouse p56 also interferes with eIF3 functions and inhibits translation. However, the murine protein binds to the “c” subunit, not the “e” subunit, of eIF3. Consequently, it has only a marginal effect on eIF3·ternary complex interaction. Instead, the major inhibitory effect of mouse p56 is manifested at a different step of translation initiation, namely the binding of eIF4F to the 40 S ribosomal subunit·eIF3·ternary complex. Thus, mouse and human p56 proteins block different functions of eIF3 by binding to its different subunits. One of the key features of the innate immune response is the induction of numerous cellular genes in response to viral stress. Viral stress conditions in cells are triggered by mechanisms commonly associated with a cell undergoing viral infection, such as the production of double-stranded RNA, the production of interferons, as well as other virus-mediated pathways that have yet to be elucidated. Previous studies from our laboratory have characterized the human viral stress-inducible protein p56, a 56-kDa protein (1Guo J. Peters K.L. Sen G.C. Virology. 2000; 267: 209-219Crossref PubMed Scopus (140) Google Scholar). Human p56 (Hup56) has been shown to act as an inhibitor of protein synthesis through its association with the “e” subunit of eukaryotic initiation factor 3 (eIF3 1The abbreviations used are: eIF, eukaryotic initiation factor; Hup56, human p56; TPR, tetratricopeptide repeat; Mup56, mouse p56; DTT, dithiothreitol; GDPNP, guanosine 5′-[β,γ-imido]triphosphate trisodium salt. ; the e subunit is also known as p48 or Int6) (2Guo J. Hui D.J. Merrick W.C. Sen G.C. EMBO J. 2000; 19: 6891-6899Crossref PubMed Scopus (178) Google Scholar, 3Asano K. Merrick W.C. Hershey J.W. J. Biol. Chem. 1997; 272: 23477-23480Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). We have shown recently (4Hui D.J. Bhasker C.R. Merrick W.C. Sen G.C. J. Biol. Chem. 2003; 278: 39477-39482Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar) that the inhibitory activity of human p56 occurs at the step of ternary complex stabilization by eIF3, a key step in the initiation pathway for protein synthesis in eukaryotes. Eukaryotic initiation factor 3 is the largest of the 11 or more factors required for the initiation of protein synthesis in eukaryotes. eIF3 is composed of 12 subunits named eIF3a to eIF3l, although the exact stoichiometry and arrangement of the subunits are poorly understood (5Hershey J.W.B. Merrick W.C. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control of Gene Expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000: 1-55Google Scholar). eIF3 has many functions in translation initiation, one of which is to serve as a ribosome dissociation factor by binding to the 40 S ribosomal subunit and preventing its re-association with 60 S subunits (6Benne R. Hershey J.W.B. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 3005-3009Crossref PubMed Scopus (110) Google Scholar, 7Merrick W.C. Lubson N.H. Anderson W.C. Proc. Natl. Acad. Sci. U. S. A. 1973; 70: 2220-2223Crossref PubMed Scopus (19) Google Scholar). eIF3 also plays a role in stabilizing interactions with other components of the initiation pathway such as the ternary complex that consists of eIF2·GTP·Met-tRNAi as well as stabilizing the formation of the 43 S complex that is formed when the ternary complex joins the 40 S ribosome (8Chadhuri J. Chowdhury D. Maitra U. J. Biol. Chem. 1999; 274: 17975-17980Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 9Gupta N.K. Roy A.L. Nag M.K. Kinzy T.G. MacMillan S. Hileman R.E. Dever T.E. Wu S. Merrick W.C. Hershey J.W.B. McCarthy J.E.G. Tuite M.F. Post-transcriptional Control of Gene Expression. Springer-Verlag, Berlin1990: 521-526Google Scholar). Finally, eIF3 is also involved in binding to eIF4G of the heterotrimeric eIF4F complex, stabilizing its association with the 43 S complex (10Imataka H. Sonenberg N. Mol. Cell. Biol. 1997; 17: 6940-6947Crossref PubMed Scopus (240) Google Scholar, 11Lamphear B.J.R. Kirchweger R. Skern T. Rhoads R.E. J. Biol. Chem. 1995; 270: 21975-21983Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar). The p56 family of proteins includes several similar sized proteins in humans (p54, p56, p58, and p60) (12Wathelet M.G. Clauss I.M. Content J. Huez G.A. FEBS Lett. 1988; 231: 164-171Crossref PubMed Scopus (32) Google Scholar, 13Niikura T. Hirata R. Weil S.C. Blood Cells Mol. Dis. 1997; 23: 337-349Crossref PubMed Scopus (17) Google Scholar, 14Yu M. Tong J.H. Mao M. Kan L.X. Liu M.M. Sun Y.W. Fu G. Jing Y.K. Yu L. Lepaslier D. Lanotte M. Wang Z.Y. Chen Z. Waxman S. Wang X.Y. Tan J.Z. Chen S.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7406-7411Crossref PubMed Scopus (72) Google Scholar, 15de Veer M.J. Sim H. Whisstock J.C. Devenish R.J. Ralph S.J. Genomics. 1998; 54: 266-277Crossref Scopus (51) Google Scholar) as well as other species including hamster (16Bluyssen H.A. van der Vlietstra R.J. Made A. Trapman J. Eur. J. Biochem. 1994; 220: 395-402Crossref PubMed Scopus (36) Google Scholar), mouse (17Bluyssen H.A. Vlietstra R.J. Faber P.W. Smit E.M. Hagemeijer A. Trapman J. Genomics. 1994; 24: 137-148Crossref PubMed Scopus (50) Google Scholar), and fish (18Zhang Y. Gui J. Gene (Amst.). 2004; 325: 43-51Crossref PubMed Scopus (34) Google Scholar). The p56 family members share a structural homology consisting of a series of loosely conserved, 34-amino acid tetratricopeptide (TPR) tandem repeats (19Sikorski R.S. Boguski M.S. Goebl M. Hieter P. Cell. 1990; 60: 307-317Abstract Full Text PDF PubMed Scopus (394) Google Scholar). TPR motifs are known to mediate protein-protein interactions (20Das A.K. Cohen P.W. Barford D. EMBO J. 1998; 17: 1192-1199Crossref PubMed Scopus (710) Google Scholar), and these motifs have been shown to be required for the interaction between human p56 and eIF3e (21Guo J. Sen G.C. J. Virol. 2000; 74: 1892-1899Crossref PubMed Scopus (58) Google Scholar). Conversely, the region of eIF3e that Hup56 interacts with is another loosely conserved structural motif known as the PCI motif. Named for Proteasome, COP9 signalosome and Initiation Factor, the three families of multi-subunit complexes that feature this motif, PCI motif-containing proteins, are potential target proteins for interaction with p56 family members based on structural homology (22Hoffman K. Bucher P. Trends Biochem. Sci. 1998; 23: 204-205Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). Three subunits of eIF3, “a,” “c,” and “e,” contain PCI motifs (23Asano 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 (146) Google Scholar, 24Kim T. von Hofmann K. Arnim A.G. Chamovitz D.A. Trends Plant Sci. 2001; 6: 379-386Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). By extending the in vitro protein synthesis studies that showed that Hup56 can inhibit initiation of translation by interfering with eIF3 function, we followed a systematic investigation to reveal that all functions of eIF3 are not blocked by Hup56; it blocks only one specific step, namely the stabilization of the ternary complex of eIF2·GTP·Met-tRNAi. Hup56 does not interfere with the interactions of eIF3 with 40 S ribosomal subunits or eIF4F. Because we are interested in studying the functions of the p56 family of proteins in interferon-infected animal models, we have extended our investigation to mouse (Mu)p56. Here we report that, like Hup56, Mup56 inhibited translation by binding to eIF3. However, unlike Hup56, it bound to the eIF3c (p110) subunit and not eIF3e. Consequently, Mup56 inhibited a function of eIF3 that is different from the one inhibited by Hup56. Constructs—The plasmid encoding full-length Hup56 was constructed by excising the full-length p56 cDNA from pBluescript KS(II) and inserting into pcDNA3 (Invitrogen). A full-length Mup56 plasmid for expression in mammalian cells was generated by PCR using an existing clone and then subcloning the cDNA sequence into Myc-pcDNA3. 2D. J. Hui, F. Terenzi, W. C. Merrick, and G. C. Sen, unpublished data. Its authenticity was confirmed by sequencing. This vector was generated by inserting six 30-nucleotide repeats of the c-Myc peptide in the N-terminal domain of the pcDNA3 expression vector. The construct encoding full-length Mup56 for expression in bacteria was generated by inserting the full-length cDNA for Mup56 into pET15b vector encoding a hexahistidine tag (Novagen). The full-length eIF3c construct was generated by reverse transcription-PCR and then inserting the cDNA sequence into pFLAG-CMV-2 (Kodak Scientific Imaging System). All constructs were confirmed by having the ligated junctions between vector and insert sequenced. Construction of a plasmid encoding full-length eIF3e was described previously (2Guo J. Hui D.J. Merrick W.C. Sen G.C. EMBO J. 2000; 19: 6891-6899Crossref PubMed Scopus (178) Google Scholar). Antibodies—The commercially available rabbit antibody (His Probe G-18) toward the His tag was used at a dilution of 1:666 for detection of the purified Mup56 in these studies (Santa Cruz Biotechnology). Transfected Mup56 was detected by the c-Myc 9E10 antibody at a 1:1000 dilution (Santa Cruz Biotechnology). A polyclonal antibody raised in goat against purified rabbit eIF3 was also used in these studies. At a 1:1000 dilution, this antibody recognized primarily the p110 (eIF3c) subunit of eIF3 and other eIF3 subunits to a lesser extent (4Hui D.J. Bhasker C.R. Merrick W.C. Sen G.C. J. Biol. Chem. 2003; 278: 39477-39482Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). A rabbit polyclonal antibody against Hup56 was used at a dilution of 1:2000 as described previously (2Guo J. Hui D.J. Merrick W.C. Sen G.C. EMBO J. 2000; 19: 6891-6899Crossref PubMed Scopus (178) Google Scholar). Purification of Recombinant Mouse p56 from Escherichia coli— Mouse p56 was purified by following the same procedure described previously for purifying Hup56 (2Guo J. Hui D.J. Merrick W.C. Sen G.C. EMBO J. 2000; 19: 6891-6899Crossref PubMed Scopus (178) Google Scholar). Briefly, the BL21 DE3 pLys S strain of E. coli was transformed with the pET15b/Mup56 construct, and the expression was induced with 1 mm isopropyl β-d-thiogalactopyranoside for 12 h at room temperature. Mup56 was purified via nickel-affinity chromatography. Protein was then dialyzed against a high glycerol buffer (20 mm Tris-HCl, pH 7.9, 150 mm KCl, 0.5 mm dithiothreitol (DTT), 0.5 mm EDTA, 50% glycerol) and stored at –20 °C. Initiation Factors—eIF2, eIF3, and eIF4F were purified from rabbit reticulocyte lysate as described previously (25Merrick W.C. Methods Enzymol. 1979; 60: 101-108Crossref PubMed Scopus (67) Google Scholar, 26Grifo J.A. Tahara S.M. Morgan M.A. Shatkin A.J. Merrick W.C. J. Biol. Chem. 1983; 258: 5804-5810Abstract Full Text PDF PubMed Google Scholar). Generation of Radiolabeled eIF4F—Radiolabeled eIF4F was generated in vitro via reductive methylation using [14C]formaldehyde (PerkinElmer Life Sciences) as described previously (27Yoder-Hill J. Pause A. Sonenberg N. Merrick W.C. J. Biol. Chem. 1993; 268: 5566-5573Abstract Full Text PDF PubMed Google Scholar). Purification of Ribosomal Subunits—Free 40 S and 60 S ribosomal subunits were purified using high salt sucrose gradients as described previously (7Merrick W.C. Lubson N.H. Anderson W.C. Proc. Natl. Acad. Sci. U. S. A. 1973; 70: 2220-2223Crossref PubMed Scopus (19) Google Scholar, 25Merrick W.C. Methods Enzymol. 1979; 60: 101-108Crossref PubMed Scopus (67) Google Scholar). Generation of [14C]Met-tRNAi—Radiolabeled Met-tRNAi was prepared by using Brewer's yeast tRNA (Ambion), E. coli aminoacyl-tRNA synthetase, and [14C]methionine (56 mCi/mmol, PerkinElmer Life Sciences) as described previously (25Merrick W.C. Methods Enzymol. 1979; 60: 101-108Crossref PubMed Scopus (67) Google Scholar). In Vitro Translation Inhibition Assay—In vitro translations in rabbit reticulocyte lysate (Promega) were programmed with luciferase mRNA in the presence of [35S]methionine. In vitro translations were performed with nuclease-treated rabbit reticulocyte lysate under conditions recommended by the manufacturer. A typical 25-μl reaction contained 17.5 μl of lysate, 0.02 mm amino acid mixture (minus methionine), 10 μCi of [35S]methionine (1200 Ci/mmol), 20 units of RNase inhibitor (Roche Applied Science), and 0.5 μg of transcript. Purified Mouse p56 was added in increasing amounts as indicated in the figure legends. Translations were allowed to proceed at 30 °C for 2 h. Following translation, a 5-μl aliquot of the reaction was resolved by 10% SDS-PAGE. Gels were dried and exposed to a PhosphorImager screen and incorporated radioactivity quantitated using the ImageQuant software (Amersham Biosciences). Cell Culture and Transfection—HT1080 human fibrosarcoma cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). Cells were transfected using FuGENE 6 (Roche Applied Science) according to the manufacturer's protocol. Four micrograms of each plasmid was transfected, and cells were harvested after 18 h. Cell lysates were prepared for either Western blots or immunoprecipitations. Immunoprecipitation and Western Blot—Immunoprecipitation of FLAG-tagged protein was performed in low salt buffer (20 mm Tris, pH 7.5, 50 mm KCl, 200 mm NaCl, 1 mm EDTA, 20% glycerol, 0.05% Triton X-100, 0.2 mm phenylmethylsulfonyl fluoride). M2 anti-FLAG-Sepharose beads (Sigma) were pre-soaked with 3 μg of bovine serum albumin for 15 min. Cell lysates were prepared as described (28Leonard G.T. Sen G.C. J. Virol. 1997; 71: 5095-5101Crossref PubMed Google Scholar), and 300 μg of whole-cell extracts were mixed with 500 μl of low salt buffer and 20 μl of preincubated anti-FLAG-Sepharose beads at 4 °C overnight. The immunocomplexes were washed with the low salt buffer and subjected to denaturing gel electrophoresis through a 10% polyacrylamide gel. Western blots were performed with a 1:2000 dilution of a polyclonal p56 antibody (2Guo J. Hui D.J. Merrick W.C. Sen G.C. EMBO J. 2000; 19: 6891-6899Crossref PubMed Scopus (178) Google Scholar). The Western blot for FLAG or Myc was performed using 1:2000 dilution of anti-FLAG M2 antibody (Kodak Scientific Imaging System) and 1:1000 dilution of c-Myc 9E10 (Santa Cruz Biotechnology), respectively. Alternatively, 300 μg of cell lysate was incubated with 6 μl of antibody against Myc (c-Myc 9E10 monoclonal antibody; Santa Cruz Biotechnology) in 500 μl of RIPA buffer (150 mm NaCl, 1% Nonidet P-40, 0.5% deoxycholic acid, 0.1% SDS, 50 mm Tris, pH 8.0, 0.4 mm phenylmethylsulfonyl fluoride, and protease inhibitors) at 4 °C overnight. 20 μl of protein A-agarose (Roche Applied Science) was then added, followed by a 4-h incubation at 4 °C. Samples were washed four times with RIPA buffer and then subjected to SDS-PAGE followed by Western blotting with an antibody against FLAG (M2-monoclonal antibody, Sigma). Gel Filtration Chromatography—The gel filtration binding assay was performed as described previously (2Guo J. Hui D.J. Merrick W.C. Sen G.C. EMBO J. 2000; 19: 6891-6899Crossref PubMed Scopus (178) Google Scholar). Briefly, an XK 16/70 column (16 mm diameter, 70 cm long, column volume ∼100 ml, V0 ∼35 ml; Amersham Biosciences) was packed with Superdex 200 resin. The column was equilibrated with the assay buffer (20 mm Tris, pH 7.9, 150 mm KCl, 1 mm DTT, 0.1 mm EDTA, and 5% glycerol). Recombinant purified p56 protein (65 μg, ∼1160 pmol) was then separately incubated in the absence or presence of eIF3 (377 μg, ∼580 pmol) for 15 min at 30 °C. Samples were applied to the column, and separation was performed at 4 °C, with a flow rate 1 ml/min, using the Akta Design fast performance liquid chromatography system (Amersham Biosciences). One-milliliter fractions were collected after the 30-ml void volume had passed, and 25 μl of each even number fraction was subjected to SDS-PAGE, transferred to nitrocellulose, and then Western-blotted using the His antibody. In Vitro Pull Down—Eighteen picomoles of purified His-Mup56 and 9 pmol of purified eIF3 were incubated for 2 h at 30 °C prior to the addition of 10 μl of eIF3 antibody in a final volume of 500 μl of RIPA buffer. Following an overnight incubation at 4 °C, 20 μl of protein A-agarose was then added to the reaction followed by a 4-h incubation at 4 °C. Samples were washed four times with RIPA buffer and then subjected to SDS-PAGE, followed by Western blotting with an antibody against His (His-G18 polyclonal antibody; Santa Cruz Biotechnology). Alternatively, 18 pmol of purified His-Mup56 and 9 mol of purified eIF3 were incubated at 30 °C for 2 h prior to the addition of 25 μl of washed nickel-nitrilotriacetic acid-agarose (Qiagen) in 400 μl of GF buffer (20 mm Tris, pH 7.9,150 mm KCl, 0.1% Triton X-100; 5% glycerol, 4 mm imidazole, and protease inhibitors). Samples were incubated for 4 h at 4 °C, and the beads were then washed four times with GF buffer containing 20 mm imidazole. Samples were then subjected to SDS-PAGE, followed by Western blotting with eIF3 antibody. Ribosome Dissociation Assay—The ribosome dissociation assay was performed as described in Ref. 7Merrick W.C. Lubson N.H. Anderson W.C. Proc. Natl. Acad. Sci. U. S. A. 1973; 70: 2220-2223Crossref PubMed Scopus (19) Google Scholar. Purified 40 S ribosomal subunits (0.7 A260 units) were incubated with 1.4 A260 units of purified 60 S ribosomal subunits to form 80 S ribosomes in a 100-μl reaction volume containing 100 mm KCl, 10 mm Tris-HCl, pH 7.5, 3 mm MgCl2, and 2 mm DTT. To dissociate ribosomes, 60 pmol of eIF3 (37.5 μg, 600 nm) was added to the reaction, incubated for 10 min at 37 °C, then layered on a 12-ml 10–25% sucrose gradient (100 mm KCl, 20 mm Hepes-KOH, pH 7.5, 5 mm MgCl2, 2 mm DTT), and centrifuged for 16 h at 20,000 rpm at 4 °C (Beckman SW28Ti rotor). To test the effect of Mup56, 60 pmol of purified p56 (3.2 μg, 600 nm) was preincubated with 60 pmol of eIF3 for 10 min at 30 °C prior to the addition of ribosomes. Gradients were unloaded via upward displacement using 60% sucrose, and UV absorbance was measured at 254 nm with an ISCO flow cell. For Western blot analysis of sucrose gradient fractions, 500 μl of each fraction was precipitated with a final concentration of 10% trichloroacetic acid, subjected to 10% SDS-PAGE, electroblotted onto a nitrocellulose membrane (Millipore), and then probed with either eIF3 antibody (1:1000) or His antibody (1:666) as described previously (2Guo J. Hui D.J. Merrick W.C. Sen G.C. EMBO J. 2000; 19: 6891-6899Crossref PubMed Scopus (178) Google Scholar). eIF3/eIF4F Interaction Assay—To observe the interaction of eIF3 and eIF4F, sucrose density centrifugation was performed as described in Ref. 27Yoder-Hill J. Pause A. Sonenberg N. Merrick W.C. J. Biol. Chem. 1993; 268: 5566-5573Abstract Full Text PDF PubMed Google Scholar. Radiolabeled [14C]eIF4F (150 pmol, 30 μg) was incubated alone or with equimolar (150 pmol, 100 μg) amounts of unlabeled eIF3 in a 100-μl volume containing 20 mm Hepes-KOH, pH 7.5, 100 mm KCl, 1 mm MgCl2, 1 mm DTT, and 0.1 mm EDTA. Reactions were then layered on a 5–18% sucrose gradient (20 mm Tris-HCl, pH 7.5, 100 mm KCl, 1 mm MgCl2, 1 mm DTT) and centrifuged at 32,000 rpm for 18.5 h at 4 °C (Beckman SW60 rotor). To test the effect of Mup56 on this interaction, purified Mup56 (150 pmol, 8.4 μg) was preincubated with eIF3 for 10 min at 30 °C prior to addition of 14C-labeled eIF4F. Gradients were unloaded via needle syringe and 1-ml fractions collected. The radioactivity in 200 μl of each fraction was determined by liquid scintillation spectrometry. Ternary Complex Assay—Ternary complex formation was performed as described previously (29Merrick W.C. Methods Enzymol. 1979; 60: 108-123Crossref PubMed Scopus (79) Google Scholar). Purified eIF2 (8 pmol, 1.0 μg) was incubated with 10 pmol of [14C]Met-tRNAi and 100 μm GTP in a 100-μl reaction volume containing 20 mm Tris-HCl, pH 7.5, 100 mm KCl, 2 mm MgCl2 1 mm DTT, 0.3 IU pyruvate kinase, and 4 mm phosphoenolpyruvate. To stimulate ternary complex formation, 30 pmol (20 μg) of purified eIF3 was also added to the reaction. Reactions were incubated for 10 min at 37 °C and then immediately quenched with 3 ml of ice-cold 20 mm Tris-HCl, pH 7.5, 100 mm KCl, and 5 mm MgCl2 (quenching buffer). Ternary complex was bound to the nitrocellulose filter (25 mm, 0.45-mm pore size, Millipore) by adding the entire reaction mixture to the filter, followed by vacuum filtration. Filters were washed twice with 5 ml of quenching buffer, followed by vacuum filtration. Filters were dried under a heat lamp, and radioactivity was then determined by liquid scintillation spectrometry. To test the effect of Mup56 on ternary complex formation, 30 pmol (1.7 μg) of purified Mup56 was preincubated with eIF3 for 10 min at 30 °C prior to their addition to the ternary complex reaction mixture. Preinitiation Complex Sucrose Gradient Analysis—Sucrose gradients (10–25%) were prepared exactly as described previously for the ribosome dissociation assay except 100 μm GDPNP was included in the mixture (30Peterson D.T. Merrick W.C. Safer B. J. Biol. Chem. 1979; 254: 2509-2516Abstract Full Text PDF PubMed Google Scholar). Thirty picomoles of eIF3 was preincubated either alone or with 30 pmol of mouse p56 for 15 min at 30 °C. One A260 unit of purified 40 S subunits was then added and incubated for 10 min at 37 °C. The ternary complex was formed separately as described previously in the ternary complex assay except amounts of 30 pmol of [14C]Met-tRNAi and 30 pmol of eIF2 (3.8 μg) were used. This reaction was added directly to the 40 S ribosome/eIF3 mixture. In some cases, where indicated, 30 pmol of radiolabeled 14C-labeled eIF4F was added to this reaction. Gradients were centrifuged for 16 h at 20,000 rpm at 4 °C (Beckman SW28Ti rotor) and unloaded via upward displacement using 60% sucrose, and UV absorbance was measured at 254 nm with an ISCO flow cell. Fractions were then directly measured for radioactivity by liquid scintillation spectrometry or subjected to a 95 °C trichloroacetic acid precipitation step after fractionation in order to remove the contribution of the radiolabel in the [14C]Met-tRNAi. Precipitated protein was then collected on nitrocellulose filters (25 mm, 0.45-mm pore size, Millipore), and radioactivity was determined by liquid scintillation spectroscopy. Mouse p56 Inhibits Translation—Mup56 and Hup56 are similar sized proteins, although they have only 50% sequence identity. However, both proteins contain six TPR motifs that are similarly spaced (Fig. 1). These motifs are defined by typical patterns of small and large hydrophobic residues, although no residues are completely invariant. The sequences within the same TPR motifs of the two proteins are highly conserved. For example, of the 34 residues of TPR2, 23 residues are identical and 6 more are similar (Fig. 1). To examine the functions of Mup56, we expressed a hexahistidine-tagged recombinant Mup56 in E. coli, and we purified it by affinity chromatography on a nickel-agarose column. The purified protein was then tested for its ability to inhibit translation in vitro. For this purpose, increasing quantities of Mup56 or Hup56 were added to a rabbit reticulocyte lysate system programmed with luciferase mRNA. Quantitation of the radiolabeled products revealed that both proteins inhibited translation in a dose-dependent fashion. Moreover, their potencies were similar: 50% inhibition was observed with about 50 nm concentration of each protein (Fig. 2). By having established that Mup56 is a potent inhibitor of translation, the underlying mechanism was further investigated.Fig. 2Recombinant Hup56 and Mup56 inhibit translation in vitro. Luciferase mRNA was translated by using a rabbit reticulocyte lysate system containing [35S]methionine in the presence of dialysis buffer (0 nm) or 50, 200, 400, and 800 nm recombinant Hup56 (A) or Mup56 protein (B). Translated luciferase was analyzed by SDS-PAGE and quantitated using a PhosphorImager (Amersham Biosciences). Inhibition is shown as a percentage of the buffer control (100% translation). Experiment was performed in duplicate. Results are shown as a percentage of the maximal level of translation achieved (0 nm of protein added), ∼17,000 cpm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Mup56 Binds to eIF3c—We first examined whether Mup56, like Hup56, binds to eIF3. Size fractionation analysis by gel filtration chromatography showed that Mup56 existed as dimers and monomers (Fig. 3A). In the presence of purified eIF3, a portion of Mup56 appeared in fractions that contained eIF3, indicating the binding of the two proteins (Fig. 3B). Similar to what was observed with Hup56 (2Guo J. Hui D.J. Merrick W.C. Sen G.C. EMBO J. 2000; 19: 6891-6899Crossref PubMed Scopus (178) Google Scholar) some Mup56 also appeared in the intermediate fractions (Fig. 3B, fractions 24–32), probably due to partial dissociation of the eIF3·Mup56 complex during gel filtration. Binding of Mup56 to eIF3 was confirmed by pull-down and immunoprecipitation assays (Fig. 4). When purified Mup56 and eIF3 were mixed in vitro, pulling down Mup56 by using its hexahistidine tag (Fig. 4A) or immunoprecipitation of eIF3 by its cognate antibody (Fig. 4B) resulted in co-purification of the associated partners (Fig. 4, A and B, lane 2). Appropriate controls (Fig. 4, A and B, lanes 1 and 3) showed that the binding was efficient and specific. Moreover, the Western blot with the whole eIF3 antibody showed that Mup56 appeared to bind specifically to the c subunit of the eIF3 complex (Fig. 4A).Fig. 4Mup56 binds to eIF3 in vitro. Six micrograms of purified eIF3 was incubated alone or in the presence of 1 μg of His-Mup56, and pull-down or immunoprecipitation assays were performed. Samples were then subjected to SDS-PAGE followed by Western blot. A, nickel-nitrilotriacetic acid-agarose was used to pull down Mup56, and Western blotting was with eIF3 antibody. Lane 1, input eIF3 without pull-down. Lane 2, eIF3 and Mup56 after pull-down. Lane 3, only eIF3 after pull-down. B, eIF3 antibody was used for immunoprecipitation, and Western blot was performed with His antibody. Lane 1, input Mup56 without immunoprecipitation. Lane 2, Mup56 and eIF3 immunoprecipitated with the eIF3 antibody. Lane 3, Mup56 and eIF3 immunoprecipitated without the eIF3 antibody.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because Hup56 binds to the e subunit of eIF3, the above result was unexpected. Hence we carried out a series of experiments to examine the specificities of in vivo interactions between p56 proteins and eIF3c/eIF3e subunits (Fig. 5). For this purpose, Myc-tagged p56 proteins were expressed in human cells along with FLAG-tagged eIF3c or eIF3e. Each partner was immunoprecipitated or affinity-purified from the cell lysates, and the presence of the other partner in the precipitate was detected by Western blotting. The levels of expression of the transfected proteins and endogenous actin were measured by straight Western blotting of cell lysates with appropriate antibodies. When Mup56 was pulled down, eIF3c co-purified with it (Fig. 5A, lane 2). Similarly, when eIF3c was immunoprecipitated, Mup56 was bound to it (Fig. 5B, lane 2) but Hup56 was not (Fig. 5B, lane 3). When eIF3e and p56 were co-expressed and eIF3e was immunoprecipitated, Hup56 interacted with it (Fig. 5C, lane 2) but Mup56 did not (Fig. 5C, lane 4). These results clearly showed that in vivo Mup56 and Hup56 specifically bound to the c and the e subunits of eIF3, respectively. Both these proteins contain the PCI domain and predicted nuclear localization signals (Fig. 5D). Mup56 Does Not Inhibit Several Functions of eIF3—We have shown previously that the inhibitory effect of Hup56 on translation initiation is primarily mediated by its ability to block the role of eIF3 in the stabilization of the eIF2·GTP·Met-tRNAi ternary complex. The assay used to demonstrate th" @default.
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