Recombinant Subunits of Mammalian Elongation Factor 1 Expressed in Escherichia coli
1997; Elsevier BV; Volume: 272; Issue: 52 Linguagem: Inglês
10.1074/jbc.272.52.33290
ISSN1083-351X
AutoresGwo‐Tarng Sheu, Jolinda A. Traugh,
Tópico(s)RNA Research and Splicing
ResumoThe first step in elongation requires two different activities; elongation factor (EF)-1α transfers aminoacyl-tRNA to the ribosome and is released upon hydrolysis of GTP, EF-1βγδ catalyzes exchange of GDP on EF-1α with GTP. To analyze the role of the individual subunits of EF-1 in elongation, the cDNAs for the β, γ, and δ subunits of EF-1 from rabbit were cloned, and proteins of 225, 437, and 280 amino acids, respectively, were expressed in Escherichia coli. The purified recombinant β subunit migrates as a dimer and the γ subunit as a trimer upon gel filtration, whereas the δ subunit forms a large aggregate. Complexes of βγ, γδ and βγδ were formed by self-association and eluted with a molecular mass of approximately 160, 530, and 670 kDa, respectively; no interaction was observed between β and δ. The activity of the recombinant subunits was determined with native EF-1α by measuring stimulation of the rate of elongation by poly(U)-directed polyphenylalanine synthesis. Recombinant β and δ alone stimulated the rate of elongation by 10-fold, with a ratio of 5α:2β or δ. The βγδ complex stimulated EF-1α activity up to 10-fold with a ratio of 20α to 1βγδ. Phosphorylation of the β and δ subunits alone or in βγδ by protein kinase CKII had no effect on the rate of elongation. The first step in elongation requires two different activities; elongation factor (EF)-1α transfers aminoacyl-tRNA to the ribosome and is released upon hydrolysis of GTP, EF-1βγδ catalyzes exchange of GDP on EF-1α with GTP. To analyze the role of the individual subunits of EF-1 in elongation, the cDNAs for the β, γ, and δ subunits of EF-1 from rabbit were cloned, and proteins of 225, 437, and 280 amino acids, respectively, were expressed in Escherichia coli. The purified recombinant β subunit migrates as a dimer and the γ subunit as a trimer upon gel filtration, whereas the δ subunit forms a large aggregate. Complexes of βγ, γδ and βγδ were formed by self-association and eluted with a molecular mass of approximately 160, 530, and 670 kDa, respectively; no interaction was observed between β and δ. The activity of the recombinant subunits was determined with native EF-1α by measuring stimulation of the rate of elongation by poly(U)-directed polyphenylalanine synthesis. Recombinant β and δ alone stimulated the rate of elongation by 10-fold, with a ratio of 5α:2β or δ. The βγδ complex stimulated EF-1α activity up to 10-fold with a ratio of 20α to 1βγδ. Phosphorylation of the β and δ subunits alone or in βγδ by protein kinase CKII had no effect on the rate of elongation. Eukaryotic elongation factor (EF) 1The abbreviations used are: EF, elongation factor; ValRS, valyl-tRNA synthetase; CKII, protein kinase CKII; IPTG, isopropyl-β-d-thiogalactopyranoside; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s); FPLC. fast protein liquid chromatography. 1The abbreviations used are: EF, elongation factor; ValRS, valyl-tRNA synthetase; CKII, protein kinase CKII; IPTG, isopropyl-β-d-thiogalactopyranoside; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s); FPLC. fast protein liquid chromatography. 1 consists of four subunits, EF-1α, β, γ, and δ. EF-1α (50 kDa) forms a ternary complex with GTP and aminoacyl-tRNA and transfers the aminoacyl-tRNA to 80 S ribosomes with the hydrolysis of GTP. EF-1βγδ facilitates the exchange of the GDP bound to EF-1α for GTP, initiating another round of elongation (1Riis B. Rattan S.I.S. Clark B.F.C. Merrick W.C. Trends Biochem. Sci. 1990; 15: 420-424Abstract Full Text PDF PubMed Scopus (281) Google Scholar, 2Moldave K. Annu. Rev. Biochem. 1985; 54: 1109-1149Crossref PubMed Scopus (357) Google Scholar). EF-1β and δ contain the GTP exchange activity (3van Damme H.T.F. Amons R. Karssies R. Timmers C.J. Janssen G.M.C. Möller W. Biochim. Biophys. Acta. 1990; 1050: 241-247Crossref PubMed Scopus (92) Google Scholar); γ is tightly associated to β and is removed only under denaturing conditions (4Janssen G.M.C. van Damme H.T.F. Kriek J. Amons R. Möller W. J. Biol. Chem. 1994; 269: 31410-31417Abstract Full Text PDF PubMed Google Scholar). The cDNAs for β and γ have been cloned and sequenced from a number of different organisms and tissues (5Chen C.-J. Traugh J.A. Biochim. Biophys. Acta. 1995; 1264: 303-311Crossref PubMed Scopus (10) Google Scholar, 6Maessen G.D.F. Amons R. Maassen J.A. Möller W. FEBS Lett. 1986; 208: 77-83Crossref Scopus (35) Google Scholar, 7Cormier P. Osborne H.B. Morales J. Bassez T. Minella O. Poulhe R. Bellé R. Mulner-Lorillon O. Nucleic Acids Res. 1993; 21: 743Crossref PubMed Scopus (23) Google Scholar, 8von der Kammer H. Klaudiny J. Zimmer M. Scheit K.H. Biochem. Biophys. Res. Commun. 1991; 177: 312-317Crossref PubMed Scopus (26) Google Scholar, 9Sanders J. Maassen J.A. Amons R. Möller W. Nucleic Acids Res. 1991; 19: 4551Crossref PubMed Scopus (32) Google Scholar, 10Maessen G.D.F. Amons R. Zeelen J.P. Möller W. FEBS Lett. 1987; 223: 181-186Crossref PubMed Scopus (28) Google Scholar, 11Cormier P. Osborne H.B. Morales J. Bassez T. Poulhe R. Mazabraud A. Mulner-Lorillon O. Bellé R. Nucleic Acids Res. 1991; 19: 6644Crossref PubMed Scopus (25) Google Scholar, 12Sanders J. Maassen J.A. Möller W. Nucleic Acids Res. 1992; 20: 5907-5910Crossref PubMed Scopus (37) Google Scholar, 13Sheu G.-T. Traugh J.A. Nucleic Acids Res. 1993; 20: 5849Crossref Scopus (6) Google Scholar). Recently, EF-1δ cDNA has been sequenced from human (14Sanders J. Raggiaschi R. Morales J. Möller W. Biochim. Biophys. Acta. 1993; 1174: 87-90Crossref PubMed Scopus (42) Google Scholar) and Xenopus (15Morales J. Cormier P. Mulner-Lorillon O. Poulhe R. Belle' R. Nucleic Acids Res. 1992; 20: 4091Crossref PubMed Scopus (38) Google Scholar), and partial amino acid sequences have been obtained from Artemia (14Sanders J. Raggiaschi R. Morales J. Möller W. Biochim. Biophys. Acta. 1993; 1174: 87-90Crossref PubMed Scopus (42) Google Scholar). A leucine zipper motif in the amino terminus of δ is found in all three species (14Sanders J. Raggiaschi R. Morales J. Möller W. Biochim. Biophys. Acta. 1993; 1174: 87-90Crossref PubMed Scopus (42) Google Scholar). EF-1β and δ have a highly homologous carboxyl terminus that contains the GDP/GTP exchange activity, whereas the amino-terminal domains differ and appear to be important for regulation of EF-1 activity. The function of the γ subunit is unknown, although there is evidence that γ can stimulate the nucleotide exchange activity of β (16Janssen G.M.C. Möller W. Eur. J. Biochem. 1988; 171: 119-129Crossref PubMed Scopus (106) Google Scholar). EF-1γ may anchor the complex to the membrane (16Janssen G.M.C. Möller W. Eur. J. Biochem. 1988; 171: 119-129Crossref PubMed Scopus (106) Google Scholar) and has been shown to contain a sequence homologous to glutathione S-transferase in the amino-terminal domain, which has been postulated to be involved in regulation of the assembly of multisubunit complexes (17Koonin E.V. Mushegian A.R. Tatusov R.L. Altschul S.F. Bryant S.H. Bork P. Valencia A. Prot. Sci. 1994; 3: 2045-2054Crossref PubMed Scopus (126) Google Scholar). Eukaryotic EF-1 has been isolated from a variety of organisms and tissues with molecular weights ranging from 50,000 to about 1 × 106. The low molecular weight form is EF-1α, the intermediate form is EF-1αβγδ, and the high molecular weight form is a complex of five polypeptides, valyl-tRNA synthetase (ValRS) and EF-1αβγδ (18Motorin Y.A. Wolfson A.D. Orlovsky A.F. Gladilin K.L. FEBS Lett. 1988; 238: 262-264Crossref PubMed Scopus (57) Google Scholar, 19Bec G. Waller J.P. J. Biol. Chem. 1989; 264: 21138-21143Abstract Full Text PDF PubMed Google Scholar, 20Venema R.C. Peters H.I. Traugh J.A. J. Biol. Chem. 1991; 266: 11993-11998Abstract Full Text PDF PubMed Google Scholar). Using purified subunits of EF-1 from Artemia, the formation of complexes of αβ (21van Damme H.T.F. Amons R. Möller W. Eur. J. Biochem. 1992; 207: 1025-1034Crossref PubMed Scopus (20) Google Scholar), αβγ, αβγδ, and αδ (4Janssen G.M.C. van Damme H.T.F. Kriek J. Amons R. Möller W. J. Biol. Chem. 1994; 269: 31410-31417Abstract Full Text PDF PubMed Google Scholar) were observed under nondenaturing conditions. β and γ, separated under denaturing conditions, were unable to reassociate. Reconstitution of a γδ complex was also unsuccessful (4Janssen G.M.C. van Damme H.T.F. Kriek J. Amons R. Möller W. J. Biol. Chem. 1994; 269: 31410-31417Abstract Full Text PDF PubMed Google Scholar). With purified subunits prepared from rabbit EF-1·ValRS, Bec et al. (22Bec G. Kerjan P. Waller J.P. J. Biol. Chem. 1994; 269: 2086-2092Abstract Full Text PDF PubMed Google Scholar) were able to reconstitute the EF-1βγδ·ValRS complex; EF-1δ was required for association of ValRS. The β subunit of EF-1 from Artemia was shown to be phosphorylated by an endogenous CKII-like protein kinase that copurified with EF-1βγ. The phosphorylation site was Ser89 in the sequence GS89DEEDEE. When EF-1βγ was treated with alkaline phosphatase to remove the phosphate, the nucleotide exchange rate was almost twice that of phosphorylated EF-1βγ (23Janssen G.M.C. Maessen G.D.F. Amons R. Möller W. J. Biol. Chem. 1988; 263: 11063-11066Abstract Full Text PDF PubMed Google Scholar). In other studies, EF-1β from Artemia, rabbit and wheat germ was shown to be phosphorylated by CKII (24Palen E. Huang T.T. Traugh J.A. FEBS Lett. 1990; 274: 12-14Crossref PubMed Scopus (18) Google Scholar). Using recombinant EF-1β from rabbit, Ser106 and Ser112 in the sequence of DLFGS106DDEEES112EEA were phosphorylated by CKII (5Chen C.-J. Traugh J.A. Biochim. Biophys. Acta. 1995; 1264: 303-311Crossref PubMed Scopus (10) Google Scholar). In this study, the cDNA for rabbit EF-1δ has been cloned and sequenced. Recombinant EF-1β, γ, and δ subunits were expressed in Escherichia coli, purified and reconstituted to form complexes of βγ, βγδ, and γδ, but not βδ, as analyzed by gel filtration. The activity of the individual subunits and reconstituted complexes was measured by stimulation of elongation with native EF-1α, and the effects of phosphorylation of the β and δ subunits and EF-1βγδ on elongation were analyzed. [γ-32P]ATP was purchased from ICN. 35S-labeled nucleotides andl-[2,3,4,5,6-3H]phenylalanine (127 Ci/mmol) were from Amersham Corp. The cDNA for EF-1δ from human was a gift from Dr. Wim Moller, University of Leiden, The Netherlands. CKII was purified from rabbit reticulocytes as described previously (25Palen E. Traugh J.A. Biochemistry. 1991; 30: 5586-5590Crossref PubMed Scopus (31) Google Scholar) and provided by William Meek. Rabbit EF-1α was a gift from Dr. William C. Merrick, Case Western Reserve University School of Medicine, Cleveland, OH. To isolate the cDNA for EF-1δ from a rabbit spleen library, human EF-1δ cDNA was digested with PstI to obtain a 374-bp amino-terminal fragment to use as a probe. The cDNA fragment was radiolabeled with the random primer labeling method (26Feinberg A.P. Vogelstein B.V. Anal. Biochem. 1984; 137: 266-267Crossref PubMed Scopus (5169) Google Scholar) and used to screen the cDNA library by in situ plaque hybridization. After the third screening, several positive clones were identified and these cDNAs were transformed into E. coli XL1-Blue cells. Recombinant plasmids purified from single colonies were cleaved with EcoRI and XhoI followed by hybridization with the radiolabeled probe. The cDNA containing the longest insert was selected for cDNA sequencing and a set of nested deletions (27Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982: 13.39-13.41Google Scholar) was constructed. The EcoRI/BstXI and XhoI/KpnI sites were chosen for the construction of sense and antisense cDNA deletion clones, respectively. Sequencing of individual cDNA was carried out using the dideoxynucleotide chain termination method (28Sanger F. Nicklen S. Coulsen A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52211) Google Scholar) with T7 and T3 primers. Rabbit EF-1β was cloned from a rabbit spleen cDNA library, sequenced, and expressed in E. coli as described by Chen and Traugh (5Chen C.-J. Traugh J.A. Biochim. Biophys. Acta. 1995; 1264: 303-311Crossref PubMed Scopus (10) Google Scholar). The cDNA for EF-1γ, cloned and sequenced from the rabbit spleen cDNA library (13Sheu G.-T. Traugh J.A. Nucleic Acids Res. 1993; 20: 5849Crossref Scopus (6) Google Scholar), and the cDNA for rabbit EF-1δ were subcloned into the pT7–7 expression vector. Synthetic oligonucleotides were prepared that contained a generated NdeI site in the 5′-end sense primer; (5′-3TTCGGCATATG GCGGCCGGGA23-3′) for γ and (5′-52AGGCATATG ACGACGAACTTCCTAG77-3′) for δ. The restriction sites are indicated by an underlineand the start codon ATG is indicated as bold. The sense and antisense primers were used for amplification of the cDNA by the polymerase chain reaction for 30 cycles (29Mullis K.B. Faloona F.A. Methods Enzymol. 1987; 155: 335-350Crossref PubMed Scopus (3785) Google Scholar). The amplified DNA products were analyzed by agarose gel electrophoresis. The purified cDNA fragment corresponding to EF-1δ was digested with NdeI and HindIII restriction enzymes to produce the sites for subcloning of the cDNA coding region into the pT7–7 vector. The recombinant plasmids were subcloned into competent BL21(DE3) cells by the CaCl2 precipitation method (30Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982: 1.82-1.84Google Scholar). Expression of recombinant EF-1β and γ in E. coli was induced with 0.4 mm IPTG for 2 h at 37 °C, and the supernatant and pellet were prepared as described by Chen and Traugh (5Chen C.-J. Traugh J.A. Biochim. Biophys. Acta. 1995; 1264: 303-311Crossref PubMed Scopus (10) Google Scholar). An overnight culture of BL21(DE3) cells transformed with pT7–7-δ was added to two flasks containing 25 ml of LB medium and 100 μg/ml ampicillin. The cells were grown at 37 °C to a density of 0.5 at A 600 and then induced with 0.4 mmIPTG. One culture was incubated at 37 °C for 2 h, and the other was incubated at 28 °C for 19 h. Samples (1 ml) were resuspended in 0.2 ml of SDS-sample buffer, and expression of the δ subunit was analyzed by SDS-PAGE on 10% polyacrylamide gels (31Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205400) Google Scholar) and stained with Coomassie Blue. The remainder of the E. coliwere harvested, and the pellet and supernatant were obtained as described above. The supernatant obtained from 250 ml of cultured E. coli containing EF-1β or γ was chromatographed on a DEAE cellulose column equilibrated with buffer (50 mm Tris-HCl, pH 7.4, 1 mm EGTA, 1 mm EDTA, 0.02% NaN3, 1 mmdithiothreitol, 10% glycerol, 0.2 mm phenylmethylsulfonyl fluoride) as described (5Chen C.-J. Traugh J.A. Biochim. Biophys. Acta. 1995; 1264: 303-311Crossref PubMed Scopus (10) Google Scholar). Elution was carried out with a linear gradient of 0–0.5 m NaCl (200 ml) in buffer for EF-1β, and 0–0.3 m NaCl (200 ml) for EF-1γ. EF-1β or γ was purified further by FPLC on a Mono Q HR5/5 column as described (5Chen C.-J. Traugh J.A. Biochim. Biophys. Acta. 1995; 1264: 303-311Crossref PubMed Scopus (10) Google Scholar) with a 10-ml gradient of 0–1.0 m NaCl in buffer. Aliquots (20 μl) of the fractions were analyzed on a 10% gel by SDS-PAGE and stained with Coomassie Blue. E. coli(500 ml) containing the pT7–7-δ insert were grown at 28 °C for 19 h and harvested, and the supernatant was chromatographed on a DEAE-cellulose column (2.5 × 15-cm). The column was washed with 120 ml of 200 mm NaCl in buffer, and δ was eluted in a single step with 700 mm NaCl in buffer. EF-1δ (0.5 ml) was chromatographed on a Superose 12 column equilibrated with buffer containing 50 mm KCl; 0.35-ml fractions were collected. EF-1δ was stored in aliquots at −70 °C for further analysis. Equimolar amounts of the β (400 μg) and γ (815 μg) subunits in a volume of 3.5 ml were incubated at 4 °C for 5 min and then diluted 5-fold with buffer to reduce the conductivity. The complex was isolated by chromatography on a DEAE-cellulose column (1.2 × 8.0 cm) and eluted with a 50-ml gradient of 0–500 mm NaCl in buffer. Fractions containing βγ were pooled, and the complex was purified by gel filtration as described below. Purified EF-1β (30 μg), γ (100 μg), and δ (40 μg) in a final volume of 0.6 ml, and purified EF-1 γ (50 μg) and δ (55 μg) in a final volume of 1.5 ml were incubated on ice for 30 min. Each sample was analyzed separately by FPLC on Mono Q with a 10-ml gradient of 0–1.0m NaCl in buffer. Fractions containing the highest amounts of βγδ or γδ were selected for further analysis by gel filtration. EF-1β and γ (0.5 ml) purified by chromatography on Mono Q were analyzed separately on a Superose 12 column equilibrated with buffer containing 100 or 500 mm NaCl. Fractions of 0.35 ml were collected. Aliquots of 20 μl were analyzed by SDS-PAGE. The protein standards for gel filtration were blue dextran (V0), thyroglobulin (670 kDa), IgG (160 kDa), β-lactoglobulin (35 kDa), and cytochrome C (12 kDa). Aliquots (0.5 ml) of the EF-1βγ complex isolated by DEAE-cellulose chromatography and EF-1βγδ and γδ isolated by FPLC on Mono Q were analyzed separately on a Superose 12 column at 4 °C. EF-1β (40 μg) and δ (17 μg) incubated together (0.5 ml) on ice were also analyzed for complex formation. The column was equilibrated with 200 mm KCl in buffer. Fractions of 0.35 ml were collected. Aliquots (20 μl) from each fraction were analyzed by gel electrophoresis, and the proteins were visualized by staining with Coomassie Blue. The stained proteins in the gels were scanned with a LKB laser scanner and quantified. Similar results were obtained when the ratio of each subunit in the complexes was determined according to the content of basic residues (lysine, arginine, and histidine) in each subunit (32Tal M. Silberstein A. Nusser E. J. Biol. Chem. 1985; 260: 9976-9980Abstract Full Text PDF PubMed Google Scholar). EF-1β contained 29 basic residues, γ contained 62, and δ contained 44. Recombinant EF-1βγδ (1.0 μg) was incubated in 0.07 ml reaction mixtures containing 50 mm Tris-HCl, pH 7.4, 50 mm KCl, 10 mm MgCl2, 0.14 mm [γ-32P]ATP (2,000 cpm/pmol) in the presence and absence of 30 units of CKII at 30 °C for 30 min. Phosphorylation of β and δ was determined by autoradiography following SDS-PAGE and 32P was quantified by excising the proteins and counting in a scintillation counter. The radiolabeled ATP was replaced with unlabeled ATP for analysis of elongation activity by poly(U)-dependent polyphenylalanine synthesis. The samples were kept on ice prior to assaying for elongation activity. The activity of native EF-1α alone and in combination with the β, γ, and δ subunits was analyzed by poly(U)-dependent [3H]polyphenylalanine synthesis as described by Venema et al. (20Venema R.C. Peters H.I. Traugh J.A. J. Biol. Chem. 1991; 266: 11993-11998Abstract Full Text PDF PubMed Google Scholar). The specific activity of [3H]phenylalanyl-tRNA was 3,100 dpm/pmol. Native EF-1α was preincubated alone and with the indicated subunits at 30 °C for 10 min, then added to the elongation assay mixture. Similar results were obtained by preincubation at 0 °C for 30 min. Protein concentrations of α, β, γ, and δ were determined by the Bradford method (33Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211519) Google Scholar) with bovine serum albumin as a standard. The cDNA for EF-1δ isolated from a rabbit spleen library was 958 bp and contained the entire 840-bp coding region for EF-1δ, a 57-bp 5′-untranslated region, a 43-bp 3′-untranslated region and a poly(A) tail of 18 nucleotides. The derived amino acid sequence consisted of 280 amino acids with a calculated molecular weight of 31,075 and pI of 4.91 (Fig. 1) and was 91% homologous with human EF-1δ, which contained an additional asparagine at position 164. Comparing the sequences of EF-1δ from rabbit with those from human, Xenopus, and the partial sequence from Artemia, the carboxyl terminus containing the nucleotide exchange activity (3van Damme H.T.F. Amons R. Karssies R. Timmers C.J. Janssen G.M.C. Möller W. Biochim. Biophys. Acta. 1990; 1050: 241-247Crossref PubMed Scopus (92) Google Scholar) was more highly conserved between species than the amino terminus, which contained a leucine zipper domain. The homologous carboxyl-terminal region started at residue Glu155 followed by a region rich in acidic amino acids. The conserved portion of the amino terminus contained a leucine zipper from Leu80 to Leu115 consisting of 6 leucine residues. A site on EF-1δ, Thr122, shown to be phosphorylated in Xenopus by p34 cdc2 kinase (34Mulner-Lorillon O. Minella O. Cormier P. Capony J.-P. Cavadore J.-C. Morales J. Poulhe R. Bellé R. J. Biol. Chem. 1994; 269: 20201-20207Abstract Full Text PDF PubMed Google Scholar), was not present in rabbit and human, which contained a glutamic acid at the corresponding residue 139 (Fig. 1). The cDNA for EF-1δ was cloned into the pT7–7 vector and expressed in E. coli. Following induction with IPTG for 2 h at 37 °C, the protein was present in significant amounts, primarily (90%) as inclusion bodies (Fig.2). Upon induction and incubation at 28 °C, the majority (75%) of EF-1δ was present as soluble protein. The supernatant obtained at 28 °C was used for purification of the soluble δ subunit by chromatography on DEAE-cellulose, and eluted at 700 mm NaCl using a step gradient (data not shown). The δ subunits were pooled and purified to apparent homogeneity on Superose 12 as shown below. EF-1β, cloned into the pT7–7 expression vector, was present primarily as a soluble form at 37 °C when analyzed by SDS-PAGE (Fig.3, left panel). EF-1β was purified from the supernatant by chromatography on DEAE-cellulose and FPLC on Mono Q. The β subunit eluted from DEAE-cellulose at 250 mm NaCl (data not shown) and from Mono Q at 560 mm NaCl. From a 250 ml culture containing 42.9 mg of soluble protein, 5.4 mg of β were recovered after chromatography on DEAE-cellulose. Further purification on Mono Q yielded 2.2 mg of pure β. EF-1γ was expressed almost exclusively as a soluble protein at 37 °C. Highly purified EF-1γ was obtained after chromatography of the supernatant on DEAE-cellulose, where it eluted at 75 mmNaCl (data not shown), and by FPLC on Mono Q, eluting at 450 mm NaCl. From a culture of 250 ml containing 46.6 mg of soluble protein, 5.6 mg of EF-1γ were recovered after DEAE-cellulose. The γ subunit was purified to apparent homogeneity by FPLC on Mono Q (Fig. 3, right panel); 1.5 mg of EF-1γ were obtained. When recombinant EF-1β (24.8 kDa) was analyzed by gel filtration on Superose 12, the protein eluted with a molecular mass of about 50 kDa (Fig. 4 A). The same elution pattern was observed at 50, 100, and 500 mm NaCl. Recombinant EF-1δ (31.0 kDa) migrated as a large aggregate in the void volume at 50, 200, and 600 mm NaCl. The addition of 0.1% Triton X-100 with 600 mm NaCl had no effect on the state of aggregation (Fig. 4 B). Unlike the β and δ subunits, the salt concentration during gel filtration was important for the γ subunit. Recombinant EF-1γ (50.0 kDa) migrated as a large aggregate when analyzed on Superose 12 at 100 mm NaCl (Fig.5, top panel). At 500 mm NaCl, the γ subunit migrated as a distinct peak of about 140 kDa (Fig. 5, bottom panel).Figure 5Gel filtration of EF-1γ expressed in E. coli. The recombinant γ subunit of EF-1 expressed in E. coli was purified as described under “Experimental Procedures” and analyzed by FPLC on Superose 12. Aliquots (20 μl) of the fractions were analyzed on 10% polyacrylamide gels, and the protein was stained with Coomassie Blue. Purified EF-1γ (0.5 ml from Mono Q) was eluted with buffer containing 100 mm NaCl (upper panel). EF-1γ was eluted with buffer containing 500 mm NaCl (lower panel).View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine whether recombinant EF-1β and γ were able to form a complex similar to that of native EF-1βγ, equimolar amounts of purified β and γ were incubated together for 5 min at 4 °C. Both subunits co-chromatographed at 200 mm NaCl on DEAE-cellulose (data not shown). On Superose 12, the βγ complex eluted with an apparent molecular mass of about 160 kDa (Fig.6 A). The ratio of β and γ subunits in the complex was 1:1 as determined by densitometric scanning of the polyacrylamide gel following electrophoresis. Because the calculated molecular mass for βγ is 74.8 kDa, the results suggested a heterodimer was formed. Purified EF-1γ and δ were combined in a ratio of 1:2 and incubated at 4 °C for 15 min. The proteins co-chromatographed during FPLC on Mono Q, eluting at 600 mm NaCl (data not shown). Upon gel filtration (Fig. 6 B), γ and δ migrated as a large molecular mass of ∼530 kDa but not in the void volume as observed with δ alone (Fig. 4 B). The ratio of γδ subunits was 2:1. The calculated molecular mass of γ2δ was 131 kDa, considerably smaller than the size determined by gel filtration, indicating the γδ complex was at least a heterodimer (γ4δ2) and possibly larger. Recombinant EF-1β and δ, combined in a ratio of 3:1 and incubated at 4 °C for 15 min, showed no interaction (Fig. 6 C). EF-1δ migrated in the void volume as a large aggregate as observed with the pure protein, whereas all of β migrated around 50 kDa apparently as a dimer. For reconstitution of the βγδ complex, the subunits were combined at a molar ratio of 1:2:1, respectively, and incubated for 15 min at 4 °C. To remove any free subunits, the βγδ complex was isolated by FPLC on Mono Q (data not shown). The peak of the complex chromatographing at 600 mm NaCl was selected for further analysis by gel filtration to confirm the existence of the subunit interactions and to determine the approximate size of the complex. EF-1βγδ migrated as a large complex of ∼670 kDa (Fig.6 D). The ratio of the β, γ, and δ subunits was 1β:2γ:1δ, as determined by densitometric scanning following SDS-PAGE. With a calculated molecular mass of 155.8 kDa for the complex, the results of gel filtration suggested that βγ2δ was a multimer. The activity of native EF-1α from rabbit reticulocytes was measured by poly(U)-directed polyphenylalanine synthesis. With 10–40 pmol of EF-1α (0.5–2.0 μg), the elongation rate was linear over a 30-min incubation period (data not shown). To measure the effect of the reconstituted βγδ complex on EF-1α activity, changes in the rate of elongation upon addition of the nucleotide exchange complex were monitored using 10 pmol of EF-1α and increasing concentrations of recombinant EF-1βγδ (0.1–0.9 pmol). In the absence of EF-1 βγδ, 1.4 pmol of polyphenylalanine were synthesized (Fig. 7). A maximal stimulation of approximately 10-fold was observed at 0.5 pmol of EF-1βγδ with the molar ratio of 20α:1βγδ. Thus, the reconstituted βγδ complex was functionally active in stimulating elongation. EF-1β and δ were analyzed individually for the ability to stimulate elongation as shown in Fig. 8. Polyphenylalanine synthesis was stimulated 10-fold with EF-1β. EF-1δ also stimulated polyphenylalanine synthesis by 10-fold. However, the amount of the individual β and δ subunits required for stimulation of elongation was significantly greater (5α:2β or 2δ) than the optimal concentration of EF-1βγδ. CKII has been shown to phosphorylate the β and δ subunits of EF-1 (35Palen E. Venema R.C. Chang Y.-W.E. Traugh J.A. Biochemistry. 1994; 33: 8515-8520Crossref PubMed Scopus (14) Google Scholar). When the purified recombinant subunits were examined separately as substrates for CKII, 0.6 and 0.8 mol/mol of phosphate were incorporated into the β and δ subunits, respectively. When these subunits were analyzed for the ability to stimulate the elongation activity of the α subunit, little or no effect of phosphorylation was observed over that obtained with the non-phosphorylated subunits (Table I).Table IStimulation of EF-1α activity by EF-1β and δ and the effects of phosphorylation by CKII on elongationSubunits of EF-1Polyphenylalanine synthesizedStimulationpmol-foldNone1.91 ± 0.11 (n = 3)1.0β14.02 ± 0.78 (n = 3)7.3β + CKII11.59 ± 1.26 (n = 3)6.1δ10.15 ± 0.67 (n = 3)5.3δ + CKII9.92 ± 0.58 (n = 3)5.2 Open table in a new tab When the reconstituted βγδ complex was phosphorylated by CKII, approximately 1.0 mol of phosphate was incorporated per mol of β and 0.7 mol/mol δ subunit. The effects of phosphorylation were monitored with the polyphenylalanine synthesis assay over a 30-min time period, using native EF-1α and phosphorylated and nonphosphorylated EF-1βγδ (Fig. 9). Polyphenylalanine synthesis with EF-1α alone was linear for up to 30 min with 1.8 pmol of polyphenylalanine synthesized at 15 min. With the addition of βγδ, polyphenylalanine synthesis was linear for 15 min. At that time, the elongation rate was 5-fold higher than that observed with EF-1α alone. Phosphorylated EF-1βγδ showed the same degree of stimulation of EF-1α as the nonphosphorylated complex. Thus, phosphorylation of EF-1βγδ by CKII appeared to have no effect on elongation. The properties of the α, β, γ, and δ subunits of EF-1 from rabbit derived from the cDNA sequences are summarized in Table II. The α subunit is basic with an isoelectric point of 9.71. The β and δ are acidic with pIs of 4.33 and 4.91, respectively, whereas γ is relatively neutral. When the amino acid sequences of EF-1α, β, γ, and δ from rabbit are compared with those of human (9Sanders J. Maassen J.A. Amons R. Möller W. Nucleic Acids Res. 1991; 19: 4551Crossref PubMed Scopus (32) Google Scholar, 12San
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