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Domain and Nucleotide Dependence of the Interaction between Saccharomyces cerevisiae Translation Elongation Factors 3 and 1A *

Domain and Nucleotide Dependence of the Interaction between Saccharomyces cerevisiae Translation... THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 43, pp. 32318 –32326, October 27, 2006 © 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Domain and Nucleotide Dependence of the Interaction between Saccharomyces cerevisiae Translation Elongation Factors 3 and 1A Received for publication, February 28, 2006, and in revised form, August 30, 2006 Published, JBC Papers in Press, September 5, 2006, DOI 10.1074/jbc.M601899200 1 1 2 Monika Anand , Bharvi Balar , Rory Ulloque, Stephane R. Gross, and Terri Goss Kinzy From the Department of Molecular Genetics, Microbiology and Immunology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854 Eukaryotic translation elongation factor 3 (eEF3) is a fungal- somes have been reported to possess a compensatory intrinsic specific ATPase proposed to catalyze the release of deacylated- ATPase activity, although they differ kinetically from the fungal tRNA from the ribosomal E-site. In addition, it has been shown eEF3 (5). Escherichia coli, on the other hand, expresses the 911 to interact with the aminoacyl-tRNA binding GTPase elonga- amino acid RbbA protein that exhibits ATPase activity and is tion factor 1A (eEF1A), perhaps linking the E and A sites. tightly associated with ribosomes (6, 7). Both pathogenic and Domain mapping demonstrates that amino acids 775–980 con- non-pathogenic fungi have been reported to contain eEF3 tain the eEF1A binding sites. Domain III of eEF1A, which is also (8–10). In Saccharomyces cerevisiae, eEF3 is encoded by a sin- involved in actin-related functions, is the site of eEF3 binding. gle copy essential YEF3 gene. A paralog of the YEF3 gene, des- The binding of eEF3 to eEF1A is enhanced by ADP, indicating ignated HEF3 or YEF3B, encodes an 84% identical protein but is the interaction is favored post-ATP hydrolysis but is not not expressed during vegetative growth (11). However, expres- dependent on the eEF1A-bound nucleotide. A temperature- sion of the HEF3 coding sequence under the YEF3 promoter sensitive P915L mutant in the eEF1A binding site of eEF3 has produces a protein that has similar ATPase activity and ribo- reduced ATPase activity and affinity for eEF1A. These results some binding properties to YEF3-encoded eEF3. support the model that upon ATP hydrolysis, eEF3 interacts eEF3 is a class 1 member of the ATP binding cassette (ABC) with eEF1A to help catalyze the delivery of aminoacyl-tRNA at family of proteins. eEF3 possesses distinct motifs including the the A-site of the ribosome. The dynamics of when eEF3 interacts HEAT repeats on the N terminus, two nucleotide binding with eEF1A may be part of the signal for transition of the post to domains with tandemly arranged bipartite (ABC) cassettes in pre-translocational ribosomal state in yeast. the middle, a conserved insertion in the intervening region of the Walker A and B motifs of ABC2, and a highly basic C ter- minus. HEAT (Huntington elongation factor 3, A subunit of The protein synthetic machinery is characterized by the protein phosphatase 2A and TOR1) repeats correspond to a interplay of different soluble factors in conjunction with ribo- tandem -helical structure that appears to serve as flexible scaf- somes to translate the mRNA into the correct sequence of folding on which other proteins can assemble. Amino acids amino acids. The three phases of translation, initiation, elonga- 98–388 within the eEF3 N-terminal HEAT domain have also tion, and termination, are driven by factors that are highly con- been shown to interact with the 18 S rRNA (12). Within the served between yeast and metazoans (1). However, a major dif- Walker A and B motifs, the nucleotide binding stretch of seven ference in elongation is the indispensability of eukaryotic amino acids in ABC1 and -2 are 100% conserved among the elongation factor 3 (eEF3) with yeast ribosomes (2, 3). eEF3 ATP-binding proteins (13). The Walker C motif is the con- catalyzes an essential step in each elongation cycle by virtue of served LSGGQ sequence, the presence of which distinguishes its ATPase activity. It has been proposed to act as an Exit-site the ABC proteins from other ATPases (14). Alterations of the (E-site) factor, facilitating the release of deacylated-tRNA and conserved glycine and lysine residues within the Walker A of simultaneously impacting on the delivery of aminoacyl-tRNA either ABC1 or -2 abolish the ATP hydrolytic activity of eEF3 (aa-tRNA) at the aminoacyl site (A-site) (4). Metazoan ribo- in vitro and are lethal for growth in vivo (15). Interestingly, a temperature-sensitive (Ts ) F650S point mutant in the intervening region of the two ABC cassettes also affects the * This research was supported by National Institutes of Health Grant GM57483 (to T. G. K.). The costs of publication of this article were defrayed catalytic ATPase activity of the protein, indicating that the in part by the payment of page charges. This article must therefore be linker region affects either ATP binding or its hydrolysis hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 (16). Crystal structures of the E. coli transporter system ABC solely to indicate this fact. These authors contributed equally. proteins HisP (17) and MalK (18) as well as the human Rad50 To whom correspondence should be addressed: Dept. of Molecular Genet- ATPase (19) and cystic fibrosis transmembrane conductance ics, Microbiology and Immunology, UMDNJ Robert Wood Johnson Medical regulator (20) demonstrate that all possess two associated School, 675 Hoes Lane, Piscataway, NJ 08854. Tel.: 732-235-5450; Fax: 732- 235-5223; E-mail: [email protected]. monomers. Each monomer harbors a single ABC cassette The abbreviations used are: eEF, eukaryotic elongation factor; GST, glutathi- and forms a homodimer in the presence of ATP to carry out one S-transferase; Ni -NTA, nickel nitrilotriacetic acid; aa-tRNA, amino- hydrolysis, although there is variation in the manner by acyl-tRNA; E-site, exit site; A-site, aminoacyl site; ABC, ATP binding cassette; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride. which each nucleotide binding domains from the two mono- This is an open access article under the CC BY license. 32318 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 43 •OCTOBER 27, 2006 eEF3 and eEF1A Interaction TABLE 1 S. cerevisiae strains used in this study Strains Genotype Source TKY554 MAT ura3-52 leu2-3, 112 trp1-7 lys2-1243 met2-1 his4-713 yef3::LEU2 2 pYEF3 URA3 Ref. 16 TKY555 MAT ura3-3 leu2-2 trp1-1 his3-3 pMA210 (GAL4 2 HIS3) This study TKY597 MAT ura3-52 leu2-3, 112 trp1-7 lys2-1243 met2-1 his4-713 yef3::LEU2 CEN pYEF3 TRP1 This study TKY616 MAT ura3-52 leu2-3, 112 trp1-1 lys2-20 met2-1 his4-713 tef1::LEU2 tef2 CEN His pTEF1 TRP1 This study TKY676 MAT ura3-52 leu2-3, 112 trp1-7 lys2-1243 met2-1 his4-713 yef3::LEU2 CEN His pYEF3 TRP1 This study TKY702 MAT ura3-52 leu2-3, 112 trp1-7 lys2-1243 met2-1 his4-713 yef3::LEU2 2 His pYEF3 TRP1 Ref. 16 TKY800 MAT ura3-52 leu2-3, 112 trp1-7 lys2-1243 met2-1 his4-713 yef3::LEU2 CEN pyef3 TRP1(P915L) This study TKY805 MAT ura3-52 leu2-3, 112 trp1-7 lys2-1243met2-1 his4-713 yef3::LEU2 2 His pyef3 TRP1 (980eEF3) This study TKY819 MAT ura3-52 leu2-3, 112 trp1-7 lys2-1243 met2-1 his4-713 yef3::LEU2 CEN His pyef3 TRP1 (P915L) This study TKY822 MAT ura3-52 leu2-3, 112 trp1-7 lys2-1243 MET2 his4-713 yef3::LEU2 CEN His pYEF3 TRP1 This study TKY824 MAT ura3-52 leu2-3, 112 trp1-7 lys2-1243 MET2 his4-713 yef3::LEU2 CEN pyef3 TRP1 (P915L) This study mers collaborate to bind ATP molecules (21). The lysine- (met2-1 and his4-713) was determined by spotting 10 lof rich C terminus of eEF3 (amino acids 980 –1044) has previ- the same dilutions onto complete medium lacking methio- ously been implicated as required for binding to the nine or histidine, respectively, and incubating for 5 days at ribosome (22, 23). 30 °C. Halo assays for sensitivity to cycloheximide, paromo- During translation elongation, delivery of aa-tRNA to the mycin, and hygromycin B were performed as previously A-site by eEF1A and the translocation of the ribosome by described (29). Total yeast translation was monitored by in eukaryotic elongation factor 2 (eEF2) require GTP hydrolysis vivo [ S]methionine incorporation as previously described (1). The unique role of eEF3 may be part of the transition of the at both 30 and 37 °C (30) using the indicated MET2 strains. post-translocational to the pre-translocational state via its ATP Isolation of the P915L eEF3 Mutant by Hydroxylamine hydrolytic activity in yeast. The allosteric three-site model sug- Mutagenesis—Ten g of plasmid DNA (pTKB594) harboring gests that only two tRNAs can occupy the ribosome at one time, YEF3 on a CEN TRP1 plasmid was added to 500 lof1 M and thus, the exit of deacylated-tRNA is a prerequisite or coreq- hydroxylamine, pH 7.0. The reaction was incubated at 37 °C for uisite for the delivery of aa-tRNA to the A-site (24). eEF3 has 20 h and stopped by adding 100 mM NaCl and 0.1 g/l bovine been proposed to aid this removal and help promote the deliv- serum albumin. DNA was ethanol-precipitated, transformed ery of only cognate aa-tRNA by eEF1A to the A-site (4). It into TKY554, and plated on C-Trp to select for the mutated yef3 remains unclear how and when eEF3 utilizes its ATP hydrolytic TRP1 plasmid at a density of150–300 cells/plate. Cells able to activity to carry out these functions. lose the wild type YEF3 URA3 plasmid were identified by To address this question the present study analyzed the growth on 5-fluoroorotic acid-containing media. The resulting regions involved in, and the nucleotide-bound state that favors strains expressing the yef3 TRP1 plasmid as the only form were eEF3 binding to eEF1A. Our results point toward an enhanced analyzed for growth at 13, 30, or 37 °C. A colony unable to grow eEF3 and eEF1A association in the presence of ADP, suggesting at 37 °C was recovered from the 30 °C plate, and the plasmid that ATP hydrolysis likely precedes eEF3 binding to eEF1A. was extracted, transformed in E. coli, recovered, and retrans- The eEF1A binding region of eEF3 has been mapped to 2 formed into TKY554. Loss of the wild type YEF3 plasmid was regions near the C terminus. A genetic screen conducted in the repeated to confirm the phenotype. The P915L eEF3 mutant current study resulted in a point mutation in one of the regions. plasmid pTKB753 isolated in this screen was also constructed A strain expressing the P915L eEF3 exhibits a temperature- with a His tag on the N terminus by site-directed mutagenesis sensitive (Ts ) growth defect and reduction in total translation. of pTKB602 by the QuikChange method (Stratagene), produc- Additionally, the protein has negligible intrinsic and ribosome- ing pTKB777. stimulated ATPase activity and shows reduced affinity for Cloning, Expression, and Purification of GST and His - eEF1A. tagged eEF3, eEF1A, and Truncations—Full-length eEF3 and EXPERIMENTAL PROCEDURES fragments containing amino acids 1–775 (85NT), 100 –367 (HEAT), 775–910 (I), 910 –1044 (15CT), and 775–1044 Yeast and Bacterial Strains, Growth, Drug Sensitivity, and (30CT) were PCR-amplified using pTKB594 as the template. Translation Assays—S. cerevisiae strains and their genotypes Fragments were cloned into pTKB544 for expression with a are listed in Table 1. E. coli DH5 was used for plasmid prep- galactose-inducible promoter (GAL1-10) and an N-terminal aration. Procedures for cell growth and genetic manipula- GST tag, resulting in plasmids pTKB705, pTKB706, tions were according to standard protocols (27). Yeast cells pTKB707, pTKB708, pTKB709, and pTKB710, respectively. were grown in either YEPD (1% Bacto-yeast extract, 2% pep- The plasmids expressing the GST-tagged eEF3 fragments tone, 2% dextrose) or in defined synthetic complete medium were transformed in TKY555 and maintained on C-Ura- (C or C) supplemented with 2% dextrose as the carbon Hisgalactose media for protein expression. Yeast cultures source unless noted. Yeast were transformed by the lithium expressing the GSTeEF3 fusions were harvested at an A of acetate method (28). Temperature sensitivity was assayed by growing strains to an A of 1.0. Serial 10-fold dilutions (5 l 1.0 –2.0, and total yeast extracts were clarified and loaded on each) were spotted on appropriate medium followed by the GST Trap column (Amersham Biosciences) in buffer A incubation at 13, 24, 30, and 37 °C for 3–7 days. Phenotypic (20 mM Tris-HCl, pH 8.0, 100 mM KCl, 1 mM DTT, and 0.2 suppression of a non-programmed 1 frameshift allele mM PMSF). The protein was eluted with buffer A plus 20 mM OCTOBER 27, 2006• VOLUME 281 • NUMBER 43 JOURNAL OF BIOLOGICAL CHEMISTRY 32319 eEF3 and eEF1A Interaction reduced glutathione (Sigma). The protein peak was dialyzed GST and His Pulldowns of eEF1A and eEF3—Yeast extracts into buffer B (20 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 10% for in vivo binding assays were prepared by glass bead lysis in glycerol, 1 mM DTT, 0.2 mM PMSF, and 100 mM KCl). TEDG buffer (10 mM Tris-HCl, pH 7.4, 2 mM EDTA, 5 mM To facilitate eEF3 purification from yeast, a His tag was DTT, 50 mM KCl, and 1 mM PMSF) from TKY555 with the added to the N terminus of S. cerevisiae eEF3 under the con- empty plasmid pTKB544, GSTeEF3 (pTKB705), or the GST- trol of its own promoter on a CEN TRP1 plasmid producing eEF3 fragments (pTKB706, pTKB707, pTKB708, pTKB709, pTKB602 (31). A yeast plasmid expressing His 980 eEF3 was 6 pTKB710). For GST and Ni -NTA pulldown assays, 200-l produced by introduction of a stop codon at amino acid 981 reactions containing 50 g of total protein (determined by by QuikChange, producing pTKB724 (31). The plasmids Bradford reagent; Bio-Rad) and 40 l of either 50% glutathione- were introduced into S. cerevisiae strain TKY554, and loss of Sepharose 4B slurry (Sigma) in KETN 150 buffer (150 m M KCl, the wild type eEF3 on a URA3 plasmid was monitored by 1mM EDTA, 20 mM Tris-HCl, pH 8.0, 0.5% Nonidet, and 1 mM growth on 5-fluoroorotic acid, producing TKY702 and PMSF) or Ni -NTA slurry (Amersham Biosciences, GE TKY805, respectively. Healthcare) in buffer C were mixed at 4 °C for 1 h. Beads were His -tagged wild type eEF1A, eEF3, 980eEF3, and P915L washed 3 times with either KETN buffer with 150 or 300 mM eEF3 proteins were purified from strains TKY616, TKY702, KCl for GST pulldown or buffer C with 100 mM imidazole for TKY805, and TKY819, respectively, on a Ni Hi Trap chelat- Ni -NTA pulldown. Samples were resolved by SDS-PAGE, ing column (Amersham Biosciences). Total yeast extracts were and were proteins were detected with a polyclonal antibody clarified and loaded on the column in buffer C (50 mM KPO , to yeast eEF1A and ECL (Amersham Biosciences) and quan- pH 7.6, 300 mM KCl, 1 mM DTT, and 0.2 mM PMSF) with 20 mM titated with the ImageQuant program (GE Healthcare). imidazole. The protein was eluted with buffer C plus 400 mM Ni -NTA pulldown of purified untagged eEF1A with His - imidazole. The protein peak was dialyzed into buffer B. tagged eEF3 or untagged eEF3 with His -tagged eEF1A were BspEI restriction sites were introduced upstream of the ATG performed with 2 g of eEF3 and 3 g of eEF1A as previously initiation codon and downstream of the TAA stop codon using described (33). the QuikChange protocol in TEF1 on pTKB731 as template, Ribosome Binding Assay—The ribosome binding assay was producing pTKB740. His -tagged eEF1A with BspEI restriction performed as described previously (34) with minor modifica- sites upstream and downstream of the open reading frame was tions. Fifty-l reactions containing 24 pM purified proteins and constructed by PCR and cloning into pTKB740, resulting in plasmid pTKB779. Domain I (amino acids 1–221) was con- 24 pM 80 S ribosomes in binding buffer (20 mM Tris-HCl, pH structed by QuikChange mutagenesis of the Lys-222 and Lys- 7.5, 50 mM ammonium acetate, 10 mM magnesium acetate, and 224 codons to TAA using pTKB779, producing pTKB852. His - 2mM DTT) were incubated for 5 min at room temperature, tagged domain III (amino acids 333–458) was obtained by layered on top of a 200-l sucrose cushion (10% sucrose in looping out domains I and II using site-directed mutagenesis binding buffer), and centrifuged at 74,000 rpm for 20 min at protocol of template pTKB779, producing pTKB785. Plasmids 4 °C in S80-AT2 (Sorvall) rotor. The pellet (bound fraction) was pTKB852 and pTKB785 were used as templates for PCR ampli- resuspended in Laemmli loading buffer and subjected to SDS- fication of His -domain I and His -domain III fragments to 6 6 PAGE and Western blot analysis using the ECL method (Amer- clone into the pET11a vector, resulting in plasmids pTKB863 sham Biosciences). and pTKB851, respectively. His -tagged domain II (amino acids Enzyme-linked Immunosorbent Assays—In vitro binding 222–316) in pET11a was constructed by QuikChange was measured by an indirect enzyme-linked immunosorbent mutagenesis of the Glu-316 and Arg-318 codons to TAA and assay. Purified GSTeEF3 (0.25 g) in 50 l of PBST (137 mM TGA, respectively, using pTKB864 as the template to produce NaCl, 2.7 mM KCl, 10 mM Na HPO ,and2mM KH PO )/ 2 4 2 4 pTKB920. well was coated overnight at room temperature in a 96-well A 1-liter culture of E. coli BL21 with each plasmid was grown ultrahigh binding polystyrene microtiter plate (Thermo- to an A of 0.6 in LB with 100 g/ml ampicillin medium. Labsystem). After blocking with 300 l of 0.1% bovine serum Protein expression was induced with 1 mM isopropyl--D-thio- albumin in PBST for 1 h at room temperature and washing 3 galactopyranoside at 37 °C for 3–4 h. Cells were harvested by times with 300 l of PBST, 50 l of 5000-fold-diluted affin- centrifugation and lysed by sonication, and the recombinant ity-purified polyclonal anti-eEF3 antibody was added to each protein was purified in accordance with the QIAexpressionist well and incubated at room temperature for 2 h. Varying protocol for His -tagged proteins under native conditions. Pro- amounts of eEF1A along with varying amounts of ATP, ADP, tein-containing fractions were dialyzed into 20 mM Tris-HCl, GTP, or GDP were added to the eEF3 antibody. After washing 3 pH 7.5, 1 mM DTT, 0.1 mM EDTA, pH 8.0, 100 mM KCl, and 20% times with 300 l with PBST, 50 l of 2500-fold-diluted sec- glycerol. ondary goat anti-rabbit antibody conjugated with alkaline ATP Hydrolysis—ATP hydrolysis was performed using puri- phosphatase was added per well (Jackson ImmunoResearch). fied proteins as previously described (32). Briefly, the assay mix- Unbound antibody was removed by three washes of 300 lof ture contained 24 pM protein, 50 pM yeast ribosomes, and 150 PBST followed by the addition of 50 lof3mM p-nitrophenyl M [- P]ATP. Hydrolysis was allowed to proceed for 5 min at phosphate (Sigma) in 50 mM Na CO and 50 M MgCl /well. 30 °C, and P release was determined. ATP hydrolysis levels i 2 3 2 The extent of p-nitrophenyl phosphate hydrolysis represents were calculated after subtracting the background for buffer alone. the antigen-antibody binding measured by A . 32320 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 43 •OCTOBER 27, 2006 eEF3 and eEF1A Interaction GST fusion fragments co-purified eEF1A at levels similar to or above that of full-length eEF3. The 85NT, GST alone, and HEAT fragments co-purified less eEF1A, although some background level of binding was observed (Fig. 1C). The same experiment was also probed for co-elution of ribosomes with the eEF3eEF1A complex. As shown in Fig. 1C, bottom panel, RPL10e, a ribosomal protein, is absent in the bound fractions. The middle panel, Fig. 1C, shows probing for phosphoglycerate kinase (PGK1) as the internal loading control. Because the fusion truncations are expressed at different levels in vivo, the 85NT, 30CT, and 15CT GST- tagged fragments were purified from yeast, and GST pulldown experiments were performed with purified untagged yeast eEF1A. The GST-HEAT and GST-I fusion were not stably expressed at sufficient levels for purification. GST-15CT FIGURE 1. C-terminal regions of eEF3 bind eEF1A. A, eEF3 fragments cloned as GST fusions under the GAL1-10 and GST-30CT co-purified with promoter in pTKB544. B, plasmids expressing the GST fusion fragments from A were transformed in TKY555 and maintained in C-Ura-Hisgalactose. Strains were grown to mid-log phase at 30 °C, yeast extracts were eEF1A at levels comparable with prepared, and equal amounts of protein (5g) were separated by SDS-PAGE and subjected to Western blotting wild type GSTeEF3, whereas the with an anti-GST monoclonal antibody. The lower panel shows eEF1A as the internal loading control. C, a GST GST-85NT was at background lev- pulldown assay was performed with the extracts (50g) from the same strains as in B, and the Western blot was developed with an anti-eEF1A antibody (top panel), anti-phosphoglycerate kinase (PGK1) antibody represent- els (GST, Fig. 1D). The results in Fig. ing internal loading control (middle panel) and anti-RPL10e antibody to detect co elution ribosomes with the 1, C and D, demonstrate that the eEF3eEF1A complex (lower panel). E, extract (10% input); S, supernatant (5%); P, pellet (100%). D, a GST pull- eEF3eEF1A interaction occurs in down assay with purified yeast eEF1A (100 pM) and GST-tagged eEF3, 30CT, 15CT, and 85NT fragments (20 pM). E, the yeast strains from B were grown to mid-log phase at 30 °C, diluted to equal A , spotted as 10-fold serial the absence of any cellular factors dilutions, and grown at 13 or 24 °C for 2–7 days. via the C-terminal region of eEF3. Dominant growth phenotypes RESULTS conferred by the truncations were monitored on C-Ura- eEF3 Interacts with eEF1A through Its C-terminal Region— Hisgalactose medium at different temperatures. The 30CT Prior studies have demonstrated that eEF3 and eEF1A inter- and 15CT fragments confer a dominant slow growth phenotype act, as monitored by both genetic and physical assays in vivo at 13 °C, whereas no effects were seen at 30 or 37 °C (Fig. 1E and and in vitro (16). To map the site of interaction, five fragments data not shown). Because there appear to be two eEF1A binding of eEF3 corresponding approximately to natural proteolytic sites, one within amino acids 775–910 and one within 910– sites were cloned into a GAL1-10-inducible expression vector 1044, fragments of eEF3 containing these amino acids may with a GST tag at the N terminus (Fig. 1A). These include full- exhibit a dominant slow growth phenotype due to the forma- length eEF3 (amino acids 1–1044), 85NT, (1–775), HEAT tion of inactive complexes with eEF1A. The I fragment (775– (100–367), 15CT (910–1044), I (775–910), and 30CT (775– 910) does not show this growth phenotype, indicating the site 1044). All the fragments are expressed in yeast although at dif- from 910 to 1044 may have a larger effect in vivo. ferent levels, as monitored by Western blot with anti-GST anti- His 980eEF3 Is Functional in Vivo and Retains Binding to body (Fig. 1B). The GST-tagged fusion proteins migrate at 140 Ribosomes and eEF1A—Prior work proposed that the C-termi- (eEF3), 105 (85NT), 57 (HEAT), 42 (15CT), 58 (30CT), and 42 nal 64 amino acids (980–1044), containing 40% basic residues, (I) kDa, with 29 kDa contributed by the GST tag. The same gel is the primary ribosome binding region of eEF3 (22). Other is also probed with anti-eEF1A antibody as the internal loading work suggests the N-terminal 98–388 amino acids binds to 18 S control. rRNA in vitro and inhibit the ribosome-dependent ATPase Because none of the eEF3 fragments can replace wild type activity of eEF3 (12). To determine the function of the basic C eEF3 in vivo (data not shown), all were co-expressed with an terminus of eEF3, His -tagged eEF3 1–980 was expressed from untagged wild type copy of eEF3 to support growth. A GST a2 TRP1 plasmid. This construct was able to function as the pulldown assay was performed to determine the binding of only form of eEF3 (Fig. 2A). Cells expressing His 980eEF3 as the eEF3 to eEF1A in total cell extracts. The 15CT, I, and 30CT only form of eEF3 have a slight slow growth phenotype (Fig. OCTOBER 27, 2006• VOLUME 281 • NUMBER 43 JOURNAL OF BIOLOGICAL CHEMISTRY 32321 eEF3 and eEF1A Interaction To determine whether the C terminus of eEF3 is dispensable for ribosome binding, purified His eEF3 and His 980eEF3 were 6 6 assayed for co-association with ribosomes through a sucrose cush- ion (Fig. 2E). The slowest migrating bands corresponding to the full- length and 1–980 proteins pellet with ribosomes. Interestingly, the same degradation products were observed for both eEF3 and 980eEF3. All three bands reacted with the anti-His antibody (data not shown), and because the His tag was located at the N terminus, this indicates the N terminus is intact. Thus, these fragments represent C-terminal truncations and imply ribosome binding occurs near the N terminus. This is consistent with work showing an N-terminal frag- ment binds 18 S rRNA (12). The negative control bovine serum albu- FIGURE 2. His 980eEF3 is functional in vivo and retains binding to eEF1A and ribosomes. A, strains con- taining plasmid-borne untagged eEF3 (2 m, TKY554), His -tagged eEF3 (CEN, TKY676), His eEF3 (2, TKY702), 6 6 min stayed in the supernatant both and His 980eEF3 (2, TKY805) were grown to mid-log phase at 30 °C, diluted to equal A , spotted as 10-fold 6 600 in the presence and absence of ribo- serial dilutions, and grown at 24 or 13 °C for 2–7 days. B, yeast extracts (2 g) were prepared from strains somes (data not shown). expressing His eEF3 (TKY702), eEF3 (TKY554), and His 980eEF3 (TKY805) and analyzed for the expression of 6 6 His 980eEF3 by Western blotting with an anti-eEF3 antibody. The lower panel shows equal loading of eEF1A as ADP Enhances the Association of internal control. C, in vivo eEF1A binding to His eEF3 and His 980eEF3 was analyzed by Ni -NTA pulldown of 6 6 eEF3 with eEF1A—Because both yeast extracts (50 g) from strains as in A. S, supernatant (5%) and P, pellet (100%) were subjected to SDS-PAGE and Western blot with anti-eEF1A and anti-His antibodies. D, in vitro binding of purified His eEF3 and 6 6 eEF3 and eEF1A bind nucleotides, His 980eEF3 in a 5-fold molar excess of untagged eEF1A was assessed by Ni -NTA pulldown and analyzed by an enzyme-linked immunosorbent SDS-PAGE and Western blot with anti-eEF1A antibody. E, association of eEF3 with purified 80 S ribosomes assay-based binding assay was through a 10% sucrose cushion is shown for His 980eEF3 and His eEF3 and analyzed as in D with an anti-eEF3 6 6 antibody. P, pellet (100%); S, supernatant (20%). developed to look at the effect of these molecules on the eEF1A-eEF3 2A). This effect is most noticeable at 13 °C. Western blot anal- interaction. Subsequent to coating the wells with purified ysis of His eEF3, wild type eEF3, and His 980eEF3 from strains eEF3, eEF1A was added to compete with an anti-eEF3 anti- 6 6 TKY702, TKY554, and TKY805 with anti-eEF3 antibody (Fig. body. Because eEF1A binding competes with antibody bind- 2B) shows that His 980eEF3 protein is expressed at similar lev- ing, the absorbance value is reduced in the presence of els as full-length-tagged and untagged eEF3. The same gel was eEF1A. Concentration-dependent eEF1A binding to eEF3 also probed with anti-eEF1A antibody as internal loading con- was observed (Fig. 3A). A 10-fold molar excess of eEF1A to trol. Therefore, although His 980eEF3 is stably expressed, its eEF3 was used for all further assays. A series of controls was function in vivo is likely partially compromised. included in this assay to validate these results. These The role of amino acids 981–1044 in binding eEF1A was included demonstrating that the anti-eEF3 antibody does determined by Ni -NTA pulldown of extracts from strains not show any affinity for eEF1A, the addition of nucleotide expressing His 980eEF3, His eEF3, or untagged eEF3. Super- alone in the absence of anti-eEF3 antibody exhibits negligi- 6 6 natant and pellet fractions were resolved by SDS-PAGE, and ble absorbance, and the addition of nucleotides alone (in the the Western blot was probed with anti-eEF1A and anti-eEF3 absence of competing factor eEF1A) along with anti-eEF3 antibodies. eEF1A associates with His 980eEF3 at levels com- antibody does not affect absorbance (data not shown). parable with full-length His eEF3 (Fig. 2C). In the negative con- To ascertain the effect of the nucleotide-bound state on the trol with untagged eEF3, minimal background eEF1A was pres- binding of the two proteins, ATP, ADP, GTP, or GDP was ent in the pellet. The Ni -NTA pulldown was also performed added with the anti-eEF3 antibody and eEF1A. Whether GTP with purified proteins, confirming that eEF1A binds to both or GDP was incubated with eEF1A and eEF3, the signal His eEF3 and His 980eEF3 directly (Fig. 2D). This indicates remained constant, and thus, binding was unaffected (Fig. 3B). 6 6 that residues 981–1044 are not required for eEF1A binding. On the other hand, there is a concentration-dependent reduc- Taken together with the truncation data (Fig. 1, C and D), it tion in signal, and hence, stimulation of eEF1A binding when appears one eEF1A binding site is located within the 205-amino ADP was added. This is shown as binding normalized to acid stretch from 775 to 910 and a second within amino acids absorbance in the presence of nucleotide alone and in the 910–980. absence of eEF1A (Fig. 3C). Furthermore, when ATP was added 32322 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 43 •OCTOBER 27, 2006 eEF3 and eEF1A Interaction (Amersham Biosciences) (Fig. 3F). His eEF3 eluted as a single sharp peak with a retention time of 29.24 min corresponding to a molecular mass of 140 kDa. The migration remains unchanged in 1 M KCl, 1 mM ATP, 1 mM ADP, or 50 mM ethylene glycol (data not shown), showing that eEF3 exists as a mon- omer in its purified form. A P915L Mutation in an eEF1A Binding Site of eEF3 Alters ATPase Activity and eEF1A Binding—A genetic screen for conditional mu- tants in eEF3 was conducted using unbiased in vitro mutagenesis of a YEF3 plasmid. A pool of hydroxy- lamine-treated plasmids was trans- formed into yeast, and plasmids able to replace the wild type YEF3 URA3 plasmid were determined by growth on 5-fluoroorotic acid. Approxi- mately 7000 colonies were screened for temperature-sensitive growth yielding a strain expressing a single eEF3 point mutation, P915L, in the C-terminal region (Fig. 4A). The doubling time of the P915L mutant strain was 5.5 h compared with 3.5 h for the wild type strain. Total pro- tein synthesis monitored by meas- uring [ S]methionine in the P915L FIGURE 3. ADP stimulates eEF1A binding to eEF3. A, microtiter 96-well plates (Falcon) were coated with purified GSTeEF3 (0.25 g), and an affinity-purified anti-eEF3 antibody was added with or without increasing strain was 20% less than a wild type amounts of eEF1A. eEF1A (1.25 g) was incubated in the GSTeEF3-coated microtiter plate as in A, with different strain at permissive temperatures concentrations of nucleotides, GTP (diamonds) or GDP (squares)in B expressed as A or ATP (diamonds), or and 22% less than wild type when ADP (squares)in C expressed as percentage bound normalized to the presence of nucleotide alone in the absence of eEF1A.D, eEF1A bound to GSTeEF3 after GST pulldown in the presence of varying amounts of ADP cells were shifted to 37 °C (Fig. 4B). were analyzed by SDS-PAGE and stained with gel code blue (Pierce). S, supernatant (5%); P, pellet (100%). E, the To determine the eEF3 defect caus- results of GST pulldown experiments as in D was analyzed with the ImageQuant program (GE Healthcare), and the ratio of pellet to supernatant was plotted. F, purified His eEF3 was subjected to gel filtration analysis by fast ing this effect, the ATPase activity of protein liquid chromatography on a Superdex 200 column (Amersham Biosciences). The elution profile of purified His P915L eEF3 was deter- His eEF3 was determined by SDS-PAGE and Western blot with an anti-eEF3 antibody. mined. The mutant lacks both intrinsic and ribosome-stimulated there was a concentration-dependent increase in signal, and ATPase activity (Fig. 4C). To assess if this loss of catalytic activ- hence, reduction in binding was observed. The experiment was ity affects eEF1A binding to the P915L eEF3 mutant, associa- done multiple times to confirm a reproducible trend. tion was assessed by Ni -NTA pulldown assay. His P915L To confirm the enzyme-linked immunosorbent assay-based eEF3 pulls down reduced levels of eEF1A as compared with wild assay, a GST pulldown of purified untagged eEF1A with type His eEF3 in both cell extracts (Fig. 4D) and with purified GSTeEF3 was performed in the presence of different concen- proteins (Fig. 4, E and F). A small amount of eEF1A is nonspe- trations of nucleotide and analyzed by SDS-PAGE followed by cifically pulled down by untagged eEF3 using Ni -NTA beads. gel code blue staining. The amount of eEF1A bound to This implies that binding of eEF3 to eEF1A is sensitive to struc- GSTeEF3 in the pellet increases in the presence of increasing tural and functional alterations caused by a point mutation in a amounts of ADP (Fig. 3, D and E). Thus, results from two inde- region proposed to bind eEF1A. pendent methods indicate that binding of eEF1A to eEF3 is eEF1A Binds eEF3 via Domain III—The co-crystal structure likely stimulated after ATP hydrolysis. of eEF1A with its guanine nucleotide exchange factor eEF1B Proteins belonging to the ABC superfamily are inter- or shows the G-protein has three domains. Domain I contains the intramolecular dimers, and the presence of two ATPase GTP binding motifs, and domains I and II contact eEF1B (33, domains is required for function (35). To confirm His eEF3 36). Domain III has been shown to interact with actin and is is a monomer, purified protein was subjected to analysis by responsible for the non-canonical functions of eEF1A in actin gel filtration chromatography on a Superdex 200 column binding and bundling (25) and the slow growth phenotype asso- OCTOBER 27, 2006• VOLUME 281 • NUMBER 43 JOURNAL OF BIOLOGICAL CHEMISTRY 32323 eEF3 and eEF1A Interaction ners to carry out its essential steps in translation elongation is still not well understood. Recent work in bacteria confirms the allosteric link between the A and E sites (38). This supports the hypothesis that a general ribosome function is the release of deacylated tRNA from the E-site preceding the GTP hydrolysis required to deposit aa- tRNA at the A-site. This step likely involves a conformational change in the 70 S ribosome. Because bac- teria lack eEF3, although the ribo- some-associated ATPase RbbA has been implicated as a bacterial counterpart of eEF3 (39), the bind- ing of the ternary complex of aa- tRNA-EF-Tu-GTP has been sug- gested to induce the required conformational change in the ribosome to catalyze the release of deacylated-tRNA from the E-site FIGURE 4. The ATP hydrolysis deficient P915LeEF3 mutant shows reduced affinity for eEF1A. A, strains (38). In mammals, the ribosome- containing wild type eEF3 (TKY597) or P915LeEF3 (TKY800) were grown to mid-log phase at 30 °C, diluted to associated ATPase activity from equal A , spotted as 10-fold serial dilutions, and grown at 30 or 37 °C for 2–7 days. B, strains expressing His P915LeEF3 (TKY824) or His eEF3 (TKY822) were monitored for total translation by [ S]methionine incor- 6 6 pig liver differs from the yeast poration after growth to mid-log phase in C-Met and labeled for varying times at both 30 and 37 °C. Total eEF3 ATPase activity in its sensi- translation is expressed as cpm/A unit. Wt, wild type. C, intrinsic and ribosome (Rbs)-stimulated ATP hydro- tivity to translation inhibitors and lytic activities of purified His P915L and His eEF3 were measured. The pM P released from [- P]ATP are 6 6 i shown after subtracting the hydrolysis in the presence of buffer alone. The results are an average of three nucleotide dependence (40). experiments and the S.D. shown. D, yeast extracts were prepared from strains containing eEF3 (TKY597), Previous reports have proposed His eEF3 (TKY702), and His P915L (TKY819), and equal amounts of total protein were incubated with 6 6 Ni -NTA beads. Extract (E, 5%), supernatant (S, 5%), and pellet (P, 100%) were separated by SDS-PAGE and two different ribosome binding analyzed by Western blot. The blot was probed with both anti-eEF3 and anti-eEF1A antibodies. E, eEF1A, regions in eEF3, the 64 amino acids His eEF3, and His P915LeEF3 proteins were purified and ran on a SDS-PAGE gel and stained with GelCode Blue 6 6 at the C terminus (22) and the (Pierce). F, a 5-fold molar excess of purified eEF1A, either alone or with purified His eEF3 or His P915L proteins, 6 6 were incubated with Ni -NTA beads. Supernatant (S, 5%) and pellet (P, 100%) were separated by SDS-PAGE N-terminal residues 98–388 (12). In and analyzed by Western blot with an anti-eEF1A antibody. the present study we report that yeast expressing eEF3 in the absence ciated with eEF1A overexpression in vivo (26). To identify the of its 64 amino acids at the C terminus are viable, and both the eEF1A region involved in binding to eEF3, purified wild type eEF1A and ribosome binding properties are retained by His 980eEF3. Thus, the N-terminal region is likely the predom- His eEF1A from yeast and His fusions of domain I (1–221, 22 6 6 6 inant ribosome binding site. kDa,), II (222–332, 11 kDa), or III (333–458, 33 kDa) purified from E. coli (Fig. 5A) was used to determine GSTeEF3 binding The family of ABC protein includes membrane-bound fac- by Ni -NTA pulldowns. GSTeEF3 was pulled down only by tors, which function in transporting solute molecules against a concentration gradient. However, the soluble members of this wild type His eEF1A and His -domain III (Fig. 5B). No 6 6 family, including Gcn20p, RL11 (41), eEF3, and the recently GSTeEF3 binding was seen by either domains I or II. reported ARB1 (42) in yeast are also implicated in functions DISCUSSION related to protein synthesis, ribosome biogenesis, and transla- Protein synthesis in yeast relies not only on the availability of tion elongation. The crystal structure of several members of the eEF1AB complex and eEF2 but also another unique fac- the class I ATPases clearly establish the phenomena of tor, eEF3. The absolute dependence of the pathogenic fungal homodimerization of two ABC proteins to sandwich two ATP translation machinery on the presence of eEF3 can be exploited molecules utilizing the Walker A and B motifs of the one mon- as a fungal-specific drug target (37). To achieve this long-term omer (43, 44) and Walker C or the conserved LSGGQ motif, goal, our primary aim is to understand the role of eEF3 in pro- characteristic of only the ABC members of the ATPases super- tein synthesis. Previously published work has assigned eEF3 family, from the other monomer. It has been shown for cystic the dual roles of removing the deacylated-tRNA from the fibrosis transmembrane conductance regulator that upon ATP E-site of the ribosome and aiding eEF1A in the delivery of the hydrolysis, the dimerized cassettes come apart, and this motor correct aa-tRNA to the A-site. eEF3 has been shown to inter- motion drives the transport across the membrane (45). Inter- act physically with both eEF1A and ribosomes. The mystery estingly, the soluble members of the ABC family harbor both of how and when eEF3 collaborates with its interacting part- the cassettes in tandem in a single molecule. Our investigation 32324 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 43 •OCTOBER 27, 2006 eEF3 and eEF1A Interaction with either eEF1A and/or ribosomes driving translation elon- gation forward, and hence, total translation is increased (16). If eEF3 competes with actin to bind eEF1A via domain III, then the increase in eEF3 may shift the balance of the cellular machinery in favor of protein synthesis rather than toward the function of eEF1A in cytoskeletal arrangements. This dynamic cross-talk between the two cellular processes of protein synthe- sis and cytoskeletal arrangement is likely mediated by the elon- gation factor eEF1A and may also be affected by the interaction of eEF3 versus actin with eEF1A. This study supports the model that the ATP hydrolysis by eEF3 stimulates the interaction with eEF1A. This observation fits in nicely with the model of eEF3 function, where ribosome- stimulated nucleotide hydrolysis of the ATP-bound eEF3 pre- cedes its interaction with eEF1A and the delivery of only cog- nate aa-tRNA at the A-site. It is still speculative if eEF1A binding occurs, whereas eEF3 is bound to or upon its release from the ribosome. The latter situation is more likely since upon ATP hydrolysis, eEF3 is likely released from the ribosome. Acknowledgment—We acknowledge the assistance of Robert Wood Johnson Medical School DNA core facility. FIGURE 5. Domain III of eEF1A binds eEF3. A, His -tagged full-length eEF1A REFERENCES was purified from yeast, whereas the His -tagged eEF1A domains I, II, and III were expressed and purified from E. coli BL21 cells. Purified proteins sepa- 1. Merrick, W. C., and Nyborg, J. (2000) in Translational Control of Gene rated by SDS-PAGE are shown by Coomassie Blue stain. B,Ni -NTA pulldown Expression (Sonenberg, N., Hershey, J. W. B., and Mathews, M. B., eds) pp. was performed with 20 pM purified GSTeEF3 and 100 pM proteins from A. 98–125, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY Shown is the pellet (100%) after pulldown. The top half of the blot was devel- 2. Skogerson, L., and Wakatama, E. (1976) Proc. Natl. Acad. Sci. U. S. 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D., Astromoff, A., Liang, H., Anderson, K., effect is lost for mutants located in domain III (26). This work Andre, B., Bangham, R., Benito, R., Boeke, J. D., Bussey, H., Chu, A. M., shows that domain III (333–458) of eEF1A has another func- Connelly, C., Davis, K., Dietrich, F., Dow, S. W., El Bakkoury, M., Foury, F., tion, the interaction with eEF3. This is also consistent with the Friend, S. H., Gentalen, E., Giaever, G., Hegemann, J. H., Jones, T., Laub, finding that neither GDP nor GTP affects eEF3 binding. Over- M., Liao, H., Liebundguth, N., Lockhart, D. J., Lucau-Danila, A., Lussier, expression of eEF3 results in enhanced growth at all tempera- M., M’Rabet, N., Menard, P., Mittmann, M., Pai, C., Rebischung, C., tures (16). This could be a result of its enhanced interaction Revuelta, J. L., Riles, L., Roberts, C. 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Domain and Nucleotide Dependence of the Interaction between Saccharomyces cerevisiae Translation Elongation Factors 3 and 1A *

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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 43, pp. 32318 –32326, October 27, 2006 © 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Domain and Nucleotide Dependence of the Interaction between Saccharomyces cerevisiae Translation Elongation Factors 3 and 1A Received for publication, February 28, 2006, and in revised form, August 30, 2006 Published, JBC Papers in Press, September 5, 2006, DOI 10.1074/jbc.M601899200 1 1 2 Monika Anand , Bharvi Balar , Rory Ulloque, Stephane R. Gross, and Terri Goss Kinzy From the Department of Molecular Genetics, Microbiology and Immunology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854 Eukaryotic translation elongation factor 3 (eEF3) is a fungal- somes have been reported to possess a compensatory intrinsic specific ATPase proposed to catalyze the release of deacylated- ATPase activity, although they differ kinetically from the fungal tRNA from the ribosomal E-site. In addition, it has been shown eEF3 (5). Escherichia coli, on the other hand, expresses the 911 to interact with the aminoacyl-tRNA binding GTPase elonga- amino acid RbbA protein that exhibits ATPase activity and is tion factor 1A (eEF1A), perhaps linking the E and A sites. tightly associated with ribosomes (6, 7). Both pathogenic and Domain mapping demonstrates that amino acids 775–980 con- non-pathogenic fungi have been reported to contain eEF3 tain the eEF1A binding sites. Domain III of eEF1A, which is also (8–10). In Saccharomyces cerevisiae, eEF3 is encoded by a sin- involved in actin-related functions, is the site of eEF3 binding. gle copy essential YEF3 gene. A paralog of the YEF3 gene, des- The binding of eEF3 to eEF1A is enhanced by ADP, indicating ignated HEF3 or YEF3B, encodes an 84% identical protein but is the interaction is favored post-ATP hydrolysis but is not not expressed during vegetative growth (11). However, expres- dependent on the eEF1A-bound nucleotide. A temperature- sion of the HEF3 coding sequence under the YEF3 promoter sensitive P915L mutant in the eEF1A binding site of eEF3 has produces a protein that has similar ATPase activity and ribo- reduced ATPase activity and affinity for eEF1A. These results some binding properties to YEF3-encoded eEF3. support the model that upon ATP hydrolysis, eEF3 interacts eEF3 is a class 1 member of the ATP binding cassette (ABC) with eEF1A to help catalyze the delivery of aminoacyl-tRNA at family of proteins. eEF3 possesses distinct motifs including the the A-site of the ribosome. The dynamics of when eEF3 interacts HEAT repeats on the N terminus, two nucleotide binding with eEF1A may be part of the signal for transition of the post to domains with tandemly arranged bipartite (ABC) cassettes in pre-translocational ribosomal state in yeast. the middle, a conserved insertion in the intervening region of the Walker A and B motifs of ABC2, and a highly basic C ter- minus. HEAT (Huntington elongation factor 3, A subunit of The protein synthetic machinery is characterized by the protein phosphatase 2A and TOR1) repeats correspond to a interplay of different soluble factors in conjunction with ribo- tandem -helical structure that appears to serve as flexible scaf- somes to translate the mRNA into the correct sequence of folding on which other proteins can assemble. Amino acids amino acids. The three phases of translation, initiation, elonga- 98–388 within the eEF3 N-terminal HEAT domain have also tion, and termination, are driven by factors that are highly con- been shown to interact with the 18 S rRNA (12). Within the served between yeast and metazoans (1). However, a major dif- Walker A and B motifs, the nucleotide binding stretch of seven ference in elongation is the indispensability of eukaryotic amino acids in ABC1 and -2 are 100% conserved among the elongation factor 3 (eEF3) with yeast ribosomes (2, 3). eEF3 ATP-binding proteins (13). The Walker C motif is the con- catalyzes an essential step in each elongation cycle by virtue of served LSGGQ sequence, the presence of which distinguishes its ATPase activity. It has been proposed to act as an Exit-site the ABC proteins from other ATPases (14). Alterations of the (E-site) factor, facilitating the release of deacylated-tRNA and conserved glycine and lysine residues within the Walker A of simultaneously impacting on the delivery of aminoacyl-tRNA either ABC1 or -2 abolish the ATP hydrolytic activity of eEF3 (aa-tRNA) at the aminoacyl site (A-site) (4). Metazoan ribo- in vitro and are lethal for growth in vivo (15). Interestingly, a temperature-sensitive (Ts ) F650S point mutant in the intervening region of the two ABC cassettes also affects the * This research was supported by National Institutes of Health Grant GM57483 (to T. G. K.). The costs of publication of this article were defrayed catalytic ATPase activity of the protein, indicating that the in part by the payment of page charges. This article must therefore be linker region affects either ATP binding or its hydrolysis hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 (16). Crystal structures of the E. coli transporter system ABC solely to indicate this fact. These authors contributed equally. proteins HisP (17) and MalK (18) as well as the human Rad50 To whom correspondence should be addressed: Dept. of Molecular Genet- ATPase (19) and cystic fibrosis transmembrane conductance ics, Microbiology and Immunology, UMDNJ Robert Wood Johnson Medical regulator (20) demonstrate that all possess two associated School, 675 Hoes Lane, Piscataway, NJ 08854. Tel.: 732-235-5450; Fax: 732- 235-5223; E-mail: [email protected]. monomers. Each monomer harbors a single ABC cassette The abbreviations used are: eEF, eukaryotic elongation factor; GST, glutathi- and forms a homodimer in the presence of ATP to carry out one S-transferase; Ni -NTA, nickel nitrilotriacetic acid; aa-tRNA, amino- hydrolysis, although there is variation in the manner by acyl-tRNA; E-site, exit site; A-site, aminoacyl site; ABC, ATP binding cassette; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride. which each nucleotide binding domains from the two mono- This is an open access article under the CC BY license. 32318 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 43 •OCTOBER 27, 2006 eEF3 and eEF1A Interaction TABLE 1 S. cerevisiae strains used in this study Strains Genotype Source TKY554 MAT ura3-52 leu2-3, 112 trp1-7 lys2-1243 met2-1 his4-713 yef3::LEU2 2 pYEF3 URA3 Ref. 16 TKY555 MAT ura3-3 leu2-2 trp1-1 his3-3 pMA210 (GAL4 2 HIS3) This study TKY597 MAT ura3-52 leu2-3, 112 trp1-7 lys2-1243 met2-1 his4-713 yef3::LEU2 CEN pYEF3 TRP1 This study TKY616 MAT ura3-52 leu2-3, 112 trp1-1 lys2-20 met2-1 his4-713 tef1::LEU2 tef2 CEN His pTEF1 TRP1 This study TKY676 MAT ura3-52 leu2-3, 112 trp1-7 lys2-1243 met2-1 his4-713 yef3::LEU2 CEN His pYEF3 TRP1 This study TKY702 MAT ura3-52 leu2-3, 112 trp1-7 lys2-1243 met2-1 his4-713 yef3::LEU2 2 His pYEF3 TRP1 Ref. 16 TKY800 MAT ura3-52 leu2-3, 112 trp1-7 lys2-1243 met2-1 his4-713 yef3::LEU2 CEN pyef3 TRP1(P915L) This study TKY805 MAT ura3-52 leu2-3, 112 trp1-7 lys2-1243met2-1 his4-713 yef3::LEU2 2 His pyef3 TRP1 (980eEF3) This study TKY819 MAT ura3-52 leu2-3, 112 trp1-7 lys2-1243 met2-1 his4-713 yef3::LEU2 CEN His pyef3 TRP1 (P915L) This study TKY822 MAT ura3-52 leu2-3, 112 trp1-7 lys2-1243 MET2 his4-713 yef3::LEU2 CEN His pYEF3 TRP1 This study TKY824 MAT ura3-52 leu2-3, 112 trp1-7 lys2-1243 MET2 his4-713 yef3::LEU2 CEN pyef3 TRP1 (P915L) This study mers collaborate to bind ATP molecules (21). The lysine- (met2-1 and his4-713) was determined by spotting 10 lof rich C terminus of eEF3 (amino acids 980 –1044) has previ- the same dilutions onto complete medium lacking methio- ously been implicated as required for binding to the nine or histidine, respectively, and incubating for 5 days at ribosome (22, 23). 30 °C. Halo assays for sensitivity to cycloheximide, paromo- During translation elongation, delivery of aa-tRNA to the mycin, and hygromycin B were performed as previously A-site by eEF1A and the translocation of the ribosome by described (29). Total yeast translation was monitored by in eukaryotic elongation factor 2 (eEF2) require GTP hydrolysis vivo [ S]methionine incorporation as previously described (1). The unique role of eEF3 may be part of the transition of the at both 30 and 37 °C (30) using the indicated MET2 strains. post-translocational to the pre-translocational state via its ATP Isolation of the P915L eEF3 Mutant by Hydroxylamine hydrolytic activity in yeast. The allosteric three-site model sug- Mutagenesis—Ten g of plasmid DNA (pTKB594) harboring gests that only two tRNAs can occupy the ribosome at one time, YEF3 on a CEN TRP1 plasmid was added to 500 lof1 M and thus, the exit of deacylated-tRNA is a prerequisite or coreq- hydroxylamine, pH 7.0. The reaction was incubated at 37 °C for uisite for the delivery of aa-tRNA to the A-site (24). eEF3 has 20 h and stopped by adding 100 mM NaCl and 0.1 g/l bovine been proposed to aid this removal and help promote the deliv- serum albumin. DNA was ethanol-precipitated, transformed ery of only cognate aa-tRNA by eEF1A to the A-site (4). It into TKY554, and plated on C-Trp to select for the mutated yef3 remains unclear how and when eEF3 utilizes its ATP hydrolytic TRP1 plasmid at a density of150–300 cells/plate. Cells able to activity to carry out these functions. lose the wild type YEF3 URA3 plasmid were identified by To address this question the present study analyzed the growth on 5-fluoroorotic acid-containing media. The resulting regions involved in, and the nucleotide-bound state that favors strains expressing the yef3 TRP1 plasmid as the only form were eEF3 binding to eEF1A. Our results point toward an enhanced analyzed for growth at 13, 30, or 37 °C. A colony unable to grow eEF3 and eEF1A association in the presence of ADP, suggesting at 37 °C was recovered from the 30 °C plate, and the plasmid that ATP hydrolysis likely precedes eEF3 binding to eEF1A. was extracted, transformed in E. coli, recovered, and retrans- The eEF1A binding region of eEF3 has been mapped to 2 formed into TKY554. Loss of the wild type YEF3 plasmid was regions near the C terminus. A genetic screen conducted in the repeated to confirm the phenotype. The P915L eEF3 mutant current study resulted in a point mutation in one of the regions. plasmid pTKB753 isolated in this screen was also constructed A strain expressing the P915L eEF3 exhibits a temperature- with a His tag on the N terminus by site-directed mutagenesis sensitive (Ts ) growth defect and reduction in total translation. of pTKB602 by the QuikChange method (Stratagene), produc- Additionally, the protein has negligible intrinsic and ribosome- ing pTKB777. stimulated ATPase activity and shows reduced affinity for Cloning, Expression, and Purification of GST and His - eEF1A. tagged eEF3, eEF1A, and Truncations—Full-length eEF3 and EXPERIMENTAL PROCEDURES fragments containing amino acids 1–775 (85NT), 100 –367 (HEAT), 775–910 (I), 910 –1044 (15CT), and 775–1044 Yeast and Bacterial Strains, Growth, Drug Sensitivity, and (30CT) were PCR-amplified using pTKB594 as the template. Translation Assays—S. cerevisiae strains and their genotypes Fragments were cloned into pTKB544 for expression with a are listed in Table 1. E. coli DH5 was used for plasmid prep- galactose-inducible promoter (GAL1-10) and an N-terminal aration. Procedures for cell growth and genetic manipula- GST tag, resulting in plasmids pTKB705, pTKB706, tions were according to standard protocols (27). Yeast cells pTKB707, pTKB708, pTKB709, and pTKB710, respectively. were grown in either YEPD (1% Bacto-yeast extract, 2% pep- The plasmids expressing the GST-tagged eEF3 fragments tone, 2% dextrose) or in defined synthetic complete medium were transformed in TKY555 and maintained on C-Ura- (C or C) supplemented with 2% dextrose as the carbon Hisgalactose media for protein expression. Yeast cultures source unless noted. Yeast were transformed by the lithium expressing the GSTeEF3 fusions were harvested at an A of acetate method (28). Temperature sensitivity was assayed by growing strains to an A of 1.0. Serial 10-fold dilutions (5 l 1.0 –2.0, and total yeast extracts were clarified and loaded on each) were spotted on appropriate medium followed by the GST Trap column (Amersham Biosciences) in buffer A incubation at 13, 24, 30, and 37 °C for 3–7 days. Phenotypic (20 mM Tris-HCl, pH 8.0, 100 mM KCl, 1 mM DTT, and 0.2 suppression of a non-programmed 1 frameshift allele mM PMSF). The protein was eluted with buffer A plus 20 mM OCTOBER 27, 2006• VOLUME 281 • NUMBER 43 JOURNAL OF BIOLOGICAL CHEMISTRY 32319 eEF3 and eEF1A Interaction reduced glutathione (Sigma). The protein peak was dialyzed GST and His Pulldowns of eEF1A and eEF3—Yeast extracts into buffer B (20 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 10% for in vivo binding assays were prepared by glass bead lysis in glycerol, 1 mM DTT, 0.2 mM PMSF, and 100 mM KCl). TEDG buffer (10 mM Tris-HCl, pH 7.4, 2 mM EDTA, 5 mM To facilitate eEF3 purification from yeast, a His tag was DTT, 50 mM KCl, and 1 mM PMSF) from TKY555 with the added to the N terminus of S. cerevisiae eEF3 under the con- empty plasmid pTKB544, GSTeEF3 (pTKB705), or the GST- trol of its own promoter on a CEN TRP1 plasmid producing eEF3 fragments (pTKB706, pTKB707, pTKB708, pTKB709, pTKB602 (31). A yeast plasmid expressing His 980 eEF3 was 6 pTKB710). For GST and Ni -NTA pulldown assays, 200-l produced by introduction of a stop codon at amino acid 981 reactions containing 50 g of total protein (determined by by QuikChange, producing pTKB724 (31). The plasmids Bradford reagent; Bio-Rad) and 40 l of either 50% glutathione- were introduced into S. cerevisiae strain TKY554, and loss of Sepharose 4B slurry (Sigma) in KETN 150 buffer (150 m M KCl, the wild type eEF3 on a URA3 plasmid was monitored by 1mM EDTA, 20 mM Tris-HCl, pH 8.0, 0.5% Nonidet, and 1 mM growth on 5-fluoroorotic acid, producing TKY702 and PMSF) or Ni -NTA slurry (Amersham Biosciences, GE TKY805, respectively. Healthcare) in buffer C were mixed at 4 °C for 1 h. Beads were His -tagged wild type eEF1A, eEF3, 980eEF3, and P915L washed 3 times with either KETN buffer with 150 or 300 mM eEF3 proteins were purified from strains TKY616, TKY702, KCl for GST pulldown or buffer C with 100 mM imidazole for TKY805, and TKY819, respectively, on a Ni Hi Trap chelat- Ni -NTA pulldown. Samples were resolved by SDS-PAGE, ing column (Amersham Biosciences). Total yeast extracts were and were proteins were detected with a polyclonal antibody clarified and loaded on the column in buffer C (50 mM KPO , to yeast eEF1A and ECL (Amersham Biosciences) and quan- pH 7.6, 300 mM KCl, 1 mM DTT, and 0.2 mM PMSF) with 20 mM titated with the ImageQuant program (GE Healthcare). imidazole. The protein was eluted with buffer C plus 400 mM Ni -NTA pulldown of purified untagged eEF1A with His - imidazole. The protein peak was dialyzed into buffer B. tagged eEF3 or untagged eEF3 with His -tagged eEF1A were BspEI restriction sites were introduced upstream of the ATG performed with 2 g of eEF3 and 3 g of eEF1A as previously initiation codon and downstream of the TAA stop codon using described (33). the QuikChange protocol in TEF1 on pTKB731 as template, Ribosome Binding Assay—The ribosome binding assay was producing pTKB740. His -tagged eEF1A with BspEI restriction performed as described previously (34) with minor modifica- sites upstream and downstream of the open reading frame was tions. Fifty-l reactions containing 24 pM purified proteins and constructed by PCR and cloning into pTKB740, resulting in plasmid pTKB779. Domain I (amino acids 1–221) was con- 24 pM 80 S ribosomes in binding buffer (20 mM Tris-HCl, pH structed by QuikChange mutagenesis of the Lys-222 and Lys- 7.5, 50 mM ammonium acetate, 10 mM magnesium acetate, and 224 codons to TAA using pTKB779, producing pTKB852. His - 2mM DTT) were incubated for 5 min at room temperature, tagged domain III (amino acids 333–458) was obtained by layered on top of a 200-l sucrose cushion (10% sucrose in looping out domains I and II using site-directed mutagenesis binding buffer), and centrifuged at 74,000 rpm for 20 min at protocol of template pTKB779, producing pTKB785. Plasmids 4 °C in S80-AT2 (Sorvall) rotor. The pellet (bound fraction) was pTKB852 and pTKB785 were used as templates for PCR ampli- resuspended in Laemmli loading buffer and subjected to SDS- fication of His -domain I and His -domain III fragments to 6 6 PAGE and Western blot analysis using the ECL method (Amer- clone into the pET11a vector, resulting in plasmids pTKB863 sham Biosciences). and pTKB851, respectively. His -tagged domain II (amino acids Enzyme-linked Immunosorbent Assays—In vitro binding 222–316) in pET11a was constructed by QuikChange was measured by an indirect enzyme-linked immunosorbent mutagenesis of the Glu-316 and Arg-318 codons to TAA and assay. Purified GSTeEF3 (0.25 g) in 50 l of PBST (137 mM TGA, respectively, using pTKB864 as the template to produce NaCl, 2.7 mM KCl, 10 mM Na HPO ,and2mM KH PO )/ 2 4 2 4 pTKB920. well was coated overnight at room temperature in a 96-well A 1-liter culture of E. coli BL21 with each plasmid was grown ultrahigh binding polystyrene microtiter plate (Thermo- to an A of 0.6 in LB with 100 g/ml ampicillin medium. Labsystem). After blocking with 300 l of 0.1% bovine serum Protein expression was induced with 1 mM isopropyl--D-thio- albumin in PBST for 1 h at room temperature and washing 3 galactopyranoside at 37 °C for 3–4 h. Cells were harvested by times with 300 l of PBST, 50 l of 5000-fold-diluted affin- centrifugation and lysed by sonication, and the recombinant ity-purified polyclonal anti-eEF3 antibody was added to each protein was purified in accordance with the QIAexpressionist well and incubated at room temperature for 2 h. Varying protocol for His -tagged proteins under native conditions. Pro- amounts of eEF1A along with varying amounts of ATP, ADP, tein-containing fractions were dialyzed into 20 mM Tris-HCl, GTP, or GDP were added to the eEF3 antibody. After washing 3 pH 7.5, 1 mM DTT, 0.1 mM EDTA, pH 8.0, 100 mM KCl, and 20% times with 300 l with PBST, 50 l of 2500-fold-diluted sec- glycerol. ondary goat anti-rabbit antibody conjugated with alkaline ATP Hydrolysis—ATP hydrolysis was performed using puri- phosphatase was added per well (Jackson ImmunoResearch). fied proteins as previously described (32). Briefly, the assay mix- Unbound antibody was removed by three washes of 300 lof ture contained 24 pM protein, 50 pM yeast ribosomes, and 150 PBST followed by the addition of 50 lof3mM p-nitrophenyl M [- P]ATP. Hydrolysis was allowed to proceed for 5 min at phosphate (Sigma) in 50 mM Na CO and 50 M MgCl /well. 30 °C, and P release was determined. ATP hydrolysis levels i 2 3 2 The extent of p-nitrophenyl phosphate hydrolysis represents were calculated after subtracting the background for buffer alone. the antigen-antibody binding measured by A . 32320 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 43 •OCTOBER 27, 2006 eEF3 and eEF1A Interaction GST fusion fragments co-purified eEF1A at levels similar to or above that of full-length eEF3. The 85NT, GST alone, and HEAT fragments co-purified less eEF1A, although some background level of binding was observed (Fig. 1C). The same experiment was also probed for co-elution of ribosomes with the eEF3eEF1A complex. As shown in Fig. 1C, bottom panel, RPL10e, a ribosomal protein, is absent in the bound fractions. The middle panel, Fig. 1C, shows probing for phosphoglycerate kinase (PGK1) as the internal loading control. Because the fusion truncations are expressed at different levels in vivo, the 85NT, 30CT, and 15CT GST- tagged fragments were purified from yeast, and GST pulldown experiments were performed with purified untagged yeast eEF1A. The GST-HEAT and GST-I fusion were not stably expressed at sufficient levels for purification. GST-15CT FIGURE 1. C-terminal regions of eEF3 bind eEF1A. A, eEF3 fragments cloned as GST fusions under the GAL1-10 and GST-30CT co-purified with promoter in pTKB544. B, plasmids expressing the GST fusion fragments from A were transformed in TKY555 and maintained in C-Ura-Hisgalactose. Strains were grown to mid-log phase at 30 °C, yeast extracts were eEF1A at levels comparable with prepared, and equal amounts of protein (5g) were separated by SDS-PAGE and subjected to Western blotting wild type GSTeEF3, whereas the with an anti-GST monoclonal antibody. The lower panel shows eEF1A as the internal loading control. C, a GST GST-85NT was at background lev- pulldown assay was performed with the extracts (50g) from the same strains as in B, and the Western blot was developed with an anti-eEF1A antibody (top panel), anti-phosphoglycerate kinase (PGK1) antibody represent- els (GST, Fig. 1D). The results in Fig. ing internal loading control (middle panel) and anti-RPL10e antibody to detect co elution ribosomes with the 1, C and D, demonstrate that the eEF3eEF1A complex (lower panel). E, extract (10% input); S, supernatant (5%); P, pellet (100%). D, a GST pull- eEF3eEF1A interaction occurs in down assay with purified yeast eEF1A (100 pM) and GST-tagged eEF3, 30CT, 15CT, and 85NT fragments (20 pM). E, the yeast strains from B were grown to mid-log phase at 30 °C, diluted to equal A , spotted as 10-fold serial the absence of any cellular factors dilutions, and grown at 13 or 24 °C for 2–7 days. via the C-terminal region of eEF3. Dominant growth phenotypes RESULTS conferred by the truncations were monitored on C-Ura- eEF3 Interacts with eEF1A through Its C-terminal Region— Hisgalactose medium at different temperatures. The 30CT Prior studies have demonstrated that eEF3 and eEF1A inter- and 15CT fragments confer a dominant slow growth phenotype act, as monitored by both genetic and physical assays in vivo at 13 °C, whereas no effects were seen at 30 or 37 °C (Fig. 1E and and in vitro (16). To map the site of interaction, five fragments data not shown). Because there appear to be two eEF1A binding of eEF3 corresponding approximately to natural proteolytic sites, one within amino acids 775–910 and one within 910– sites were cloned into a GAL1-10-inducible expression vector 1044, fragments of eEF3 containing these amino acids may with a GST tag at the N terminus (Fig. 1A). These include full- exhibit a dominant slow growth phenotype due to the forma- length eEF3 (amino acids 1–1044), 85NT, (1–775), HEAT tion of inactive complexes with eEF1A. The I fragment (775– (100–367), 15CT (910–1044), I (775–910), and 30CT (775– 910) does not show this growth phenotype, indicating the site 1044). All the fragments are expressed in yeast although at dif- from 910 to 1044 may have a larger effect in vivo. ferent levels, as monitored by Western blot with anti-GST anti- His 980eEF3 Is Functional in Vivo and Retains Binding to body (Fig. 1B). The GST-tagged fusion proteins migrate at 140 Ribosomes and eEF1A—Prior work proposed that the C-termi- (eEF3), 105 (85NT), 57 (HEAT), 42 (15CT), 58 (30CT), and 42 nal 64 amino acids (980–1044), containing 40% basic residues, (I) kDa, with 29 kDa contributed by the GST tag. The same gel is the primary ribosome binding region of eEF3 (22). Other is also probed with anti-eEF1A antibody as the internal loading work suggests the N-terminal 98–388 amino acids binds to 18 S control. rRNA in vitro and inhibit the ribosome-dependent ATPase Because none of the eEF3 fragments can replace wild type activity of eEF3 (12). To determine the function of the basic C eEF3 in vivo (data not shown), all were co-expressed with an terminus of eEF3, His -tagged eEF3 1–980 was expressed from untagged wild type copy of eEF3 to support growth. A GST a2 TRP1 plasmid. This construct was able to function as the pulldown assay was performed to determine the binding of only form of eEF3 (Fig. 2A). Cells expressing His 980eEF3 as the eEF3 to eEF1A in total cell extracts. The 15CT, I, and 30CT only form of eEF3 have a slight slow growth phenotype (Fig. OCTOBER 27, 2006• VOLUME 281 • NUMBER 43 JOURNAL OF BIOLOGICAL CHEMISTRY 32321 eEF3 and eEF1A Interaction To determine whether the C terminus of eEF3 is dispensable for ribosome binding, purified His eEF3 and His 980eEF3 were 6 6 assayed for co-association with ribosomes through a sucrose cush- ion (Fig. 2E). The slowest migrating bands corresponding to the full- length and 1–980 proteins pellet with ribosomes. Interestingly, the same degradation products were observed for both eEF3 and 980eEF3. All three bands reacted with the anti-His antibody (data not shown), and because the His tag was located at the N terminus, this indicates the N terminus is intact. Thus, these fragments represent C-terminal truncations and imply ribosome binding occurs near the N terminus. This is consistent with work showing an N-terminal frag- ment binds 18 S rRNA (12). The negative control bovine serum albu- FIGURE 2. His 980eEF3 is functional in vivo and retains binding to eEF1A and ribosomes. A, strains con- taining plasmid-borne untagged eEF3 (2 m, TKY554), His -tagged eEF3 (CEN, TKY676), His eEF3 (2, TKY702), 6 6 min stayed in the supernatant both and His 980eEF3 (2, TKY805) were grown to mid-log phase at 30 °C, diluted to equal A , spotted as 10-fold 6 600 in the presence and absence of ribo- serial dilutions, and grown at 24 or 13 °C for 2–7 days. B, yeast extracts (2 g) were prepared from strains somes (data not shown). expressing His eEF3 (TKY702), eEF3 (TKY554), and His 980eEF3 (TKY805) and analyzed for the expression of 6 6 His 980eEF3 by Western blotting with an anti-eEF3 antibody. The lower panel shows equal loading of eEF1A as ADP Enhances the Association of internal control. C, in vivo eEF1A binding to His eEF3 and His 980eEF3 was analyzed by Ni -NTA pulldown of 6 6 eEF3 with eEF1A—Because both yeast extracts (50 g) from strains as in A. S, supernatant (5%) and P, pellet (100%) were subjected to SDS-PAGE and Western blot with anti-eEF1A and anti-His antibodies. D, in vitro binding of purified His eEF3 and 6 6 eEF3 and eEF1A bind nucleotides, His 980eEF3 in a 5-fold molar excess of untagged eEF1A was assessed by Ni -NTA pulldown and analyzed by an enzyme-linked immunosorbent SDS-PAGE and Western blot with anti-eEF1A antibody. E, association of eEF3 with purified 80 S ribosomes assay-based binding assay was through a 10% sucrose cushion is shown for His 980eEF3 and His eEF3 and analyzed as in D with an anti-eEF3 6 6 antibody. P, pellet (100%); S, supernatant (20%). developed to look at the effect of these molecules on the eEF1A-eEF3 2A). This effect is most noticeable at 13 °C. Western blot anal- interaction. Subsequent to coating the wells with purified ysis of His eEF3, wild type eEF3, and His 980eEF3 from strains eEF3, eEF1A was added to compete with an anti-eEF3 anti- 6 6 TKY702, TKY554, and TKY805 with anti-eEF3 antibody (Fig. body. Because eEF1A binding competes with antibody bind- 2B) shows that His 980eEF3 protein is expressed at similar lev- ing, the absorbance value is reduced in the presence of els as full-length-tagged and untagged eEF3. The same gel was eEF1A. Concentration-dependent eEF1A binding to eEF3 also probed with anti-eEF1A antibody as internal loading con- was observed (Fig. 3A). A 10-fold molar excess of eEF1A to trol. Therefore, although His 980eEF3 is stably expressed, its eEF3 was used for all further assays. A series of controls was function in vivo is likely partially compromised. included in this assay to validate these results. These The role of amino acids 981–1044 in binding eEF1A was included demonstrating that the anti-eEF3 antibody does determined by Ni -NTA pulldown of extracts from strains not show any affinity for eEF1A, the addition of nucleotide expressing His 980eEF3, His eEF3, or untagged eEF3. Super- alone in the absence of anti-eEF3 antibody exhibits negligi- 6 6 natant and pellet fractions were resolved by SDS-PAGE, and ble absorbance, and the addition of nucleotides alone (in the the Western blot was probed with anti-eEF1A and anti-eEF3 absence of competing factor eEF1A) along with anti-eEF3 antibodies. eEF1A associates with His 980eEF3 at levels com- antibody does not affect absorbance (data not shown). parable with full-length His eEF3 (Fig. 2C). In the negative con- To ascertain the effect of the nucleotide-bound state on the trol with untagged eEF3, minimal background eEF1A was pres- binding of the two proteins, ATP, ADP, GTP, or GDP was ent in the pellet. The Ni -NTA pulldown was also performed added with the anti-eEF3 antibody and eEF1A. Whether GTP with purified proteins, confirming that eEF1A binds to both or GDP was incubated with eEF1A and eEF3, the signal His eEF3 and His 980eEF3 directly (Fig. 2D). This indicates remained constant, and thus, binding was unaffected (Fig. 3B). 6 6 that residues 981–1044 are not required for eEF1A binding. On the other hand, there is a concentration-dependent reduc- Taken together with the truncation data (Fig. 1, C and D), it tion in signal, and hence, stimulation of eEF1A binding when appears one eEF1A binding site is located within the 205-amino ADP was added. This is shown as binding normalized to acid stretch from 775 to 910 and a second within amino acids absorbance in the presence of nucleotide alone and in the 910–980. absence of eEF1A (Fig. 3C). Furthermore, when ATP was added 32322 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 43 •OCTOBER 27, 2006 eEF3 and eEF1A Interaction (Amersham Biosciences) (Fig. 3F). His eEF3 eluted as a single sharp peak with a retention time of 29.24 min corresponding to a molecular mass of 140 kDa. The migration remains unchanged in 1 M KCl, 1 mM ATP, 1 mM ADP, or 50 mM ethylene glycol (data not shown), showing that eEF3 exists as a mon- omer in its purified form. A P915L Mutation in an eEF1A Binding Site of eEF3 Alters ATPase Activity and eEF1A Binding—A genetic screen for conditional mu- tants in eEF3 was conducted using unbiased in vitro mutagenesis of a YEF3 plasmid. A pool of hydroxy- lamine-treated plasmids was trans- formed into yeast, and plasmids able to replace the wild type YEF3 URA3 plasmid were determined by growth on 5-fluoroorotic acid. Approxi- mately 7000 colonies were screened for temperature-sensitive growth yielding a strain expressing a single eEF3 point mutation, P915L, in the C-terminal region (Fig. 4A). The doubling time of the P915L mutant strain was 5.5 h compared with 3.5 h for the wild type strain. Total pro- tein synthesis monitored by meas- uring [ S]methionine in the P915L FIGURE 3. ADP stimulates eEF1A binding to eEF3. A, microtiter 96-well plates (Falcon) were coated with purified GSTeEF3 (0.25 g), and an affinity-purified anti-eEF3 antibody was added with or without increasing strain was 20% less than a wild type amounts of eEF1A. eEF1A (1.25 g) was incubated in the GSTeEF3-coated microtiter plate as in A, with different strain at permissive temperatures concentrations of nucleotides, GTP (diamonds) or GDP (squares)in B expressed as A or ATP (diamonds), or and 22% less than wild type when ADP (squares)in C expressed as percentage bound normalized to the presence of nucleotide alone in the absence of eEF1A.D, eEF1A bound to GSTeEF3 after GST pulldown in the presence of varying amounts of ADP cells were shifted to 37 °C (Fig. 4B). were analyzed by SDS-PAGE and stained with gel code blue (Pierce). S, supernatant (5%); P, pellet (100%). E, the To determine the eEF3 defect caus- results of GST pulldown experiments as in D was analyzed with the ImageQuant program (GE Healthcare), and the ratio of pellet to supernatant was plotted. F, purified His eEF3 was subjected to gel filtration analysis by fast ing this effect, the ATPase activity of protein liquid chromatography on a Superdex 200 column (Amersham Biosciences). The elution profile of purified His P915L eEF3 was deter- His eEF3 was determined by SDS-PAGE and Western blot with an anti-eEF3 antibody. mined. The mutant lacks both intrinsic and ribosome-stimulated there was a concentration-dependent increase in signal, and ATPase activity (Fig. 4C). To assess if this loss of catalytic activ- hence, reduction in binding was observed. The experiment was ity affects eEF1A binding to the P915L eEF3 mutant, associa- done multiple times to confirm a reproducible trend. tion was assessed by Ni -NTA pulldown assay. His P915L To confirm the enzyme-linked immunosorbent assay-based eEF3 pulls down reduced levels of eEF1A as compared with wild assay, a GST pulldown of purified untagged eEF1A with type His eEF3 in both cell extracts (Fig. 4D) and with purified GSTeEF3 was performed in the presence of different concen- proteins (Fig. 4, E and F). A small amount of eEF1A is nonspe- trations of nucleotide and analyzed by SDS-PAGE followed by cifically pulled down by untagged eEF3 using Ni -NTA beads. gel code blue staining. The amount of eEF1A bound to This implies that binding of eEF3 to eEF1A is sensitive to struc- GSTeEF3 in the pellet increases in the presence of increasing tural and functional alterations caused by a point mutation in a amounts of ADP (Fig. 3, D and E). Thus, results from two inde- region proposed to bind eEF1A. pendent methods indicate that binding of eEF1A to eEF3 is eEF1A Binds eEF3 via Domain III—The co-crystal structure likely stimulated after ATP hydrolysis. of eEF1A with its guanine nucleotide exchange factor eEF1B Proteins belonging to the ABC superfamily are inter- or shows the G-protein has three domains. Domain I contains the intramolecular dimers, and the presence of two ATPase GTP binding motifs, and domains I and II contact eEF1B (33, domains is required for function (35). To confirm His eEF3 36). Domain III has been shown to interact with actin and is is a monomer, purified protein was subjected to analysis by responsible for the non-canonical functions of eEF1A in actin gel filtration chromatography on a Superdex 200 column binding and bundling (25) and the slow growth phenotype asso- OCTOBER 27, 2006• VOLUME 281 • NUMBER 43 JOURNAL OF BIOLOGICAL CHEMISTRY 32323 eEF3 and eEF1A Interaction ners to carry out its essential steps in translation elongation is still not well understood. Recent work in bacteria confirms the allosteric link between the A and E sites (38). This supports the hypothesis that a general ribosome function is the release of deacylated tRNA from the E-site preceding the GTP hydrolysis required to deposit aa- tRNA at the A-site. This step likely involves a conformational change in the 70 S ribosome. Because bac- teria lack eEF3, although the ribo- some-associated ATPase RbbA has been implicated as a bacterial counterpart of eEF3 (39), the bind- ing of the ternary complex of aa- tRNA-EF-Tu-GTP has been sug- gested to induce the required conformational change in the ribosome to catalyze the release of deacylated-tRNA from the E-site FIGURE 4. The ATP hydrolysis deficient P915LeEF3 mutant shows reduced affinity for eEF1A. A, strains (38). In mammals, the ribosome- containing wild type eEF3 (TKY597) or P915LeEF3 (TKY800) were grown to mid-log phase at 30 °C, diluted to associated ATPase activity from equal A , spotted as 10-fold serial dilutions, and grown at 30 or 37 °C for 2–7 days. B, strains expressing His P915LeEF3 (TKY824) or His eEF3 (TKY822) were monitored for total translation by [ S]methionine incor- 6 6 pig liver differs from the yeast poration after growth to mid-log phase in C-Met and labeled for varying times at both 30 and 37 °C. Total eEF3 ATPase activity in its sensi- translation is expressed as cpm/A unit. Wt, wild type. C, intrinsic and ribosome (Rbs)-stimulated ATP hydro- tivity to translation inhibitors and lytic activities of purified His P915L and His eEF3 were measured. The pM P released from [- P]ATP are 6 6 i shown after subtracting the hydrolysis in the presence of buffer alone. The results are an average of three nucleotide dependence (40). experiments and the S.D. shown. D, yeast extracts were prepared from strains containing eEF3 (TKY597), Previous reports have proposed His eEF3 (TKY702), and His P915L (TKY819), and equal amounts of total protein were incubated with 6 6 Ni -NTA beads. Extract (E, 5%), supernatant (S, 5%), and pellet (P, 100%) were separated by SDS-PAGE and two different ribosome binding analyzed by Western blot. The blot was probed with both anti-eEF3 and anti-eEF1A antibodies. E, eEF1A, regions in eEF3, the 64 amino acids His eEF3, and His P915LeEF3 proteins were purified and ran on a SDS-PAGE gel and stained with GelCode Blue 6 6 at the C terminus (22) and the (Pierce). F, a 5-fold molar excess of purified eEF1A, either alone or with purified His eEF3 or His P915L proteins, 6 6 were incubated with Ni -NTA beads. Supernatant (S, 5%) and pellet (P, 100%) were separated by SDS-PAGE N-terminal residues 98–388 (12). In and analyzed by Western blot with an anti-eEF1A antibody. the present study we report that yeast expressing eEF3 in the absence ciated with eEF1A overexpression in vivo (26). To identify the of its 64 amino acids at the C terminus are viable, and both the eEF1A region involved in binding to eEF3, purified wild type eEF1A and ribosome binding properties are retained by His 980eEF3. Thus, the N-terminal region is likely the predom- His eEF1A from yeast and His fusions of domain I (1–221, 22 6 6 6 inant ribosome binding site. kDa,), II (222–332, 11 kDa), or III (333–458, 33 kDa) purified from E. coli (Fig. 5A) was used to determine GSTeEF3 binding The family of ABC protein includes membrane-bound fac- by Ni -NTA pulldowns. GSTeEF3 was pulled down only by tors, which function in transporting solute molecules against a concentration gradient. However, the soluble members of this wild type His eEF1A and His -domain III (Fig. 5B). No 6 6 family, including Gcn20p, RL11 (41), eEF3, and the recently GSTeEF3 binding was seen by either domains I or II. reported ARB1 (42) in yeast are also implicated in functions DISCUSSION related to protein synthesis, ribosome biogenesis, and transla- Protein synthesis in yeast relies not only on the availability of tion elongation. The crystal structure of several members of the eEF1AB complex and eEF2 but also another unique fac- the class I ATPases clearly establish the phenomena of tor, eEF3. The absolute dependence of the pathogenic fungal homodimerization of two ABC proteins to sandwich two ATP translation machinery on the presence of eEF3 can be exploited molecules utilizing the Walker A and B motifs of the one mon- as a fungal-specific drug target (37). To achieve this long-term omer (43, 44) and Walker C or the conserved LSGGQ motif, goal, our primary aim is to understand the role of eEF3 in pro- characteristic of only the ABC members of the ATPases super- tein synthesis. Previously published work has assigned eEF3 family, from the other monomer. It has been shown for cystic the dual roles of removing the deacylated-tRNA from the fibrosis transmembrane conductance regulator that upon ATP E-site of the ribosome and aiding eEF1A in the delivery of the hydrolysis, the dimerized cassettes come apart, and this motor correct aa-tRNA to the A-site. eEF3 has been shown to inter- motion drives the transport across the membrane (45). Inter- act physically with both eEF1A and ribosomes. The mystery estingly, the soluble members of the ABC family harbor both of how and when eEF3 collaborates with its interacting part- the cassettes in tandem in a single molecule. Our investigation 32324 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 43 •OCTOBER 27, 2006 eEF3 and eEF1A Interaction with either eEF1A and/or ribosomes driving translation elon- gation forward, and hence, total translation is increased (16). If eEF3 competes with actin to bind eEF1A via domain III, then the increase in eEF3 may shift the balance of the cellular machinery in favor of protein synthesis rather than toward the function of eEF1A in cytoskeletal arrangements. This dynamic cross-talk between the two cellular processes of protein synthe- sis and cytoskeletal arrangement is likely mediated by the elon- gation factor eEF1A and may also be affected by the interaction of eEF3 versus actin with eEF1A. This study supports the model that the ATP hydrolysis by eEF3 stimulates the interaction with eEF1A. This observation fits in nicely with the model of eEF3 function, where ribosome- stimulated nucleotide hydrolysis of the ATP-bound eEF3 pre- cedes its interaction with eEF1A and the delivery of only cog- nate aa-tRNA at the A-site. It is still speculative if eEF1A binding occurs, whereas eEF3 is bound to or upon its release from the ribosome. The latter situation is more likely since upon ATP hydrolysis, eEF3 is likely released from the ribosome. Acknowledgment—We acknowledge the assistance of Robert Wood Johnson Medical School DNA core facility. FIGURE 5. Domain III of eEF1A binds eEF3. A, His -tagged full-length eEF1A REFERENCES was purified from yeast, whereas the His -tagged eEF1A domains I, II, and III were expressed and purified from E. coli BL21 cells. Purified proteins sepa- 1. Merrick, W. C., and Nyborg, J. (2000) in Translational Control of Gene rated by SDS-PAGE are shown by Coomassie Blue stain. B,Ni -NTA pulldown Expression (Sonenberg, N., Hershey, J. W. B., and Mathews, M. B., eds) pp. was performed with 20 pM purified GSTeEF3 and 100 pM proteins from A. 98–125, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY Shown is the pellet (100%) after pulldown. The top half of the blot was devel- 2. Skogerson, L., and Wakatama, E. (1976) Proc. Natl. Acad. Sci. U. S. 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J. Biochem. 268, 278–286 bound state of eEF3. The ATPase inactive eEF3 mutants F650S 8. DiDomenico, B., Lupisella, J., Sandbaken, M. G., and Chakraburtty, K. (16) and P915L (present study) are in different regions of the (1992) Yeast 8, 337–352 protein but have a similar effect on eEF1A binding. The F650S is 9. Blakely, G., Hekman, J., Chakraburtty, K., and Williamson, P. R. (2001) J. in the region between the two ATP binding domains. The Bacteriol. 183, 2241–2248 10. Ypma-Wong, M., Fonzi, W., and Sypherd, P. S. (1992) Infect. Immun. 60, P915L mutant is in one of the eEF1A binding regions. Both lose 4140–4145 the ability to interact with eEF1A, pointing toward hydrolysis of 11. Sarthy, A. V., McGonigal, T., Capobianco, J. O., Schmidt, M., Green, S. R., ATP as a critical event in eEF1A binding. Consistent with these Moehle, C. M., and Goldman, R. C. (1998) Yeast 14, 239–253 findings, the presence of ADP stimulates eEF1A binding. This 12. Gontarek, R. 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Journal

Journal of Biological Chemistry – American Society for Biochemistry and Molecular Biology

Published: Oct 27, 2006

References

  • Methods in Yeast Genetics: A Laboratory Course Manual
    J.B. Hicks