Báo cáo khoa học: Characterization of eIF3k A newly discovered subunit of mammalian translation initiation factor eIF3 potx

Characterization of eIF3k A newly discovered subunit of mammalian translation initiation factor eIF3 Greg L. Mayeur, Christopher S. Fraser, Franck Peiretti, Karen L. Block and John W. B. Hershey Department of Biological Chemistry, School of Medicine, University of California, Davis, CA, USA Mammalian translation initiation factor 3 (eIF3) is a multisubunit complex containing at least 12 subunits with an apparent aggregate mass of  700 kDa. eIF3 binds to the 40S ribosomal subunit, promotes the binding of methionyl- tRNA i and mRNA, and interacts with several other initi- ation factors to form the 40S initiation complex. Human cDNAs encoding 11 of the 12 subunits have been isolated previously; here we report the cloning and characterization of a twelfth subunit, a 28-kDa protein named eIF3k. Evi- dence that eIF3k is present in the eIF3 complex was obtained. A monoclonal anti-eIF3a (p170) Ig coimmuno- precipitates eIF3k with the eIF3 complex. Affinity purifica- tion of histidine-tagged eIF3k from transiently transfected COS cells copurifies other eIF3 subunits. eIF3k colocalizes with eIF3 on 40S ribosomal subunits. eIF3k coexpressed with five other ÔcoreÕ eIF3 subunits in baculovirus-infected insect cells, forms a stable, immunoprecipitatable, complex with the ÔcoreÕ. eIF3k interacts directly with eIF3c, eIF3g and eIF3j by glutathione S-transferase pull-down assays. Sequences homologous with eIF3k are found in the genomes of Caenorhabitis elegans, Arabidopsis thaliana and Droso- phila melanogaster, and a homologous protein has been reported to be present in wheat eIF3. Its ubiquitous expression in human tissues, yet its apparent absence in Saccharomyces cerevisiae and Schizosaccharomyces pombe, suggest a unique regulatory role for eIF3k in higher organ- isms. The studies of eIF3k complete the characterization of mammalian eIF3 subunits. Keywords: protein synthesis; translation initiation; initiation factor; eIF3; eIF3k. Initiation of protein synthesis in eukaryotic cells involves formation of an 80S ribosomal complex containing the initiator methionyl-tRNA i bound to the initiation codon in a mRNA. It is a multistep process promoted by a number of proteins called eukaryotic translation initiation factors (eIFs). Currently, at least 12 eIFs, minimally composed of 29 distinct subunits, have been identified [1]. eIF3, the largest initiation factor, is a multisubunit complex with an apparent molecular mass of  700 kDa. eIF3 plays an essential role in translation by binding directly to the 40S ribosomal subunit and promoting formation of the 40S pre- initiation complex consisting of the Met-tRNA i eIF2GTP ternary complex, eIF1, eIF1A and the 40S ribosomal subunit [2]. eIF3 also promotes the binding of 5¢-m 7 G- capped mRNA through its interaction with eIF4G, the largest member of the eIF4F cap-binding complex [3,4]. The 40S preinitiation complex then scans the mRNA in a 5¢ to 3¢ direction, until the initiation codon AUG is selected. Upon recognition of the initiation codon, eIF5 stimulates the hydrolysis of the GTP bound to eIF2, and the eIFs are ejected from the ribosome. The 60S ribosomal subunit then joins the 40S initiation complex aided by eIF5B [5]. Mammalian eIF3 is known to interact with other initiation factors including eIF1 [6], eIF4B [7] and eIF5 [2]. Clearly, it plays a central role in the initiation pathway, possibly as an organizer of other initiation factors on the surface of the 40S ribosomal subunit. Eleven nonidentical subunits have been identified in mammalian eIF3 and their cDNAs have been cloned and characterized: a (p170) [8], b (p116) [9], c (p110) [10], d (p66) [11], e (p48) [12], f (p47) [11], g (p44) [13], h (p40) [11], i (p36) [10], j (p35) [13] and l (p69) [14]. This contrasts with eIF3 from Saccharomyces cerevisiae, which contains only six subunits, all homologous with the mammalian subunits of the same letter designation: a (TIF32) [15], b (PRT1) [16], c (NIP1) [17], g (TIF35) [18], i (TIF34) [19] and j (HCR1) [20]. The presence of at least six additional subunits in mamma- lian eIF3 suggests a potentially important regulatory role for these subunits. Interactions of the various yeast subunits with one another and with other eIFs have been extensively characterized and mapped [21], but a high-resolution, three- dimensional structure for neither yeast nor mammalian eIF3 is yet available. The knowledge of eIF3 subunit protein sequences provided by their cDNAs has proved useful in our understanding of the structure and function of eIF3. We therefore turned out attention to an uncharacterized protein of 28 kDa that is found in preparations of eIF3 derived from HeLa cells. In this work, we describe the isolation and characterization of a cDNA encoding this 28 kDa protein, which we have named eIF3k. Evidence is presented that demonstrates that eIF3k is indeed the twelfth subunit of mammalian eIF3. Correspondence to J. Hershey, Department of Biological Chemistry, School of Medicine, University of California, Davis, CA 95616, USA. Fax: + 1 530 752 3516, Tel.: + 1 530 752 3235, E-mail: jwhershey@ucdavis.edu Abbreviations: eIF3, mammalian translation initiation factor 3; GST, glutathione S-transferase. (Received 23 July 2003, revised 20 August 2003, accepted 26 August 2003) Eur. J. Biochem. 270, 4133–4139 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03807.x Materials and methods Materials eIF3 was prepared from human HeLa cells as described previously [22]. Polyclonal antiserum against rabbit eIF3 was prepared in a goat and characterized as previously [23]. Monoclonal antibody specific for eIF3a was a generous gift of J. T. Parsons (University of Virginia, USA). The monoclonal antibody against c-myc was obtained from Santa Cruz Biotechnology, Inc. Anti-FLAG agarose and FLAG peptides were obtained from Sigma-Aldrich Co. All cell culture materials were from Mediatech. Cloning of cDNAs encoding eIF3k The 28 kDa protein in highly purified eIF3 was separated from the other subunits by SDS/PAGE. The protein was digested in the gel with Lys-C protease and thefragments were fractionated by high-performance liquid chromatography. N-terminal sequences of internal peptides were obtained by automated Edman degradation in the Protein Structure Laboratory (University of California, Davis, USA). A peptide sequence, YNPENLATLER, matched 11 of 11 pre- dicted residues from the human EST clone zb98c06.r1. Clone zb98c06.r1 (GenBank nucleotide accession number W38661) was obtained from IMAGE (ATCC) and its 931-bp cDNA insert was sequenced on both strands. The cDNA sequence predicts a protein of 218 amino acid residues, consistent with it encoding the 28 kDa eIF3k subunit. Additional BLAST searches identified more than 20 overlapping human ESTs. The cDNA insert in zb98c06.r1 was amplified by PCR with the forward primer 5¢-CCCATATGGCC ATGGCGA TGTTTGAGCAG-3¢ and reverse primer 5¢-GCTCGAGA AGC TTACTGGGAGGAGGCCATG-3¢ (restriction sites are in bold and eIF3k start and stop codons are underlined). The NdeI- and XhoI-digested PCR product was ligated into pET28c (Novagen) to generate a histidine-tagged bacterial expression construct, pET-Hisp28. An untagged construct, pETp28, was generated by digesting the PCR product with NcoIandXhoI and ligating it into the equivalent sites in pET28c. Similarly, the eIF3k cDNA was cloned into pcDNA3.1/myc-His A (Invitrogen) for expression in COS-1 cells. PCR amplification from zb98c06.r1 with the forward primer 5¢-CCTCGAGC ATGGCGATGTTTGA GCAG-3¢ and reverse primer 5¢-GGAAGCTTGGGAG GAGGCCATGAT-3¢ (restriction sites are in bold and eIF3k startcodonisunderlined;thestopcodonhas beenremovedfor C-terminal c-myc tag) followed by digestion with XhoIand SfuI allowed construction of pcDNA/myc-His:p28. Synthesis of recombinant eIF3k in vitro Radiolabeled eIF3k was synthesized in the TnT system (Promega) programmed with pETp28 in the presence of [ 35 S]Met. The radiolabeled protein product was used without further purification. Transient transfection of COS-1 cells COS-1 cells were routinely grown in Dulbecco’s medium with 10% fetal bovine serum. Cells at approximately 60% confluence were transiently transfected by the DEAE- Dextran method [24] with 10 lg pcDNA/myc-His:p28. In some cases, proteins were radiolabeled by incubating the transfected cells in Met- and Cys-free DMEM (Mediatech) containing 0.2 mCiÆmL )135 S-Trans Label (NEN) for 18 h prior to lysis at 48 h. Cells were lysed in Lysis Buffer containing 10 m M Tris/HCl pH 7.4, 100 m M KCl, 5 m M MgCl 2, 0.5% NP-40, 0.5% deoxycholate, 0.1% Tri- ton X-100, and Complete Protease Inhibitor (Promega). Lysates were clarified by centrifugation at 10 000 g for 10 min. eIF3k expression in baculovirus-infected insect cells The FASTBAC vector (Invitrogen) was modified so that expressed proteins would contain the FLAG epitope fused at their N-termini (C. S. Fraser, J. Y. Lee, M. Bushell, G. L. Mayeur, & J. W. B Hershey, unpublished observa- tion). The NdeI-XhoI DNA fragment encoding untagged eIF3k derived from pETp28 above was inserted into the same restriction sites of the FLAG-FASTBAC vector to generate FLAG-eIF3k. FASTBAC vectors for the expres- sion of untagged eIF3a, eIF3b, eIF3c, eIF3g and eIF3i were constructed similarly as described elsewhere (C. S. Fraser, J. Y. Lee, M. Bushell, G. L. Mayeur, & J. W. B Hershey, unpublished observation). The six recombinant FASTBAC vectors were recombined individually with baculovirus DNA using DH10BAC Escherichia coli (Invitrogen) and the high molecular mass DNA (ÔbacmidÕ) was purified according to the manufacturer’s guidelines. Sf9 cells were transfected with bacmid DNA by using the calcium phosphate method (Promega) and viral stocks were pre- pared by three-step growth amplification according to the manufacturer’s guidelines. Sf9 cells (1 · 10 7 ) were coinfected with a mixture of the six baculovirus strains and grown for 24 h according to procedures in the Life Technologies Bac- to-Bac manual. Cells supplemented with 0.5 mCi [ 35 S]Met for an additional 36 h were harvested, after placing on ice, by washing once with NaCl/P i (50 m M Na phosphate, pH 7.0, 150 m M NaCl) and scraping into 1 mL of Buffer A [20 m M Tris/HCl (pH 7.5), 120 m M KCl, 10 m M 2-merca- ptoethanol, 1% (v/v) Triton X-100, 10% glycerol]. Follow- ing a 5-min incubation on ice with occasional vortexing, extracts were centrifuged for 10 min at 12 000 g in a cooled microcentrifuge. The supernatant was either used immedi- ately or frozen in liquid nitrogen and stored at )70 °C. Glutathione S -transferase (GST) pulldowns For GST fusions, DNA encoding each of the subunits of eIF3 was inserted into pGEX4T-1 (Amersham Pharmacia) essentially as described [10, 13]. Expression of the con- structs generated GST fused in-frame at the N-terminus of the individual eIF3 subunits. GST-fusion proteins were expressed in E.coli BL21 and purified according to manufacturer’s instructions (Amersham Pharmacia). The amount of cell lysate incubated with the beads was adjusted so that a similar amount of each subunit was bound to the beads. The beads were washed once and then resuspended in 500 lL of binding buffer [75 m M KCl, 20 m M Hepes (pH 7.5), 0.1 m M EDTA, 2.5 m M MgCl 2 ,1m M dithiothre- itol, 1% nonfat dry milk and 0.05% NP-40]. In vitro 4134 G. L. Mayeur et al. (Eur. J. Biochem. 270) Ó FEBS 2003 translated, [ 35 S]Met-eIF3kwasincubatedwiththebeads containing the GST fusion proteins for 2 h at 4 °C. Samples were washed and bound proteins were eluted with 2· SDS sample buffer and analyzed by SDS/PAGE. Results Preparations of eIF3 purified from HeLa cells routinely contain a previously uncharacterized protein with a mobility in SDS gels of 28 kDa. To clone the cDNA encoding the 28 kDa protein, a partial peptide sequence, YNPENLA TLER, was obtained and used to identify human cDNA sequences (ESTs) as described in Materials and methods. A plasmid carrying a 921-bp insert was obtained from IMAGE (ATCC). The insert contained an open reading frame from nucleotides 1–637, with an 84 nucleotide 3¢-UTR containing a polyadenylation signal (AAUAAA) at position 835. The first AUG was found at nucleotide 165–167, with two downstream, in-frame AUGs at 171–173 and 183–185. The first AUG probably serves as the initiator codon, as it alone matches the Kozak consensus sequence for initiation [25]. Furthermore, attempts to sequence eIF3k indicated that its N-terminus is blocked, suggesting that the amino acid following the initiator Met is small [26,27]. The protein sequence starting at the first AUG is MAM FEQMRA. Thus, the Met encoded by the first AUG is followed by Ala, whereas those encoded by the downstream AUGs are followed by Phe and Arg, residues that do not normally follow acylated Mets. Finally, initiation at the first AUG generates a protein with 218 residues and a mass of 25.1 kDa, in agreement with its migration in SDS gels. Initiation at a possible AUG codon located upstream from the DNA insert is unlikely, even though no in-frame termination codon exists in the 164 nucleotide 5¢-UTR, as the protein would contain over 270 residues, and thus would be considerably larger than 28 kDa. We conclude that the cloned cDNA (GenBank accession number AY245432) encodes eIF3k, the 28 kDa subunit of eIF3. The cloned cDNA appears to be nearly full length, as Northern blot analysis of mRNAs from a number of cell lines (Fig. 1) produces a single hybridization signal at approximately 1.1 kb. While eIF3k mRNA expression is ubiquitous, as determined by multiple tissue Northern blots, the brain, testis and kidney express the highest levels of eIF3k mRNA (data not shown). The eIF3k sequence contains no obvious RNA binding motif, nor does the protein bind RNA when analyzed by North-Western blotting (results not shown). A number of putative phos- phorylation sites for PKC and CK2 are present, but eIF3k does not appear to be phosphorylated in vivo (data not shown). The subunit is Leu-rich (10.6%), but the Leu are distributed throughout the protein and do not show characteristics of Leu zippers. The eIF3k gene [28] is comprised of eight exons located on chromosome 19q13.2. A putative TATA box is located 747 nucleotides upstream from the identified start codon and 581 nucleotides upstream from the beginning of the cloned insert. Three retropseudogenes with sequence identities greater than 85% are located on chromosomes 3, 4, and 5; however, the longest translatable region only generates a peptide 60% that of full-length eIF3k. A cDNA containing an ORF with a sequence (GenBank acc. no. AF085358) identical to that encoding eIF3k was cloned as the product of HSPC029,an uncharacterized gene; however, the protein was neither identified or characterized other than showing that its mRNA is expressed ubiquitously [29]. A number of methods was used to demonstrate that eIF3k is a true subunit of the eIF3 complex. First, eIF3k cDNA was expressed in vitro and the product was compared to the 28 kDa protein in eIF3. The eIF3k coding region was subcloned into the E.coli expression vector pET28c to create pETp28, as described in Materials and methods. The vectorwasusedasatemplateforin vitro transcription and translation in a rabbit reticulocyte lysate system. The resulting 35 S-labeled protein comigrates with the 28 kDa protein found in purified eIF3 (Fig. 2A). Second, we asked if eIF3k is associated with eIF3 subunits partially purified by immunoprecipitation. Lysates derived from COS-1 cells metabolically labeled with 35 S were immunoprecipitated with an anti-eIF3a monoclonal Ig in the absence or presence of RNase T1 and the precipi- tates were fractionated by SDS/PAGE (Fig. 2B). A radio- labeled band of 28 kDa in the immunoprecipitate comigrates with recombinant eIF3k synthesized in vitro from its cDNA. This result indicates that the 28 kDa protein is a true component of eIF3, rather than a copurifying contaminant. However, because we have been unsuccessful in obtaining antibodies specific for eIF3k, we cannot definitively prove that the immunoprecipitated 28 kDa protein actually corresponds to the eIF3k subunit in this experiment. Therefore, to better demonstrate that the 28-kDa protein associated with eIF3 is eIF3k, the anti- eIF3a immunoprecipitate was analyzed by two-dimensional isoelectric focusing and SDS/PAGE. Other known eIF3 subunits are identified readily, as their migration positions Fig. 1. Northern analysis of human eIF3k. Total RNA was prepared from five cell lines by using the Qiashredder/RNeasy Kit (Qiagen) and 5 lg of RNA was loaded into each lane of a denaturing formaldehyde agarose gel. Following electrophoresis, the RNA was transferred to a nitrocellulose membrane and probed with a 32 P-labeled cDNA probe constructed using the Random Primers (Life Technologies) system and full-length eIF3k cDNA as template. Cell lines from which the RNA was isolated are indicated at the top of the figure and the migration positions of RNA size markers (kb) are shown on the left. Ó FEBS 2003 Characterization of eIF3k (Eur. J. Biochem. 270) 4135 Fig. 2. Analysis of human eIF3 and eIF3k. (A) Purified HeLa eIF3 (lane 1) and in vitro synthesized recombinant eIF3k (lane 2) were subjected to 10% SDS/PAGE and Coomassie Blue staining (lane 1) or autoradiography (lane 2). Plasmid pETp28 carrying untagged eIF3k DNA behind a T7 promoter was expressed in the TnT-coupled transcription/translation system (Promega) containing [ 35 S]Met. The migration positions of molecular mass markers (kDa) are indicated on the left and eIF3 subunits are identified on the right with eIF3k shown in bold. (B) COS-1 cells were labeled with [ 35 S]Met and lysed as described in Materials and methods. RNase T1 (200 000 UÆmL )1 ) was included as indicated in the figure. Gamma- Bind G Plus beads (Santa Cruz Biotechnology) were preloaded with nonimmune antibodies (control) or monoclonal anti-eIF3a Igs (anti eIF3a). The beads were mixed with the radiolabeled COS-1 cell lysate for 30 min and washed three times in Lysis Buffer. Bound proteins were eluted in SDS/PAGE loading buffer and fractionated by 10% SDS/PAGE. Radioactivity was detected by analysis on a phosphorimager (Molecular Dynamics). The migration position of eIF3k was determined by examining the radiolabeled TnT product described in A (TNT p28). eIF3 subunits are identified on the right. (C) Radiolabeled COS cells were lysed in Lysis Buffer and immunoprecipitated with anti-eIF3a monoclonal Igs as described in B. The immunoprecipitate (right panel) or TnT-expressed radiolabeled eIF3k (left panel) was suspended in IPG strip rehydration buffer and isoelectric focusing on 3–10 NL strips proceeded according to the IPGphor (Amersham Pharmacia) protocol. The second dimension was 10% SDS/PAGE. Molecular mass markers are identified on the right, approximate pH is identified on the bottom and individual eIF3 subunits are identified on the right panel. (D) COS-1 cells were transiently transfected with pcDNA3.1/myc-His:p28 or were mock-transfected as described under Materials and methods. At 48 h post-transfection, the nonradiolabeled cells were lysed into Lysis Buffer and the lysate was diluted 1 : 10 into Extraction Buffer (50 m M sodium phosphate pH 7.0, 300 m M NaCl) and loaded onto a Talon IMAC Column (Clontech). The column was washed and eluted following the procedure recommended by the manufacturer. The load (10%) and entire TCA-precipitated eluate were analyzed by SDS/ PAGE and immunoblotted with anti-eIF3a and anti-c-myc monoclonal Igs. (E) Insect Sf9 cells were coinfected with recombinant baculovirus strains expressing eIF3a, FLAG-tagged eIF3b, eIF3c, eIF3g and eIF3i with (lane 2) and without (lane 1) a virus strain expressing eIF3k, 35 S-labeled and lysed as described in Materials and methods. Lysates were immunoprecipitated with anti-FLAG agarose, eluted with FLAG peptide and analyzed by 10% SDS/PAGE and autoradiography. Radiolabeled eIF3 subunits are identified on the right. The asterisks mark degradation products of higher molecular mass subunits. 4136 G. L. Mayeur et al. (Eur. J. Biochem. 270) Ó FEBS 2003 are known [30]. The 28 kDa protein exhibits an apparent pI of 4.8 in the IEF dimension (Fig. 2C) and is located in the same position in the gel as the in vitro synthesized, radiolabeled eIF3k whose theoretical pI is 4.81. These results provide strong support for the view that the 28 kDa protein is eIF3k. Additional evidence showing that eIF3k is present in eIF3 was generated by expressing recombinant eIF3k in mam- malian cells. When DNA encoding eIF3k double-tagged with (His) 6 and c-myc is transiently transfected into COS-1 cells and proteins are purified by Cobalt IMAC chroma- tography, eIF3a is retained on the column along with the (His) 6 -tagged eIF3k (Fig. 2D). A control where c-myc- tagged eIF3k lacking the (His) 6 tagwasexpressedinCOS-1 cells does not result in retention of eIF3a. This supports the view that eIF3k is truly associated with the eIF3 complex. However, the apparent low yield of eIF3 associated with tagged eIF3k is surprising, but could be explained by an inefficient exchange of recombinant tagged eIF3k with the endogenous eIF3k in eIF3. Similar results have been observed in previous attempts to exchange other eIF3 subunits in our hands. We suggest that eIF3 is a highly stable complex, a view consistent with results obtained by pulse-chase labeling of cells followed by a time-course of eIF3 immunoprecipitation [31]. Recent experiments in our laboratory have established the baculovirus-infected insect cell expression system as a suitable way to obtain large eIF3 subcomplexes (C. S. Fraser, J. Y. Lee, M. Bushell, G. L. Mayeur, & J. W. B Hershey, unpublished observation). Co-infection with up to seven recombinant virus strains, each expressing a different eIF3 subunit, has enabled us to generate and isolate a variety of eIF3 subcomplexes. We asked if eIF3k might associate with one or more of such subcomplexes, in particular eIF3a,b,c,g,i, whose subunits correspond to homologous yeast subunits found in the yeast eIF3 ÔcoreÕ [32]. To this end, recombinant virus strains designed to express untagged human eIF3a, eIF3c, eIF3g, and eIF3i, along with FLAG-tagged eIF3b, were used to coinfect insect cells in the presence or absence of recombinant eIF3k virus as described in Materials and methods. Infected insect cells were labeled with [ 35 S]Met, and lysates were immuno- precipitated with anti-FLAG agarose. The bound proteins were subjected to SDS/PAGE and autoradiography. Figure 2E shows the ÔcoreÕ complex of eIF3a, FLAG- eIF3b, eIF3c, eIF3g and eIF3i has formed, and that eIF3k copurifies with this complex. Proteins expressed in a control experiment, in which none of the expressed proteins is FLAG-tagged, do not bind to the affinity beads (data not shown). The results indicate that eIF3k binds to one or more of the subunits found in the eIF3 ÔcoreÕ complex. GST-pulldown experiments (Fig. 3) were used to identify the direct binding partners of eIF3k. Eleven subunits were expressed as GST fusion proteins and used as bait for [ 35 S]Met-eIF3k synthesized in vitro. Direct interactions are seen between eIF3k and eIF3c, eIF3g and eIF3j. We expected that at least one member of the eIF3 ÔcoreÕ complex (Fig. 2E) would interact with eIF3k in the GST pull-down experiments, and indeed both eIF3c and eIF3g were identified. It is likely, therefore, that eIF3k associates with the eIF3c and eIF3g subunits, and possibly also with the eIF3j subunit which is known to associate with eIF3b,g,i (C. S. Fraser, J. Y. Lee, M. Bushell, G. L. Mayeur, & J. W. B Hershey, unpublished observation). If eIF3k is a subunit of eIF3, the portion of overexpressed myc-tagged eIF3k that exchanges into endogenous eIF3 in transiently transfected COS-1 cells is expected to bind to 40S ribosomal subunits. We therefore examined the ribosome binding of tagged eIF3k in relation to eIF3. COS-1 cells transiently transfected with pcDNA3/myc-His:p28 were Fig. 3. Identification of eIF3 subunits interacting with eIF3k. GST- fusion proteins were expressed and purified as described in Materials and methods. (Top panel) Coomassie Blue staining of purified GST- fusion proteins. The eIF3 subunit present in the pulldown is identified below, and the right-most lane contains GST alone. (Bottom panel) Autoradiograph of in vitro [ 35 S]Met-labeled eIF3k, produced using pETp28 as a template, interacting with eIF3 subunits identified below. Left-most lane indicates eIF3k input. Fig. 4. eIF3k binds to 40S ribosomal subunits. COS-1 cells were transiently transfected with pcDNA3.1/myc-His:p28 and lysed as described under Materials and methods with the addition of 0.1mgmL )1 cycloheximide, 0.2 mg mL )1 heparin, 1 m M dithiothre- itol and 24 UÆmL )1 RNAguard (Promega) to the lysis buffer. Cleared lysates were loaded onto a 10–50% sucrose gradient and centrifuged at 40 000 g in a Beckman SW40 rotor for 150 min. The gradient was fractionated on an ISCO fractionator, 1 mL fractions were collected and protein was precipitated with methanol. The proteins in the fractions were analyzed by 10% SDS/PAGE and transferred onto a poly(vinylidene difluoride) membrane. A portion of the membrane was probed with anti-c-myc and anti-eIF3a monoclonal Igs and developed with ECL (lower panels). The migration positions in the gradient of 40S, 60S and 80S ribosomes and polysomes, as identified by the A 260 scan (upper panel), are indicated above the immunoblot. Ó FEBS 2003 Characterization of eIF3k (Eur. J. Biochem. 270) 4137 analyzed by sucrose gradient centrifugation, SDS/PAGE and immunoblotting with anti-eIF3a and anti-c-myc Igs. eIF3, as identified by anti-eIF3a Ig, was found mostly associated with 40S ribosomal subunits (Fig. 4). A small amount of eIF3k colocalizes with eIF3a in the 40S region of the gradient, indicating an ability to bind to the ribosomal subunit. However, most of the eIF3k is found at the top of the gradient, as expected for an overproduced subunit of eIF3. The low amount of 40S binding is consistent with the coimmunoprecipitation experiments (Fig. 2B), again sug- gesting that recombinant tagged eIF3k does not exchange efficiently with endogenous eIF3k in the eIF3 complex. Discussion The cloning of a cDNA encoding eIF3k completes the characterization of the known subunits of mammalian eIF3. The calculated mass of the protein, 25.1 kDa, is consistent with the apparent mass determined by SDS/PAGE, 28 kDa. The cDNA obtained from the partial peptide sequence generates a polypeptide that migrates in one- dimensional SDS/PAGE and two-dimensional IEF/SDS/ PAGE at exactly the same position as the human purified eIF3k. However, the band corresponding to eIF3k does not stain as heavily with Coomassie Blue as expected for a subunit present in stoichiometric amounts. Similar to eIF3j, the amount of eIF3k varies from preparation to prepar- ation; perhaps due to the multistep purification process required to obtain highly purified eIF3. Immunoprecipita- tion from [ 35 S]Met-labeled cells shows eIF3k to be closer to stoichiometric than observed in the Coomassie stained gel. Considerable additional data support the conclusion that the cloned eIF3k cDNA codes for a true subunit of mammalian eIF3. Recombinant eIF3k in transiently trans- fected COS-1 cell lysates coimmunoprecipitates with other eIF3 subunits by using a monoclonal antieIF3a antibody, and this association is not RNA-dependent as shown by insensitivity to RNase treatment. Similarly, other eIF3 subunits copurify with recombinant histidine-tagged eIF3k following fractionation of the transiently transfected COS-1 cell lysates on a nickel affinity column. The protein also associates in vivo with a ÔcoreÕ group of eIF3 subunits when coexpressed in baculovirus-infected insect cells, and direct interactions with eIF3c, eIF3g and eIF3j have been observed. Recombinant eIF3k also binds to 40S ribosomal subunits, presumably through eIF3. Finally, a protein corresponding to eIF3k has been identified in preparations of purified wheat and Arabidopsis thaliana eIF3 [33]. Its identification as eIF3k was based on the similarity of its sequence with that of the eIF3k protein described here; no other characterization of the plant protein was reported. In many of the experiments above, the exchange of recombin- ant eIF3k into endogenous eIF3 appears to be inefficient, making detection of the subunit’s association with eIF3 difficult. However, the sum of all of the results indicates that eIF3k is a true subunit of eIF3. The fact that eIF3k is not present in either S. cerevisiae or S. pombe may indicate a specialized regulatory role in the higher eukaryotic eIF3 complex. Further studies are required to elucidate the precise location of eIF3k in the structure of eIF3, and how this subunit contributes to the activity of the factor. Acknowledgements We thank Susan MacMillan for preparations of purified human eIF3 and Chris Bradley for critically reading the manuscript. The work was supported by National Institutes of Health. Grant GM22135 from the U.S. Public Health Service. References 1. Hershey, J.W.B. & Merrick, W.C. (2000) The Pathway and Mechanism of Initiation of Protein Synthesis. In Translational Control of Gene Expression (Sonenberg, N. Hershey, J.W.B. & Mathews, M.B., eds), pp. 33–88, Cold Spring. Harbor Laboratory Press, Cold Spring Harbor, NY. 2. Chaudhuri, J., Chowdhury, D. & Maitra, U. (1999) Distinct functions of eukaryotic translation initiation factors eIF1A and eIF3 in the formation of the 40S ribosomal preinitiation complex. J. Biol. Chem. 274, 17975–17980. 3. Lamphear, B.J., Kirchweger, R., Skern, T. & Rhoads, R.E. (1995) Mapping of functional domains in eukaryotic protein synthesis initiation factor 4G (eIF4G) with picornaviral proteases. J. Biol. Chem. 270, 21975–21983. 4. Korneeva, N.L., Lamphear, B.J., Hennigan, F.L. & Rhoads, R.E. (2000) Mutually cooperative binding of eukaryotic translation initiation factor (eIF) 3 and eIF4A to human eIF4G-1. J. Biol. Chem. 275, 41369–41376. 5. Pestova,T.V.,Lomakin,I.B.,Lee,J.H.,Choi,S.K.,Dever,T.E.& Hellen, C.U.T. (2000) The joining of ribosomal subunits in eukaryotes requires eIF5B. Nature 403, 332–335. 6. Fletcher, C.M., Pestova, T.V., Hellen, C.U.T. & Wagner, G. (1999) Structure and interactions of the translation initiation fac- tor eIF1. EMBO J. 18, 2631–2639. 7. Me ´ thot, N., Song, M.S. & Sonenberg, N. (1996) A region rich in aspartic acid, arginine, tyrosine and glycine (DRYG) mediates eIF4B self association and interaction with eIF3. Mol. Cell. Biol. 16, 5328–5334. 8.Johnson,K.E.,Merrick,W.C.,Zoll,W.L.&Zhu,Y. (1997) Identification of cDNA clones for the large subunit of eukaryotic translation initiation factor 3. Comparison of homologues from human, Nicotiana tabacum, Caenorhabditis elegans and Saccharomyces cerevisiae. J. Biol. Chem. 272, 7106– 7113. 9. Me ´ thot, N., Rom, E., Olsen, H. & Sonenberg, N. (1997) The human homologue of the yeast Prt1 protein is an integral part of the eukaryotic initiation factor 3 complex and interacts with p170. J. Biol. Chem. 272, 1110–1116. 10. Asano,K.,Kinzy,T.G.,Merrick,W.C.&Hershey,J.W.B.(1997) Conservation and diversity of eukaryotic translation initiation factor eIF3. J. Biol. Chem. 272, 1101–1109. 11. Asano, K., Vornlocher, H.P., Richter-Cook, N.J., Merrick, W.C., Hinnebusch, A.G. & Hershey, J.W.B. (1997) Structure of cDNAs encoding human eukaryotic initiation factor 3 subunits. Possible roles in RNA binding and macromolecular assembly. J. Biol. Chem. 272, 27042–27052. 12. Asano, K., Merrick, W.C. & Hershey, J.W.B. (1997) The trans- lation initiation factor eIF3-p48 subunit is encoded by int-6, a site of frequent integration by the mouse mammary tumor virus genome. J. Biol. Chem. 272, 23477–23480. 13. Block, K.L., Vornlocher, H.P. & Hershey, J.W.B. (1998) Char- acterization of cDNAs encoding the p44 and p35 subunits of human translation initiation factor eIF3. J. Biol. Chem. 273, 31901–31908. 14. Morris-Desbois,C.,Rety,S.,Ferro,M.,Garin,J.&Jalinot,P. (2001) The human protein HSPC021 interacts with Int 6 and is associated with eukaryotic translation initiation factor 3. J. Biol. Chem. 276, 45988–45995. 4138 G. L. Mayeur et al. (Eur. J. Biochem. 270) Ó FEBS 2003 15. Vornlocher,H.P.,Hanachi,P.,Ribeiro,S.&Hershey,J.W.B. (1999) A 110-kilodalton subunit of translation initiation factor eIF3 and an associated 135-kilodalton protein are encoded by the Saccharomyces cerevisiae TIF32 and TIF31 genes. J. Biol. Chem. 274, 16802–16812. 16. Keierleber, C., Wittekind, M., Qin, S.L. & McLaughlin, C.S. (1986) Isolation and characterization of PRT1, a gene required for the initiation of protein biosynthesis in Saccharomyces cerevi- siae. Mol. Cell. Biol. 6, 4419–4424. 17. Gu, Z., Moerschell, R.P., Sherman, F. & Goldfarb, D.S. (1992) NIP1, a gene required for nuclear transport in yeast. Proc. Natl Acad. Sci. USA 89, 10355–10359. 18. Hanachi, P., Hershey, J.W.B. & Vornlocher, H.P. (1999) Char- acterization of the p33 subunit of eukaryotic translation initiation factor-3 from Saccharomyces cerevisiae. J. Biol. Chem. 274, 8546– 8553. 19. Naranda,T.,Kainuma,M.,MacMillan,S.E.&Hershey,J.W.B. (1997) The 39-kilodalton subunit of eukaryotic translation initiation factor 3 is essential for the complex’s integrity and for cell viability in Saccharomyces cerevisiae. Mol. Cell. Biol. 17, 145–153. 20. Valasek,L.,Hasek,J.,Trachsel,H.,Imre,E.M.&Ruis,H.(1999) The Saccharomyces cerevisiae HCR1 gene encoding a homologue of the p35 subunit of human translation initiation factor 3 (eIF3) is a high copy suppressor of a temperature-sensitive mutation in the Rpg1p subunit of yeast eIF3. J. Biol. Chem. 274, 27567–27572. 21. Valasek,L.,Mathew,A.,Shing,B.S.,Nielsen,K.H.,Szamecz,B. & Hinnebusch, A.G. (2003) Yeast eIF3 Subunits TIF32/a and NIP1/c and eIF5 Make Critical Connections with the 40S Ribo- some in vivo. Genes Dev. 17, 786–799. 22. Brown-Luedi,M.L.,Meyer,L.J.,Milburn,S.C.,Yau,M P.P., Corbett, S. & Hershey, J.W.B. (1982) Protein synthesis initiation factors from human HeLa cells and rabbit reticulocytes are similar: comparison of protein structure, activities and immuno- chemical properties. Biochemistry 21, 4202–4206. 23. Meyer, L.J., Milburn, S.C. & Hershey, J.W.B. (1982) Immuno- chemical characterization of mammalian protein synthesis initia- tion factors. Biochemistry 21, 4206–4212. 24. Kaufman, R.J. (1997) DNA transfection to study translational control in mammalian cells. Methods 11, 361–370. 25. Kozak, M. (1987) At least 6 nucleotides preceding the AUG ini- tiator codon enhance translation in mammalian cells. J. Mol. Biol. 196, 947–950. 26. Boissel, J P., Kasper, T.J. & Bunn, H.F. (1988) Cotranslational Amino-terminal Processing of Cytosolic Proteins. J. Biol. Chem. 263, 8443–8449. 27. Huang, S., Elliott, R.C., Liu, P.S., Koduri, R.K., Weickmann, J.L.,Lee,J.H.,Blair,L.C.,Ghosh-Dastidar,P.,Bradshaw,R.A., Bryan, K.M., Einarson, B., Kendall, R.L., Kolacz, K.H. & Saito, K. (1987) Specificity of cotranslational amino-terminal processing of proteins in yeast. Biochemistry 26, 8242–8246. 28. UCSC Genome Broswer (2002), http://genome.ucsc.edu (genome, Human; assembley, Nov.2002; position, AF085358). 29. Zhang, Q H. Ye, M., Wu, X Y., Ren, S X., Zhao, M., Zhao, C J., Fu, G., Shen, Y., Fan, H Y., Lu, G., Zhong, M., Xu, X R., Han,Z G.,Zhang,J W.,Tao,J.,Huang,Q H.,Zhou,J., Hu, G X., Gu, J., Chen, S J. & Chen, Z. (2000) Cloning and functional analysis of cDNAs with open reading frames for 300 previously undefined genes expressed in CD34+ hematgopoietic stem/progenitor cells. Genome Res. 10, 1546–1560. 30. Duncan, R., Etchison, D. & Hershey, J.W.B. (1983) Protein synthesis eukaryotic initiaiton factors 4A and 4B are not altered by poliovirus infection of HeLa cells. J. Biol. Chem. 258, 7236–7239. 31. Scholler, J.K. & Kanner, S.B. (1997) The human p167 gene encodes a unique structural protein that contains a centrosomin A homology and associates with a multicomponent complex. DNA Cell. Biol. 16, 515–531. 32. Phan, L., Zhang, X., Asano, K., Anderson, J., Vornlocher, H P., Greenberg, J.R., Qin, J. & Hinnebusch, A.G. (1998) Identification of a translation initiation factor 3 (eIF3) core complex, conserved in yeast and mammals, that interacts with eIF5. Mol. Cell. Biol. 18, 4935–4946. 33. Burks, E.A., Bezerra, P.P., Le, H., Gallie, D.R. & Browning, K.S. (2001) Plant initiation factor 3 subunit composition resembles mammalian initiation factor 3 and has a novel subunit. J. Biol. Chem. 276, 2122–2131. Ó FEBS 2003 Characterization of eIF3k (Eur. J. Biochem. 270) 4139 . Characterization of eIF3k A newly discovered subunit of mammalian translation initiation factor eIF3 Greg L. Mayeur, Christopher S. Fraser, Franck. cloning of a cDNA encoding eIF3k completes the characterization of the known subunits of mammalian eIF3. The calculated mass of the protein, 25.1 kDa, is