Complex Formation by All Five Homologues of Mammalian Translation Initiation Factor 3 Subunits from Yeast Saccharomyces cerevisiae*

Katsura AsanoDagger , Lon Phan, James Anderson, and Alan G. Hinnebusch§

From the Laboratory of Eukaryotic Gene Regulation, NICHD, National Institutes of Health, Bethesda, Maryland 20892

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The PRT1, TIF34, GCD10, and SUI1 proteins of Saccharomyces cerevisiae were found previously to copurify with eukaryotic translation initiation factor 3 (eIF3) activity. Although TIF32, NIP1, and TIF35 are homologous to subunits of human eIF3, they were not known to be components of the yeast factor. We detected interactions between PRT1, TIF34, and TIF35 by the yeast two-hybrid assay and in vitro binding assays. Discrete segments (70-150 amino acids) of PRT1 and TIF35 were found to be responsible for their binding to TIF34. Temperature-sensitive mutations mapping in WD-repeat domains of TIF34 were isolated that decreased binding between TIF34 and TIF35 in vitro. The lethal effect of these mutations was suppressed by increasing TIF35 gene dosage, suggesting that the TIF34-TIF35 interaction is important for TIF34 function in translation. Pairwise in vitro interactions were also detected between PRT1 and TIF32, TIF32 and NIP1, and NIP1 and SUI1. Furthermore, PRT1, NIP1, TIF34, TIF35, and a polypeptide with the size of TIF32 were specifically coimmunoprecipitated from the ribosomal salt wash fraction. We propose that all five yeast proteins homologous to human eIF3 subunits are components of a stable heteromeric complex in vivo and may comprise the conserved core of yeast eIF3.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

In the initiation phase of eukaryotic protein synthesis, mRNA and Met-tRNAiMet are placed in their proper positions on the 40 S ribosomal subunit to form the 48 S preinitiation complex (1). Binding of the 60 S ribosomal subunit to this complex forms the 80 S initiation complex that can enter the translation elongation cycle. These complex reactions in the initiation phase depend on numerous protein factors called initiation factors (eIFs),1 many of which bind directly or indirectly to 40 S ribosomes. Two well characterized heteromeric protein complexes, eIF2 and eIF4F, play important roles in the binding of Met-tRNAiMet and mRNA to 40 S subunits, respectively, in both yeast and mammals (2-5). The largest of the initiation factors, eIF3, is thought to have an important role in formation of the 48 S preinitiation complex, as early studies on the mammalian factor showed that it promoted dissociation of 80 S ribosomes and stimulated binding of both eIF2-GTP-Met-tRNAiMet ternary complex and mRNA to 40 S subunits (6, 7). Mammalian eIF3 binds to the 40 S ribosome independently of other eIFs at a location separate from the P site where ternary complex is presumed to bind (8, 9). Physical interactions between eIF4B and eIF3-p170 (10) and between eIF4F and the whole eIF3 complex (11, 12) have been demonstrated in mammalian systems, suggesting that eIF3 serves as a scaffold for these mRNA binding factors.

A mammalian eIF3 complex containing nine different subunits was purified by its ability to promote methionyl puromycin (Met-puromycin) synthesis by an 80 S initiation complex in an assay containing other purified eIFs and ribosomal subunits (13). The cDNAs encoding all nine subunits of this complex were recently cloned and sequenced (14-19).2 By comparing the sequences of these nine cDNAs to the entire Saccharomyces cerevisiae genome, it became apparent that only five of the known mammalian eIF3 subunits have identifiable homologues encoded in yeast; the products of the yeast genes TIF32, PRT1 (20), NIP1 (21), TIF34 (22), and TIF35 have similar molecular weights and show strong sequence similarities to the p170, p116, p110, p36, and p44 subunits, respectively, of mammalian eIF3. The 90- and 39-kDa products of PRT1 and TIF34, respectively, were shown to copurify with yeast eIF3 isolated by its ability to substitute for human eIF3 in the Met-puromycin synthesis assay (20, 22). The products of TIF32, NIP1, and TIF35 were not known to be components of yeast eIF3 purified in this fashion, although it contained a polypeptide similar in size to the predicted molecular weight of TIF35 (20).

The 62- and 16-kDa products of GCD10 (23) and SUI1 (24) also copurified with yeast eIF3 activity using the Met-puromycin synthesis assay. SUI1 was first identified genetically by mutations that permit increased utilization of UUG triplets as translation initiation codons (25) and is homologous to a poorly characterized mammalian factor called eIF1 (26). GCD10 was thought to be the yeast counterpart of human eIF3-p66 because of their similar molecular weights and in vitro RNA binding activities (16, 23); however, they do not have strong sequence similarity. Moreover, a putative human homologue of GCD10 can be predicted from human EST (expressed sequence tag) sequences3 that does not correspond to one of the known subunits of mammalian eIF3.

The above comparison of yeast and human eIF3 complexes suggests differences in subunit composition between the two species. In addition, a five-subunit complex containing PRT1 has been purified from yeast (27) with a composition quite different from that purified using the Met-puromycin assay (20). In view of these apparent discrepancies between different eIF3 preparations, we decided to pursue an alternative strategy for investigating the subunit composition of yeast eIF3. By using the cloned genes for all of the known or potential subunits of eIF3, we attempted to construct a protein linkage map (28) of the eIF3 complex with the yeast two-hybrid system. By combining this approach with in vitro binding assays, we identified protein-protein interactions which link together all five yeast homologues of human eIF3 subunits, TIF32, PRT1, NIP1, TIF34, and TIF35. Genetic suppressor analysis provided independent evidence that TIF34 and TIF35 interact with one another in vivo. In addition, we showed that PRT1, NIP1, TIF34, TIF35, and possibly TIF32 were the major polypeptides specifically coimmunoprecipitated with TIF34 from the ribosomal salt wash fraction. Based on these findings, we conclude that all five yeast proteins with sequence similarity to human eIF3 subunits physically interact with one another in vivo.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Plasmid Construction-- Plasmids carrying different yeast genes fused in-frame to the coding sequences for the DNA binding domain or activation domain of GAL4 in pGBT9 (TRP1 vector) or pGAD424 (LEU2 vector) (purchased from CLONTECH, Ref. 29), respectively, were constructed as follows. Oligonucleotides corresponding to the 5' and 3' ends of the ORFs in TIF32, NIP1 (21), PRT1 (30), TIF34 (22), TIF35, SUI1 (25), GCD10 (23) and GCD14,4 listed in Table I, were synthesized (Life Technologies, Inc.) and employed for PCR using yeast genomic DNA as template and the eLONGase kit, (Life Technologies, Inc.). Amplified DNA fragments were digested with the appropriate restriction enzymes corresponding to the terminal sequences (BamHI or SalI at 5' end, and PstI or BglII at 3' end) and subcloned into pGAD424 or pGBT9. The resulting plasmids are listed in Table II. For insertions into pGBT9, the BglII site at the 3' end of the amplified fragments was filled in with Klenow enzyme and ligated to the PstI site of pGBT9, after trimming the latter with mung bean nuclease.

                              
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Table I
List of oligonucleotides used in this study

                              
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Table II
Plasmids employed in this study

To construct plasmid pGEX-TIF34 for bacterial expression of the GST-TIF34 fusion, DNA containing the TIF34 ORF was amplified by PCR with oligonucleotides 7 and 20 (Table I), introducing terminal NdeI and SalI sites, respectively. The 0.9-kb NdeI-EcoRI and 0.5-kb EcoRI-SalI fragments of the amplified DNA were subcloned together between the NdeI and SalI sites of pGEXp66Delta E (16), a derivative of pGEX-4T-1 (Amersham Pharmacia Biotech, see Ref. 31), generating pGEX-TIF34. pGEX-TIF35 was constructed by subcloning the 1.6-kb EcoRI-EcoRV fragment of pGAD-TIF35 between the EcoRI and BsaAI sites of pGEX-4T-1. pGEX-PRT1/A1, pGEX-PRT1/B1, and pGEX-TIF35/C3 were constructed by subcloning the PRT1 or TIF35 insert portions of pGAD-PRT1/A1, pGAD-PRT1/B1, and pGAD-TIF35/C3 between the EcoRI and SalI sites of pGEX-4T-1. pGEX-TIF32 and pGEX-NIP1 were constructed by subcloning the 2.9- and 2.4-kb BamHI-BglII fragments of pGAD-TIF32 and pGAD-NIP1, respectively, into the BamHI site of pGEX-5X-3 (Amersham Pharmacia Biotech, Ref. 31). As BglII cleaves in the middle of these genes, BamHI-digested pGAD-TIF32 or pGAD-NIP1 DNA was digested partially with BglII to isolate the intact ORF DNA.

To insert the TIF34, TIF35, or NIP1 ORF behind the T7 promoter, the 1.0-, 0.8-, or 2.4-kb NdeI-PstI fragments of pGAD-TIF34, pGAD-TIF35, or pGAD-NIP1 were subcloned into pT7-7 (33) to generate pT7-TIF34, pT7-TIF35, or pT7-NIP1, respectively. As NdeI cleaves in the middle of TIF35 and NIP1, intact ORF DNA was isolated by partial NdeI digestion of pGAD-TIF35 or pGAD-NIP1 DNA, respectively. tif34-HA mutant derivatives of pT7-TIF34 were constructed by replacing the 0.9-kb Asp718I-PstI fragment of pT7-TIF34 with the corresponding DNA fragment that was PCR-amplified using oligonucleotides 7-8 (Table I) and tif34-HA mutant derivatives of YCpL-TIF34-HA (see below) as template, followed by digestion with Asp718I and PstI. The resulting plasmids were called pT7-TIF34-1 (tif34-HA-1), pT7-TIF34-2 (tif34-HA-2), and pT7-TIF34-3 (tif34-HA-3). pT7-PRT1 was constructed by subcloning the 2.1-kb SalI-BglII fragment of pGAD-PRT1, after the SalI site was filled in with Klenow enzyme, between the NdeI and BamHI sites of pT7-7 after trimming the NdeI site with mung bean nuclease. pT7-PRT1Delta S and pT7-PRT1Delta M were constructed by removing from pT7-PRT1 the 0.31-kb StyI-SalI and 0.14-kb MscI-SalI fragments, respectively, encoding the C-terminal segments of PRT1, followed by filling-in and self-ligation. pT7-SUI1 was generated by subcloning the 0.7-kb SalI-HindIII fragment of pGBT-SUI1 into pT7-7. The SalI site of the resulting plasmid was filled-in and religated so that the SUI1 ORF is in-frame with the initiation codon at the NdeI site in the vector.

Plasmids YCpL-TIF34 (TIF34 LEU2) and YCpU-TIF34 (TIF34 URA3) were constructed by subcloning the 1.6-kb BamHI-HindIII TIF34 fragment of pNOTA-TIF34 (22) into YCplac111 and YCplac33 (33), respectively. To generate the TIF34 deletion plasmid pTZ-tif34Delta , 0.5-kb fragments corresponding to the 5' and 3' ends of the TIF34 insert in pNOTA-TIF34 were amplified by PCR using pNOTA-TIF34 as template and oligonucleotides 17-18 and 19-20 (Table I), respectively. The amplified 5' and 3' fragments were digested at terminal sequences with the appropriate enzymes and subcloned together between the EcoRI and SalI sites of pTZ19R (34). The resulting plasmid, pTZ-tif34Delta , carries a tif34Delta allele lacking codons 34 to 253 with a BamHI site at the deletion junction. pTZ-tif34Delta -URA was constructed by subcloning the 3.8-kb hisG-URA3-hisG BamHI-BglII fragment of pNKY51 (35) into the unique BamHI site of pTZ-tif34Delta . To construct the TIF34-HA allele encoding TIF34 tagged at its C terminus with three copies of the HA epitope (36), YCpL-TIF34-Bm was first generated by subcloning the 1.4-kb SacI-BamHI (TIF34 5'-UTR plus ORF) and 0.25-kb BamHI-HindIII (TIF34 3'-UTR) fragments, amplified by PCR with oligonucleotides 21-22 and 23-24 (Table I), respectively, into YCplac111. YCpL-TIF34-HA was constructed by inserting the 123-bp BglII fragment of p22965 encoding a triple HA peptide sequence into the BamHI site of YCpL-TIF34-Bm.

Plasmid YEp-TIF35 used for overexpression of TIF35 in yeast was constructed by subcloning the 2.0-kb BamHI fragment of pNOp33 (a gift from Parisa Hanachi and John Hershey) into YEp352 (37). CR52 containing PRT1 inserted in YEp352 was employed for overexpression of PRT1 (38). YEp-TIF34 carries an 8-kb fragment of chromosome XIII encompassing TIF34 and was isolated from a yeast genomic library (39) as a high copy suppressor of the temperature-sensitive phenotype of the tif34-HA-1 mutation. To overexpress TIF35 with the FLAG epitope attached to its C terminus, the 0.8-kb HindIII fragment of YEp-TIF35 containing the 3'-terminal TIF35 sequence (encoding C-terminal segment of TIF35 ORF plus 3'-UTR of 0.6 kb) was replaced with the 107-bp HindIII fragment, amplified by PCR using oligonucleotides 29-30, encoding the C terminus of TIF35 fused in-frame to the FLAG epitope (DYKDDDDK), followed by a stop codon and the HindIII site, generating YEp-TIF35-FLAG.

Yeast Strains-- Strains Y190 (MATa leu2-3,-112 ura3-52 trp1-901 his3-Delta 200 ade2-101 gal4Delta gal80Delta URA3::GAL-lacZ LYS::GAL-HIS3) and Y187 (MATalpha gal4 gal80 his3 trp1-901 ade2-101 ura3-52 leu2-3,-112 met- URA3::GAL-lacZ) were employed as hosts for the pGBT9 and pGAD424 derivatives, respectively, used in yeast two-hybrid analysis (40).

TIF34 was deleted in strain H1485 (MATalpha his1-29 gcn2-508 ura3-52 leu2-3,-112 <HIS4-lacZ ura3-52>) as follows. A transformant of strain H1485 carrying plasmid YCpL-TIF34 (TIF34 LEU2) was transformed to Ura+ with the 4.8-kb EcoRI-SalI fragment of pTZ-tif34Delta -URA that contains the tif34Delta ::hisG::URA3::hisG disruption allele. Ura+ transformants were purified by streaking and screened for the tif34Delta disruption allele by PCR amplification of total yeast DNA in extracts of the transformants permeabilized with Zymolyase-20T (ICN Biomedicals), using oligonucleotides 25-27 and 26-28 (Table I) as primers. Primers 25 and 26 correspond to sequences 5' and 3' to TIF34, respectively, located outside the TIF34 sequences present in pTZ-tif34Delta , whereas primers 27 and 28 correspond to hisG sequences at the tif34 deletion junction. Subsequently, Ura- derivatives of the confirmed deletion mutants, in which URA3 was excised from the tif34Delta ::hisG::URA3::hisG allele by homologous recombination between the flanking hisG repeats, were isolated on synthetic complete (SC) medium containing 5-FOA (41). The Ura- derivatives were again subjected to PCR analyses to confirm the presence of the tif34Delta deletion allele. The resulting strain was called KAY1 (MATalpha his1-29 gcn2-508 ura3-52 leu2-3,-112 tif34Delta -1 <HIS4-lacZ ura3-52> YCpL-TIF34 [TIF34 LEU2]). Strain KAY4 is a derivative of KAY1 in which YCpL-TIF34 was replaced by YCpU-TIF34 (TIF34 URA3). Strain KAY8, in which YCpL-TIF34-HA (TIF34-HA LEU2) replaces YCpU-TIF34, was constructed from KAY4 by plasmid shuffling (41). We verified that KAY8, KAY4, and their parental strain H1485 had identical growth rates as judged by rates of colony formation on YPD medium.

Construction of Libraries for Yeast Two-hybrid Screening for Interactions between Potential eIF3 Subunits-- Usage of libraries containing gene fragments for yeast two-hybrid screening has been described (28, 42). Two libraries of possible eIF3 subunit gene fragments fused to coding sequences for the GAL4 activation domain were constructed in pGAD424 as follows. DNA fragments containing the complete ORFs of the relevant genes were PCR-amplified using oligonucleotides listed in Table I, as described above. Equimolar amounts of the appropriate fragments were sheared by sonication, end-filled with T4 DNA polymerase, and inserted at the SmaI site of pGAD424. The ligation mixture was used for transformation of Escherichia coli DH10B cells on LB plates supplemented with 100 µg/ml ampicillin, and DNA was purified from pools of transformants by using a plasmid isolation kit (Qiagen) according to the vendor's instructions. For library 1, an aliquot of plasmid DNA derived from a pool of 104 transformants bearing a mixture of inserts from PRT1, TIF34, GCD10, and GCD14 with an average size of 0.3 kb was mixed with 1/20 of the same amount of plasmid DNA derived from 3 × 103 transformants containing SUI1 inserts with an average size of 0.15 kb. For library 2, plasmid DNA was prepared from a pool of 104 transformants containing a mixture of inserts from NIP1, TIF32, and TIF35 with an average size of 0.45 kb. In libraries 1 and 2, ~50% and 30% of the clones, respectively, were found to contain inserts. Taking into account the total numbers of independent clones comprising each library and the number of base pairs of coding sequence represented by the genes in the library, we estimated that the end points of overlapping clones for each gene in library 1 are spaced an average of 25 bp apart. The corresponding estimate for library 2 is an average spacing of 13 bp.

Interactions between Recombinant Proteins in Vitro-- Cell extracts were prepared from transformants of E. coli BL21(DE3) carrying the different GST fusion plasmids (Table II), and GST fusion proteins were purified on glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech), all as recommended by the vendor. After extensive washing, beads were resuspended in 0.75 ml of binding buffer (20 mM HEPES (pH 7.5), 75 mM KCl, 0.1 mM EDTA, 2.5 mM MgCl2, 1% skim milk, 1 mM dithiothreitol, 0.05% Nonidet P-40), to which 10 µl of rabbit reticulocyte lysate containing S-labeled proteins was added. The 35S proteins were synthesized by in vitro translation using [35S]methionine and pT7-7 derivatives (Table II) as template with the TnT transcription/translation kit (Promega). Binding assays were conducted as described in Fig. 2.

Isolation of Temperature-sensitive and Lethal Point Mutations in TIF34-- YCpL-TIF34-HA, the single copy LEU2 plasmid encoding HA-tagged TIF34 was randomly mutagenized through transformation of E. coli XL1-Red cells (Promega) as recommended by the manufacturer. Plasmid DNA was isolated from a pool of 20,000 E. coli transformants and used to transform to Leu+ strain KAY4, the tif34Delta -1 yeast strain, bearing the URA3 TIF34 plasmid YCpU-TIF34. Two thousand Ura+ Leu+ transformants were picked and replica-plated to two 5-FOA plates that were incubated at 30 or 36 °C. Clones that did not grow on 5-FOA medium at both 30 and 36 °C after 5 days of incubation were selected as candidates for containing lethal mutations in the TIF34 allele carried on plasmid YCpL-TIF34-HA, whereas those that grew at 30 °C but not at 36 °C were selected as candidates for containing temperature-sensitive (Ts-) tif34-HA alleles. Total yeast DNA was isolated from the candidate strains, and used to transform E. coli MC1066 (leuB trpC pyrF) (43) to Leu+ on M9 medium (44) supplemented with tryptophan and uracil, thereby isolating in E. coli the putative YCpL-TIF34-HA derivatives containing tif34-HA mutant alleles. Plasmid DNA was prepared from these E. coli transformants and reintroduced into yeast strain KAY4 to verify the phenotype after evicting YCpU-TIF34 with attendant growth on 5-FOA medium. The sites of mutations in all 19 tif34-HA alleles thus obtained were determined by sequencing the entire TIF34 ORF in each plasmid.

Preparation of Extracts for Immunoprecipitation of Epitope-tagged TIF34-- Yeast cells from transformants of KAY1 (tif34Delta -1 YCpL-TIF34 [TIF34]) and KAY8 (tif34Delta -1 YCpL-TIF34-HA [TIF34-HA]) carrying YEp-TIF35-FLAG (URA3 TIF35-FLAG) were grown in 50 ml of SC medium lacking uracil to early log phase, harvested, and suspended in 0.1 ml of ice-cold lysis buffer (20 mM Tris-HCl, pH 7.4, 1 mM magnesium acetate, 100 mM KCl, 0.1% Triton X-100, CompleteTM protease inhibitor (Boehringer Mannheim)) after washing with <FR><NU>1</NU><DE>5</DE></FR> volume of the same buffer. Whole cell extracts (WCE) were prepared by homogenizing the washed cells by vortexing with glass beads in a microcentrifuge tube for 15 s three times. Cells were placed on ice for 30 s between each cycle of vortexing. Cell lysates were clarified by centrifigation in a microcentrifuge tube at maximum speed for 10 min at 4 °C. Ribosomal salt wash (RSW) was prepared as described (27) except for a slight modification (56) from 2-liter cultures (A600 = ~10) of the same yeast strains employed above for WCE preparation, grown in SC medium lacking uracil.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Two-hybrid Interactions between Possible eIF3 Subunits-- TIF32, TIF35, and NIP1 are homologous to mammalian eIF3 subunits but were not known to copurify with the yeast factor. We used the yeast two-hybrid assay (20, 23, 24) to investigate whether these three proteins can physically interact with the four previously reported subunits of yeast eIF3, PRT1, TIF34, GCD10, and SUI1. We also included in this analysis GCD14, which was recently linked both genetically and physically to GCD10.4,5 The complete coding sequences for these eight proteins were fused to the DNA binding and activation domains of the transcriptional activator GAL4, and the resulting constructs were tested in all combinations for two-hybrid interactions in yeast. As shown in Table III, we detected three groups of interactions, one between NIP1 and SUI1, a second between GCD10 and GCD14, and a third involving PRT1, TIF34, and TIF35. This last group of interactions indicated that TIF35, the putative yeast homologue of human eIF3-p44, is capable of interacting with two known subunits of yeast eIF3. We failed to detect any other interaction outside of these three groups. In particular, we detected no interaction involving TIF32, even though the TIF32 fusion proteins were expressed in amounts comparable to the other fusion proteins, based on immunoblot analysis of cell extracts with anti-GAL4 antibodies (data not shown). As described later, however, we show that TIF32 binds to PRT1 and NIP1, by in vitro binding assays (see Fig. 6).

                              
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Table III
Two-hybrid interactions between possible eIF3 subunits
Strain Y187 transformants bearing pGAD424 derivatives were mated with transformants of strain Y190 bearing pGBT9 derivatives, and the resulting diploid clones were replica-plated to SC-Leu-Trp-His with different concentrations of 3-AT. The amount of growth was judged after 5 days of incubation at 30 °C; -, no growth on 5 mM 3-AT; ±, growth on 5 mM 3-AT; +, growth on 10 mM 3-AT; ++, growth on 15 mM 3-AT; +++, growth on 30 mM 3-AT.

Intersubunit Binding Domains within PRT1, TIF34, and TIF35-- Failure to see interactions between full-length proteins by two-hybrid assay is not uncommon and can occur for a variety of reasons (29, 45). In an effort to overcome this limitation, and also to identify protein segments responsible for the two-hybrid interactions detected in Table III, we constructed two activation domain libraries containing random fragments of these eight genes and screened them for two-hybrid interactions with the DNA binding domain constructs containing full-length TIF34 and TIF35 coding sequences (see "Materials and Methods" and Refs. 28 and 42). By screening an activation domain library containing fragments of TIF34, PRT1, GCD10, GCD14, and SUI1 against the full-length TIF34 DNA binding domain fusion, we identified interactions between seven C-terminal segments of PRT1 (labeled A1-A16 in Fig. 1A) and full-length TIF34. An overlapping set of C-terminal segments of PRT1 from the same library (labeled B1-B17 in Fig. 1A) was found to interact with full-length TIF35. By screening the second activation domain library containing fragments of NIP1, TIF32, and TIF35 against the TIF34 DNA binding domain fusion, we also identified interactions between TIF34 and five N-terminal segments of TIF35 (labeled C1-C8 in Fig. 1B). These results confirmed the two-hybrid interactions involving full-length TIF34, PRT1, and TIF35 (Table III) and suggested that TIF34 and TIF35 both interact in vivo with the C-terminal ~80 amino acids of PRT1. In addition, they show that a binding domain for TIF34 resides within the N-terminal half of TIF35, upstream from the putative RNA-recognition motif in TIF35 (Fig. 1B). We also screened the fragment libraries with the full-length SUI1-, NIP1-, PRT1-, and GCD10-DNA binding domain fusions as bait; however, no additional interacting plasmids were obtained. This may indicate that the NIP1-SUI1 and GCD10-GCD14 interactions detected using full-length fusion proteins (Table III) are dependent on multiple non-contiguous segments in these proteins. Alternatively, fusions containing segments of these proteins may be too unstable in yeast cells to permit a detectable two-hybrid interaction (29, 45).


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Fig. 1.   Two-hybrid screening for segments of eIF3 subunits that interact with full-length TIF34 or TIF35. Two different libraries of DNA fragments encoding segments of known or potential eIF3 subunits inserted in a yeast two-hybrid activation domain vector were screened for interactions with pGBT-TIF34 or pGBT-TIF35 (Table II) encoding full-length eIF3 subunits inserted in a DNA binding domain vector. Y190 (GAL-HIS3) transformants bearing pGBT-TI34 or -TIF35 and a library plasmid were isolated on SC-Leu-Trp-His plates containing 25 mM 3-AT after incubating 3-4 days at 30 °C. The number of transformants screened on 3-AT medium was several times larger than the size of the libraries. The library plasmids from the 3-AT-resistant transformants were isolated from total yeast DNA by transformation of bacterial strain MC1066 (leuB) (43) to Leu+. After confirming that the isolated plasmids conferred 3-AT resistance in Y190 carrying pGBT-TIF34 or -TIF35, their inserts were sequenced using an Applied Biosystems type 373 automatic sequencer. A, interactions detected between segments of PRT1 and full-length TIF34 and TIF35. At the top is shown a schematic of the 724-amino acid PRT1 polypeptide (GenBankTM accession number P06103), shaded to indicate its sequence similarities to human eIF3-p116 (GenBankTM accession number U62853) and the putative S. pombe (GenBankTM accession number Q10425) homologue, as determined by the Pileup program (Wisconsin PackageTM, GCG). Dark gray boxes indicate regions of 21-34% identity; light gray boxes indicate regions of 9-11% identity, and unshaded regions denote regions of 0-4% identity. Segments of PRT1 that interacted with full-length TIF34 or TIF35 are listed below the schematic along with clone designations, A1-A16 (for PRT1 segments that interacted with TIF34) or B1-B17 (for segments that interacted with TIF35), and the amino acid positions in PRT1 at the boundaries of the interacting segments. The dashed lines connect segments with identical N- or C-terminal end points. The location of the RNA recognition motif is indicated by thick bars below the schematic and locations of prt1 temperature-sensitive point mutations are shown by asterisks above the schematic (52). B, interactions detected between segments of TIF35 and full-length TIF34. The 274-amino acid TIF35 polypeptide is shown schematically, shaded as in A to indicate sequence (GenBankTM accession number S69710) similarities to mouse eIF3-p442 and the putative Caenorhabditis elegans homologue (GenBankTM accession number Z50044). The locations of a zinc finger motif (CCHC zinc finger) and RNA recognition motif are shown below. The segments of TIF35 that interacted with TIF34 are shown below the schematic with their clone designations and locations in the sequence.

In Vitro Binding between PRT1, TIF34, and TIF35-- The two-hybrid interactions between the C-terminal segment of PRT1, the N-terminal half of TIF35, and full-length TIF34 were confirmed by in vitro protein binding experiments. Recombinant GST fusions were constructed containing full-length TIF34 or TIF35 or the minimal interacting fragments of PRT1 or TIF35 identified by the two-hybrid analysis summarized in Fig. 1. The GST fusion proteins were expressed in bacteria and purified by immobilization on glutathione-Sepharose beads. As shown in Fig. 2A (upper panel), the slowest migrating species for each purified fusion had the predicted apparent molecular weight; however, a sizable proportion of the GST-TIF35 fusion proteins was truncated at various positions from the C terminus (the GST moiety is N-terminal), presumably due to proteolytic degradation (Fig. 2A, lane 6). The GST fusion proteins were mixed with 35S-labeled TIF34, PRT1, or TIF35 proteins synthesized by in vitro translation. After extensive washing, the radiolabeled proteins still bound to the beads were eluted, fractionated by SDS-PAGE, and visualized by autoradiography. In these reactions, the GST fusions are present in large molar excess over the radiolabeled protein. Therefore, at similar concentrations of the GST fusion proteins, the proportions of a given labeled protein recovered in complexes with the GST fusion proteins reflects its relative binding affinities for the different GST fusions (45).


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Fig. 2.   Binding between recombinant TIF34, PRT1, and TIF35 in vitro. A, GST fusion proteins listed across the top were immobilized on glutathione-Sepharose beads and incubated with 35S-labeled full-length TIF35, TIF34, or PRT1 at 4 °C for 2 h. After washing the beads 5 times with 0.5 ml of phosphate-buffered saline, bound proteins were eluted in Laemmli buffer (54) at 95 °C for 2 min and separated by SDS-PAGE. Gels were stained with Coomassie Blue (top panel) followed by autoradiography (bottom three panels, lanes 2-7). Lane 1 contains 50% of the input amounts of in vitro translated proteins added to each reaction (In). Lanes 2 and 3, GST fusions containing C-terminal PRT1 segments including amino acids 641-724 and 643-711, encoded by clones A1 and B1 (Fig. 1), respectively. Lane 4, GST fusion containing the N-terminal 152 amino acids of TIF35 encoded by clone C3 (Fig. 1). Lanes 5 and 6, GST fusions containing full-length TIF35 and TIF34, respectively. B, binding of 35S-labeled PRT1, either full-length or with C-terminal deletions (PRT1Delta M or PRT1Delta S) to the GST fusions listed across the top (lanes 1-3; same as lanes 2, 7, and 5, in A, respectively). Lane 4, 50% of the input amounts of labeled proteins added to each reaction. C, model of complex formation by TIF34, PRT1, and TIF35. TIF34 is depicted as circle representing a beta -propeller structure, by analogy with the crystal structure of beta -transducin. The evolutionarily conserved domains of TIF35 and PRT1 are shown as ovals; the nonconserved regions as thick lines. The arrow indicates direct interaction between the N-terminal half of TIF35 and the C-terminal region of PRT1.

As shown in Fig. 2A, in vitro translated TIF34 bound to the GST fusions bearing the C-terminal PRT1 segments identified in clones A1 and B1 from the two-hybrid library (35S-TIF34, lanes 3 and 4). 35S-TIF34 also interacted with GST fusions to either full-length TIF35 or just the N-terminal half of this protein represented in the two-hybrid library clone C3 (lanes 5 and 6). In contrast, S-TIF34 did not bind to GST-TIF34 or to GST alone (lanes 2 and 7). Quantification of the proportions of the input labeled proteins bound to the GST fusions in these experiments (Table IV) indicated that nearly all of the 35S-TIF34 was recovered in complexes with the different GST-PRT1 and GST-TIF35 fusions, whereas only 0.5% or less of the labeled protein bound to GST-TIF34 or GST alone. These results confirm the conclusions reached from two-hybrid analysis that TIF34 can stably interact with 80 residues or less located at the C-terminal end of PRT1 and also with the N-terminal half of TIF35.

                              
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Table IV
Quantitation of in vitro binding experiments
The amounts of labeled proteins, shown in Figs. 2 and 6, were quantitated using STORM model 860 (Molecular Dynamics). Numbers in parentheses indicate values obtained in independent experiments. NT, not tested.

As expected from the two-hybrid analysis, in vitro translated TIF35 showed specific binding to GST-TIF34 (Fig. 2A, 35S-TIF35, lane 7); however, it failed to interact with the GST-PRT1 fusions containing the C-terminal segments encoded in clones A1 and B1 (lanes 3 and 4), even though it interacted with these small PRT1 segments in the two-hybrid assays. One explanation for this discrepancy is that the two-hybrid interactions observed between TIF35 and the C-terminal segments of PRT1 could be indirect; the TIF35 two-hybrid construct would interact with native TIF34 which, in turn, would interact with the PRT1 segments. The idea that TIF34 bridges an interaction between TIF35 and the C-terminal tail of PRT1 is depicted schematically in Fig. 2C. Bridging of this type in yeast two-hybrid analysis has been reported previously (46).

Although TIF35 did not interact with GST fusions containing the extreme C-terminal fragments of PRT1, as just described, full-length S-PRT1 interacted specifically with the GST fusions bearing the full-length or N-terminal half of TIF35 in addition to its expected interaction with GST-TIF34 (Fig. 2A, 35S-PRT1, lanes 5-7). (The weaker binding of 35S-PRT1 to GST-TIF35 versus GST-TIF35/C3-(1-152) seen in lane 6 versus lane 5 probably reflects the lesser amount of the former GST fusion present in the reaction.) Binding of 35S-PRT1 with GST-TIF35 and GST-TIF34 proteins was abolished by truncating PRT1 after residue 682 (Fig. 2B, Delta S-(1-624) and Delta M-(1-682), lanes 2 and 3; see Table IV for quantitative binding data). Together, these results suggest that PRT1 can interact with the N-terminal half of TIF35, as well as with TIF34, and that PRT1 residues C-terminal to position 682 are required for both interactions. Whereas only ~70 residues of PRT1 C-terminal to position 643 are sufficient for a stable interaction with TIF34 (Fig. 2A), PRT1 residues N-terminal of position 641 are additionally required for strong binding to TIF35 (Fig. 2B). These conclusions are depicted schematically in Fig. 2C. The N-terminal boundary of the minimal TIF35 binding domain in PRT1 remains to be identified. From the results of two-hybrid and in vitro protein binding experiments shown in Table III and Figs. 1 and 2, we conclude that PRT1 and TIF34, known subunits of yeast eIF3, are capable of stable interactions with the N-terminal half of TIF35, the yeast homologue of mammalian eIF3-p44.

Isolation of Unconditional Lethal and Temperature-sensitive Mutations in TIF34-- TIF34, and its human homologue eIF3-p36, consist of 7 WD repeats (14, 22). Although WD repeats 3 and 6 in TIF34 are degenerate, their occurrence is supported by a sequence alignment of TIF34, human eIF3-p36, and three putative homologues of these proteins from Arabidopsis thaliana, Drosophila melanogaster, and Schizosaccharomyces pombe identified in GenBankTM (Fig. 3A). This alignment also reveals two regions of high sequence similarity (38-39% identity) spanning WD repeats 1-2 at the N-terminal ends (residues 1-84) and repeats 6-7 at the C-terminal ends (residues 232-332) of all five proteins. As other WD proteins are known to be involved in protein-protein interactions (47), we set out to isolate point mutations in TIF34 that would weaken its ability to interact with PRT1 or TIF35.


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Fig. 3.   Isolation of tif34-HA lethal and temperature-sensitive mutations. A, alignment of TIF34-related sequences and positions of tif34-HA missense mutations. The polypeptide sequence of TIF34 (GenBankTM P40217) was aligned with the human (GenBankTM accession number U39067), Drosophila (GenBankTM accession number U90930), Arabidopsis (GenBankTM accession number S60256), and S. pombe (GenBankTM accession number D89187) related sequences using the GCG Pileup program. Identical residues were highlighted in black, and similar residues were highlighted in gray. The locations of 7 WD40 repeats and a WD40 consensus sequence, defined as below (47), are shown above the Drosophila sequence. phi, for hydrophobic amino acids; G for V, A, S, or G; H, for H; Phi  for I, L, W, Y, or F; P for D, G, N, or P; t for C, A, T, S, or G; T for any amino acid of t or Y; d for D; W for W, F, or Y; D for K, R, N, or D. Positions and amino acid changes of the following tif34-HA missense or deletion mutations are indicated below the sequences: 8 lethal mutations changed Tyr-18 (TAC) to His (CAC), Cys-28 (TGT) to Tyr (TAT), His-51 (CAC) to Tyr (TAC), Asp-61 (GAC) to Tyr (TAC), Gly-257 (GGT) to Asp (GAT), Gly-257 (GGT) to Ser (AGT), Gly-294 (GGT) to Asp (GAT), and Asp-314 (GAT) to Tyr (TAT). The ninth lethal mutation deleted the entire Gly-257 codon. The two Ts- mutations changed Gly-311 (GGT) to Ser (AGT) (tif34-HA-1) and Pro-247 (CCA) to Leu (CTA) (tif34-HA-2). The allele tif34-HA-3 changed Gln-258 (CAA) to Arg (CGA). Besides mutations listed here, two of the lethal mutations changed the first Met codon to ATA, and five others contained premature stop codons introduced by altering codon 56 (TGG) to TAG, codon 79 (TGG) to TGA, codon 92 (TCG) to TAG, codon 161 (TGG) to TGA, and codon 290 (CAA) to TAA. B, temperature-sensitive and slow-growth phenotypes of tif34-HA mutations. Derivatives of strain KAY8 (tif34Delta -1 YCpL-TIF34-HA) bearing the tif34-HA alleles on low copy plasmids in place of wild-type TIF34-HA were streaked on YPD plates and incubated at 30 °C or 36 °C for 2 days.

TIF34 is an essential protein required for translation initiation (22, 48); therefore, we mutagenized a plasmid-borne TIF34 allele at random sites and screened the mutagenized plasmids for unconditional or temperature-sensitive lethal phenotypes in a yeast strain deleted for chromosomal TIF34 (KAY4) by the technique of plasmid shuffling (see "Materials and Methods"). We mutagenized a modified allele of TIF34, encoding three tandem copies of the influenza hemagglutinin (HA) epitope inserted at the C-terminal end of the ORF. This TIF34-HA allele was indistinguishable from wild-type TIF34 in its ability to complement the lethal phenotype of a tif34 chromosomal deletion, as judged by comparing isogenic TIF34 and TIF34-HA strains for the rate of colony formation on rich medium at various temperatures (data not shown). Among 16 unconditionally lethal mutations we isolated, two altered the initiation codon to ATA and five introduced a premature stop codon in TIF34, confirming the proposed ORF and start codon (22). Nine additional lethal mutations were obtained that altered or deleted a single amino acid in the TIF34 protein, as indicated in Fig. 3A below the sequence alignment. We also obtained two temperature-sensitive (Ts-) alleles (tif34-HA-1 and tif34-HA-2) and a third allele (tif34-HA-3) that confers slow growth at all temperatures (Slg-) (Fig. 3B). Each of these three mutations alters a single amino acid of the TIF34 protein (Fig. 3A).

Interestingly, all the missense and deletion mutations we obtained in TIF34-HA mapped within WD repeats 1, 2, 6, and 7, the most highly conserved regions among the TIF34-related proteins aligned in Fig. 3A. This finding suggests that these four repeats are the most critically required segments of the TIF34 family of proteins. If TIF34 is folded into a structure resembling the beta -propeller structure described for beta -transducin (49), these repeats would comprise a contiguous surface to which other proteins could bind, such as TIF35 or PRT1.

Genetic Interactions between TIF34 and TIF35-- To test whether the tif34-HA-1, -2, and -3 mutations weaken an essential interaction between TIF34 and either TIF35 or PRT1 in vivo, we asked whether overexpression of TIF35 or PRT1 could suppress the growth defects of the tif34-HA mutant strains. The TIF35 and PRT1 genes were inserted into a high copy vector, and the resulting plasmids were introduced into the tif34-HA mutant strains. As shown in Fig. 4, the Ts- phenotypes of tif34-HA-1 and tif34-HA-2 were fully suppressed by multiple copies of TIF35. Thus, transformants of these mutants containing the high copy TIF35 plasmid grew as rapidly at 36 °C as did the wild-type TIF34 strain (TIF34-HA) transformed with empty vector, or the tif34 mutants transformed with a plasmid bearing TIF34. The Slg- phenotype of the tif34-HA-3 mutant was only weakly suppressed by the high copy TIF35 plasmid, even though its growth defect at 36 °C was no more severe than that of the tif34-HA-1 mutant (Fig. 4). In contrast to the effects of the high copy TIF35 plasmid, introducing a multicopy PRT1 plasmid did not suppress the phenotypes of any of the tif34-HA mutants; however, it should be noted that high copy PRT1 conferred an Slg- phenotype in the wild-type TIF34-HA strain (Fig. 4). This effect could complicate the ability of overexpressed PRT1 to compensate for the growth defects conferred by the tif34-HA mutations. To explain these results, we suggest that the tif34-HA-1 and tif34-HA-2 mutations weaken the physical interaction between TIF34 and TIF35 at the non-permissive temperature due to an altered conformation of the mutant TIF34 proteins and that overexpression of TIF35 can restore the TIF34-TIF35 interaction by mass action. According to this model, the tif34-HA-3 allele is defective for a function of TIF34 besides complex formation with TIF35 and thus cannot be fully suppressed by overexpression of TIF35.


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Fig. 4.   Multiple copies of TIF35 suppresses the growth defects of tif34-HA mutants. Strain KAY8 (tif34Delta -1 YCpL-TIF34-HA) and its tif34-HA-1, -2, and -3 mutant derivatives were transformed with the high copy number plasmids YEp352 (Vector), YEp-TIF35 (hc TIF35), CR52 (hc PRT1), and YEp-TIF34 (hc TIF34) and streaked on SD + His plates for 2 days (wild-type, tif34-HA-1, and tif34-HA-2) or for 3 days (tif34-HA-3). The amino acid substitution for each tif34-HA mutation is shown below the allele name.

Effects of Point Mutations in TIF34 on Binding to TIF35 and PRT1 in Vitro-- To test our model that the mutations in tif34-HA-1 and tif34-HA-2 weaken a physical interaction between TIF34 and TIF35, we synthesized 35S-labeled TIF34 proteins bearing these mutations and compared them to wild-type TIF34 for binding to the GST fusion containing the N-terminal half of TIF35. Because the tif34-HA-1 and tif34-HA-2 alleles have a Ts- phenotype at 36 °C in vivo, these in vitro binding reactions were carried out at 36 °C. As shown in Fig. 5, A and B, binding of wild-type 35S-TIF34 increased with increasing amounts of the GST-TIF35 fusion protein added to the reactions. Binding of the 35S-labeled Tif34-HA-1 and Tif34-HA-2 proteins to the GST-TIF35 fusion protein was greatly reduced compared with wild-type 35S-TIF34 binding at all concentrations of the GST fusion protein tested. These results support the idea that the tif34-HA-1 and tif34-HA-2 mutations reduce the affinity between TIF34 and TIF35 proteins and thus can be suppressed by increasing the concentration of TIF35. In contrast, binding of 35S-Tif34-HA-3 showed only a small reduction in binding relative to wild-type 35S-TIF34, consistent with our conclusion that tif34-HA-3 has additional defects that cannot be corrected simply by overexpression of TIF35. The fact that tif34 point mutations which are suppressed in vivo by high copy TIF35 were found to weaken binding between TIF34 and TIF35 in vitro strongly suggests that the interactions we detected between these proteins are relevant to the in vivo function of TIF34. The results in Fig. 5, C and D, indicate that the Tif34-HA-1 and Tif34-HA-2 proteins also were very defective for binding to the GST-PRT1 fusion containing the C-terminal 83 residues of PRT1, whereas Tif34-HA-3 again showed only a small defect in binding relative to wild-type TIF34. The implication of these last findings in relation to complex formation by TIF34, PRT1, and TIF35 in vivo will be discussed later.


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Fig. 5.   Effect of tif34-HA mutations on in vitro interactions between TIF34 and TIF35 or PRT1. 35S-Labeled TIF34 protein or its tif34-HA-1, -2, or -3 mutant derivatives, synthesized in vitro, was incubated at 36 °C for 1 h with increasing amounts of GST-TIF35/C3 (A) or GST-PRT1/A1 (C), defined in Fig. 2, and immobilized on glutathione-Sepharose beads. TIF34 was stable in this condition irrespective of the tif34-HA mutations. Lane 1, glutathione-Sepharose beads only; lanes 2-4, 1-, 2-, and 4-fold relative amounts of GST-TIF35/C3 (A) or GST-PRT1/A1 (C); lane 5, 50% of the input amounts of labeled proteins in the reactions. B and D, the percentage of mutant and wild-type TIF34 protein bound to the GST fusions were plotted against the relative amount of fusion proteins, added to the binding reactions.

PRT1 Binds Specifically to TIF32-- Although PRT1, TIF34, and TIF35 did not interact with NIP1 or TIF32 in the two-hybrid assay, we reinvestigated their possible interactions in vitro using GST-TIF32 and GST-NIP1 fusions. SDS-PAGE analyses of these purified fusion proteins revealed full-length products of the expected sizes plus numerous degradation products truncated at the C terminus (Fig. 6, top panel). As shown in Fig. 6, 35S-labeled full-length PRT1 and the Delta M-(1-682) and Delta S-(1-624) PRT1 proteins truncated at the C terminus bound strongly to the GST-TIF32 fusion proteins but showed little or no binding to the GST-NIP1 proteins (Fig. 6, panels 2-4 from top, lanes 3 and 4; Table IV). In contrast, neither 35S-TIF34 nor 35S-TIF35 interacted with either set of GST fusion proteins (Fig. 6, panels 5 and 6 from top). The Delta M-(1-682) and Delta S-(1-624) C-terminal truncations of PRT1 were shown above to abolish the binding of PRT1 to GST-TIF34 and GST-TIF35 (Fig. 2B). Thus, it appears that the binding domain for TIF32 in PRT1 is located N-terminal to the binding domains for TIF35 and TIF34 (summarized in Fig. 8A).


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Fig. 6.   Binding involving recombinant TIF32 and NIP1 in vitro. Full-length GST-TIF32 or GST-NIP1 fusion proteins or GST alone was immobilized on glutathione-Sepharose beads and incubated with 35S-labeled PRT1 (full-length or deletion derivatives), TIF34, TIF35, NIP1, or SUI1, as described in Fig. 2. Coomassie Blue staining (top panel) shows the GST fusion proteins that were bound to the beads, as listed across the top. The remaining panels show the amounts of each 35S-labeled protein that were recovered with the GST fusions (lanes 2-4). Lane 1, 50% of the input amounts of labeled proteins in the reactions. For the reactions involving 35S-labeled NIP1, incubation was conducted in a volume of 0.10 ml instead of 0.75 ml to increase the concentration of interacting components. With the standard volume of 0.75 ml, 0.5% of the input amount of 35S-NIP1 was recovered with GST-TIF32 compared with 0.1% recovered with GST alone (data not shown).

NIP1 Binds Specifically to TIF32 and SUI1-- Fig. 6 also shows that 35S-labeled NIP1 bound specifically to GST-TIF32 (panel labeled 35S-NIP1, lane 3), although the binding was weak compared with that seen between 35S-PRT1 and GST-TIF32 (see Table IV). This could indicate that NIP1 has a lower affinity than PRT1 for TIF32. Alternatively, it is possible that NIP1 can interact only with full-length GST-TIF32, whereas PRT1 is capable of binding to GST-TIF32 degradation products in addition to full-length GST-TIF32. These alternative possibilities remain to be addressed in the future. 35S-Labeled SUI1 bound to GST-NIP1 but not to GST-TIF32 or GST alone (Fig. 6, bottom panel, lanes 2-4; Table IV), confirming the interaction between SUI1 and NIP1 observed in the two-hybrid assay (Table III). Taken together, the results in Fig. 6 suggest that PRT1 binds to TIF32 and that NIP1 is capable of bridging an interaction between TIF32 and SUI1 (see Fig. 8A).

Complex Formation by TIF35, PRT1, NIP1, TIF34, and a 120-kDa Protein in the Ribosomal Salt Wash Fraction-- To provide more direct evidence for complex formation by the five yeast proteins homologous to subunits of mammalian eIF3 in vivo, we asked whether they could be coimmunoprecipitated from WCE. Toward this end, we constructed an allele of TIF35 encoding a FLAG epitope-tagged form of the protein on a high copy plasmid. This tagged allele was judged to be functional because in high copy number it suppressed the Ts- phenotype of the tif34-HA-1 mutant to the same extent observed for high copy number wild-type TIF35 (data not shown). We reasoned that overexpression of FLAG-tagged-TIF35 would favor its incorporation into the eIF3 complex in place of wild-type TIF35. Accordingly, the high copy plasmid bearing TIF35-FLAG was introduced into strains containing wild-type TIF34 or TIF34-HA, and WCEs from the resulting transformants were immunoprecipitated with anti-HA antibodies. When the immune complexes were probed by immunoblot analysis for PRT1, NIP1, TIF35-FLAG, and TIF34-HA, we found that all four proteins were immunoprecipitated only from the strain containing HA-tagged TIF34 (Fig. 7A, panels 1-4, lanes 2 and 5). The yields of PRT1 and NIP1 in the immune complexes from the TIF34-HA strain were similar to that observed for TIF34-HA itself (~50% of the input amounts), suggesting that the majority of these three proteins was present in the same heteromeric complexes in vivo. The yield of TIF35-FLAG in the immune complexes was less (15-20%), presumably because it was overexpressed and the excess protein was not incorporated into the complex. In contrast, we found that little or no GCD10 or the gamma -subunit of eIF2 (encoded by GCD11) was coimmunoprecipitated with the other four proteins (Fig. 7A, bottom two panels).


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Fig. 7.   Coimmunoprecipitation of PRT1, NIP1, and TIF35 with epitope-tagged TIF34. The tif34Delta strains KAY1 and KAY8 containing TIF34 or TIF34-HA, respectively, on single copy plasmids were transformed with the high copy plasmid YEp-TIF35-FLAG bearing TIF35-FLAG. 200 µg of whole cell extracts (A, WCE) or 100 µg of a ribosomal salt wash (B, RSW) fraction prepared from the transformants were immunoprecipitated with 2.5 µl of mouse monoclonal 12CA5 anti-HA antibodies (Boehringer Mannheim) bound to 25 µl of protein A-Sepharose beads CL-4B (Amersham Pharmacia Biotech) in 300 µl of binding buffer for 1 h at 4 °C. Binding buffer was the cell lysis buffer (see "Materials and Methods") for WCE or buffer B (20 mM Tris-HCl, pH 7.5, 0.35 M KCl, 5 mM MgCl2, 0.1 mM EDTA, 7 mM beta -mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 1 ×CompleteTM protease inhibitor (Boehringer Mannheim)) for RSW. Immune complexes attached to the beads were washed 4 times with 0.8 ml of lysis buffer or 4 times with buffer B containing 0.1% Triton X-100 and once with phosphate-buffered saline, respectively. Bound proteins were released by heating for 2 min at 90 °C in Laemmli buffer and separated by SDS-PAGE (8-16% gradient polyacrylamide) for immunoblot analyses with anti-PRT1 antibodies (38), anti-NIP1 antibodies (obtained from David Goldfarb), affinity purified anti-GCD10 antibodies (23), anti-GCD11 antibodies (55), anti-SUI1 antibodies (25), the mouse monoclonal anti-FLAG antibodies (Kodak), and the anti-HA antibodies. Immunodetection was performed by chemiluminescence (ECLTM, Amersham Pharmacia Biotech) using horseradish peroxidase-conjugated secondary antibodies (Amersham Pharmacia Biotech). A, the immunoprecipitated fractions were subjected to immunoblot analysis using the antibodies listed on the left of each panel along with the input and supernatant fractions. In, 10% of the input WCE used for immunoprecipitation (lanes 1 and 4 from KAY1 and KAY8, respectively; Ppt, the entire immunoprecipitated fraction (lanes 2 and 5 from the same cells as in lanes 1 and 4); Sup, 15% of the supernatant fractions from the immunoprecipitations (lanes 3 and 6 from the same cells as in lanes 1 and 4). B, the immunoprecipitated fractions from the RSW were resolved by SDS-PAGE along with molecular weight standards (lane 1) and stained with Coomassie Blue (lanes 2 and 3 from the TIF34 and TIF34-HA strains, respectively). The assignments of the six polypeptides listed to the right of lane 3 were based on the results of immunoblot analysis (shown in lanes 4 and 5) of the same samples analyzed in lanes 2 and 3. The band which reacted with anti-HA antibodies in lane 5 appeared as a doublet in lighter exposures. The 120-kDa protein (lane 3) was tentatively assigned as TIF32 based on its size.

Encouraged by these findings, we asked whether a heteromeric complex containing these proteins could be detected in the RSW fraction. The RSW fraction is prepared by stripping proteins from concentrated ribosomes in a high salt buffer and is generally the starting material for isolation of initiation factors. RSW fractions were prepared from the isogenic strains containing either wild-type TIF34 or TIF34-HA and multicopy TIF35-FLAG described above, immunoprecipitated with anti-HA antibodies, and the resulting immunoprecipitates were stained with Coomassie Blue. As shown in Fig. 7B (lanes 2 and 3), seven major polypeptides were coimmunoprecipitated from the TIF34-HA RSW fraction, and all seven were absent from the corresponding sample prepared from the TIF34 RSW fraction. By immunoblot analysis, we identified six of the seven polypeptides as follows (from largest to smallest in apparent molecular mass): NIP1, PRT1, a degradation product of NIP1 (NIP1*), a doublet of TIF34-HA, and TIF35-FLAG (Fig. 7B, lanes 4 and 5). The largest polypeptide had an apparent molecular mass of 120 kDa, close to that predicted for TIF32 (110 kDa); however, lacking antibodies against this protein, we could not confirm this assignment. SUI1 and GCD10 were not coimmunoprecipitated with TIF34-HA from the RSW fraction (data not shown). From these results, we conclude that at least four of the five proteins homologous to human eIF3 subunits are stably associated with TIF34-HA in both whole cell extracts and in the ribosomal high salt wash fraction.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

PRT1 and TIF34 are essential proteins in S. cerevisiae known to be required for the initiation of protein synthesis (22, 50, 51). They are homologous to the 116- and 36-kDa subunits, respectively, of the nine-subunit mammalian eIF3 complex purified by its activity in the Met-puromycin synthesis assay, and they were shown to be subunits of a yeast eIF3 complex purified with the same assay (20, 22). Thus, there is strong evidence that PRT1 and TIF34 carry out their essential functions in yeast as components of eIF3. The other three yeast proteins homologous to subunits of human eIF3, TIF32, NIP1, and TIF35 were not known to be subunits of yeast eIF3, although a 33-kDa polypeptide close in molecular weight to that of TIF35 copurified with eIF3 activity (20).

We considered the possibility that all five yeast proteins homologous to human eIF3 subunits are components of a high molecular weight complex in yeast cells, and we obtained several lines of evidence supporting this hypothesis. We detected interactions between PRT1, TIF34, and TIF35 in the yeast two-hybrid assay (Table III and Fig. 1), and these interactions were confirmed by in vitro binding assays using recombinant forms of the proteins (Fig. 2). We were able to localize binding domains responsible for these interactions within the C-terminal portion of PRT1 and the N-terminal half of TIF35 (Figs. 1 and 2). In addition to these physical interactions, we showed that certain Ts- mutations in TIF34 could be suppressed in vivo by increasing the dosage of TIF35 (Fig. 4). The suppressible tif34 mutations were found to weaken the interaction between recombinant TIF34 and TIF35 proteins in vitro (Fig. 5). These findings suggested that an unstable interaction between TIF34 and TIF35 in vivo was at least partially responsible for the inability of the mutant tif34 proteins to carry out their essential functions in translation. Together, these data provide strong evidence that two known subunits of eIF3, PRT1 and TIF34, interact in vivo with TIF35, the yeast homologue of mammalian eIF3-p44.

We also found that recombinant forms of PRT1 and TIF32 interacted strongly in vitro and that NIP1 showed a weaker but significant interaction with TIF32 (Fig. 6). These data suggested that all five yeast homologues of mammalian eIF3 subunits might interact with one another in vivo. We obtained biochemical evidence for this hypothesis by showing that NIP1, PRT1, TIF34, and TIF35 were specifically coimmunoprecipitated from both WCEs and the RSW fraction using antibodies against an HA-tagged form of TIF34 (Fig. 7). In the coimmunoprecipitations shown in Fig. 7B, these four polypeptides (or degradation products of them), and a protein with the predicted molecular weight of TIF32, were the major proteins that specifically coimmunoprecipitated with HA-tagged TIF34. We conclude that at least four of the five homologues of human eIF3 subunits, and probably all five, interact with one another in vivo and are likely to be components of the same high molecular weight complex. These results support the physical interactions linking together all five yeast homologues of human eIF3 subunits summarized in Fig. 8A.


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Fig. 8.   Complex formation by S. cerevisiae proteins homologous to subunits of human eIF3. A, interactions between yeast proteins detected by yeast two-hybrid analysis or in vitro protein binding assays are indicated with arrows or by contact between the schematic representations of the proteins. We found that the C-terminal end of PRT1 is sufficient for a strong interaction with TIF34 and necessary but not sufficient for interaction with the N-terminal half of TIF35. More N-terminally located residues in PRT1 mediate its interaction with TIF32. TIF34 also interacts strongly with the N-terminal half of TIF35. NIP1 interacts with TIF32, but this interaction appeared to be weaker than the others summarized here. Genetic evidence for TIF34-TIF35 interactions came from the fact that certain tif34 point mutations which weaken in vitro binding of TIF35 to TIF34 were suppressed in vivo by high copy number TIF35. The five yeast proteins with sequence similarity to human eIF3 subunits (shown in B) are encircled to represent their presence in a stable heteromeric complex predicted from their coimmunoprecipitation from the RSW fraction with HA-TIF34, although the presence of TIF32 in this complex remains hypothetical. We detected interactions between NIP1 and SUI1, another subunit of yeast eIF3 (24) and the yeast counterpart of mammalian eIF1 (26); however, SUI1 did not coimmunoprecipitate with HA-TIF34 from the RSW fraction and may be less tightly associated with the complex than are the other five proteins. GCD10, which copurified with yeast eIF3 (23), has no homologue in human eIF3 and appears to reside in a distinct complex with GCD14 (see "Discussion" for details). B, the p170, p116, p110, p44, and p36 subunits of human eIF3 and eIF1 are depicted using the same shapes and locations as in A to indicate their sequence similarity to the yeast homologues analyzed in this study. Human eIF3 contains four additional proteins without obvious counterparts encoded in the yeast genome. The mammalian eIF3 complex is encircled by a solid line to reflect the results of coimmunoprecipitation experiments carried out for the subunits (except p44) which confirm their physical association with the p170 subunit (see text for details).

The C-terminal portion of PRT1 (residues 641-724) was sufficient for both two-hybrid and in vitro interactions with TIF34. Full-length PRT1 interacted directly with TIF35 in vitro in a manner that depended on residues C-terminal to position 624 in PRT1 (Fig. 2B). As the C-terminal segment of PRT1 alone did not interact with TIF35 in vitro, we presume that more N-terminally located residues in PRT1 are additionally required for a direct interaction with TIF35 in the absence of TIF34 (Fig. 8A). PRT1 also showed direct binding to TIF32 in vitro, and this interaction was not dependent on the C-terminal segments of PRT1 required for its interaction with TIF35 and TIF34 (Fig. 6). Thus, the binding domain for TIF32 in PRT1 is probably located N-terminal to those for TIF35 and TIF34 (Fig. 8A). There is evidence that the mammalian homologues of PRT1 and TIF32, namely eIF3-p116 and eIF3-p170, also interact directly with one another (15) (Fig. 8B).

We obtained genetic and biochemical evidence that the WD repeat elements of TIF34 are important for its interactions with TIF35 and PRT1. The Ts- phenotypes of point mutations in the 6th or 7th WD repeats in tif34-HA-2 and tif34-HA-1, respectively, were suppressed by increased TIF35 gene dosage (Fig. 4). Because the tif34-HA-3 mutant was not efficiently suppressed by high copy TIF35, it is unlikely that overexpression of TIF35 simply bypassed the function of TIF34 in translation initiation. Based on the fact that both Tif34-HA-1 and Tif34-HA-2, but not Tif34-HA-3, were very defective for interaction with TIF35 in vitro (Fig. 5), we proposed that overexpression of TIF35 compensated for its reduced affinity for mutant Tif34-HA-1 and Tif34-HA-2 and restored the TIF35-TIF34 interaction by mass action. The tif34-HA-1 and tif34-HA-2 mutations also impaired binding between TIF34 and PRT1 in vitro; in fact, there was a greater reduction in binding to PRT1 versus TIF35 (Fig. 5); however, increasing PRT1 dosage did not suppress the Ts- phenotypes of the tif34 mutations (Fig. 4). To explain these observations, it could be proposed that conformational changes caused by the Ts- tif34 mutations led to dissociation of both PRT1 and TIF35 from TIF34, but the reduction in PRT1 binding affinity was too great to be overcome by PRT1 overexpression. In contrast, TIF35 overexpression would restore TIF34-TIF35 interaction and this, in turn, would recruit PRT1 to the complex because PRT1 can interact with TIF35 as well as with TIF34 (Fig. 8A). It was shown previously that a deletion in the C-terminal 147 amino acids of PRT1 destabilized the protein in vivo and had a lethal phenotype (52). These previous findings are consistent with the idea that the physical interactions we detected between TIF34 and the C-terminal end of PRT1 are important for the integrity of the eIF3 complex in vivo.

While this manuscript was in preparation, Verlhac et al. (48) also identified TIF35 as a protein that interacts with TIF34 in vivo by using yeast two-hybrid assay and coimmunoprecipitation analysis and by showing that increased TIF35 dosage suppressed the Ts- phenotype of a TIF34 allele containing two substitutions in WD repeats 3 and 6 but not one containing three substitutions in WD repeats 2, 4, and 5. They also cited unpublished data indicating that direct peptide sequencing of the 33-kDa polypeptide that copurified with yeast eIF3 activity in the Met-puromycin assay (20) had established its identity as TIF35. These findings, in combination with the coimmunoprecipitation results in Fig. 8, provide strong evidence that TIF35 is an integral subunit of yeast eIF3.

The eIF3 complex purified using the Met-puromycin synthesis assay contains PRT1 (20), TIF34 (22), GCD10 (23), SUI1 (24), and apparently TIF35 (48); however, it lacked NIP1 and TIF32 (53). Our finding that a large percentage of the NIP1 in whole cell extracts was coimmunoprecipitated with HA-TIF34 (Fig. 7A) and that this association was maintained in high salt buffers (Fig. 7B) suggests that NIP1 is stably associated with the yeast eIF3 complex in vivo. The in vitro interaction between GST-TIF32 and 35S-PRT1 was among the strongest we detected (Table IV). In addition, a protein with molecular weight similar to that of TIF32 was coimmunoprecipitated with HA-TIF34 from the RSW at roughly the same stoichiometry as the other four yeast homologues of mammalian eIF3 subunits (Fig. 7B). These findings suggest that TIF32 is also associated with yeast eIF3. Supporting this conclusion, we recently purified a high molecular mass complex of ~600 kDa from a strain expressing a polyhistidine-tagged form of PRT1 and showed by mass spectrometric analysis that it contains TIF32, NIP1, TIF34, and TIF35 as the major proteins specifically associated with the tagged PRT1 protein (56). A PRT1-containing complex has been purified previously by conventional chromatography containing major polypeptides with relative molecular weights similar to those of TIF32, NIP1, TIF34, and TIF35 (27). This complex was not tested in the Met-puromycin assay; however, it was shown to rescue binding of Met-tRNAiMet to 40 S ribosomes in extracts prepared from a temperature-sensitive prt1 mutant (27), consistent with a known function of eIF3. We recently succeeded in demonstrating the same activity for the complex we purified containing His-tagged PRT1 and the other four yeast homologues of mammalian eIF3 subunits.6

It was shown previously that a temperature-sensitive sui1 mutant grown at the nonpermissive temperature lacks SUI1 protein and eIF3 activity (24), suggesting that SUI1 is required for eIF3 function in the Met-puromycin assay. We detected interactions between NIP1 and SUI1 with both yeast two-hybrid and in vitro protein binding assays. Unlike NIP1, however, we found that SUI1 did not coimmunoprecipitate with HA-TIF34 from the RSW fraction (Fig. 7B). Thus, SUI1 may dissociate more readily from the yeast eIF3 complex than do other eIF3 subunits, consistent with the fact that the mammalian homologue of SUI1 (eIF1) and eIF3 were purified as separate factors from the RSW fractions of rabbit reticulocytes and HeLa cells (6, 13). It is noteworthy that the active fraction of mammalian eIF3 apparently does not contain eIF1 (13, 16).

GCD10 was found previously to coimmunoprecipitate with PRT1 using polyclonal antibodies against PRT1 or GCD10 (23); however, GCD10 did not coimmunoprecipitate with HA-TIF34 in our experiments (Fig. 7). In addition, we found that GCD10 did not copurify with a polyhistidine-tagged form of PRT1 (56). These recent results suggest that GCD10 is not tightly bound to other subunits of yeast eIF3. On the other hand, GCD10 interacted with GCD14 in the two-hybrid assay, confirming observations that GCD10 is stably associated with GCD14 in a nuclear complex that functions in the expression of initiator tRNAiMet.5 More work is needed to determine whether GCD10 also functions in the cytoplasm in association with eIF3.

The results presented in this report suggest that all five yeast homologues of human eIF3 subunits are physically associated with the eIF3 complex of yeast. It appears that NIP1 and TIF32 are not required for eIF3 activity in the Met-puromycin assay. In addition, it was recently shown that the mammalian counterpart of TIF32 (eIF3-p170) is dispensable for stimulating tRNAiMet binding to 40 S ribosomes (19). Thus, NIP1 and TIF32 may be required only for the function of eIF3 in promoting ribosomal subunit dissociation or mRNA binding. A high degree of evolutionary conservation has been observed for most of the translation initiation factors characterized in both yeast and mammals (1). Nevertheless, human eIF3 contains four subunits, p40, p47, p48, and p66, with no apparent counterparts in yeast. In particular, eIF3-p48 is encoded by int-6, a site of frequent integration by mouse mammary tumor viruses and, therefore, is suspected to be a regulatory subunit of mammalian eIF3 (18). It will be interesting to learn whether the functions of these unique subunits in mammalian eIF3 are carried out in yeast by other nonconserved proteins not tightly associated with the eIF3 complex.

    ACKNOWLEDGEMENTS

We are indebted to John Hershey and Parisa Hanachi for pNOTAp39 and pNOp33 and for communicating results prior to publication and to David Goldfarb for anti-NIP1 antibodies. We also thank Tom Dever for critical reading of the manuscript; Weimin Yang for sharing materials used in two-hybrid screening; and Hanse Chung and Fan Zhang for technical assistance.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Supported by the Japan Society for the Promotion of Science Research Fellowships for Japanese Biomedical and Behavioral Researchers at the National Institutes of Health.

§ To whom correspondence should be addressed. Tel.: 301-496-4480; Fax: 301-496-6828; E-mail: ahinnebusch{at}nih.gov.

1 The abbreviations used are: eIF, eukaryotic translation initiation factor; PCR, polymerase chain reaction; ORF, open reading frame; kb, kilobase(s); GST, glutathione S-transferase; UTR, untranslated region; SC, synthetic complete (medium); 5-FOA, 5-fluoroorotic acid; 3-AT, 3-aminotriazole; bp, base pair(s); WCE, whole cell extract(s); RSW, ribosomal salt wash; PAGE, polyacrylamide gel electrophoresis; HA, hemagglutinin.

2 K. Block, H.-P. Vornlocher, K. Asano, and J. Hershey, manuscript in preparation. The amino acid sequence of human eIF3-p44 was made available by J. Hershey.

3 J. Anderson, K. Asano, and A. G. Hinnebusch, unpublished observations.

4 R. Cuesta, O. Calvo, J. Anderson, M. T. Garcia Barrio, A. G. Hinnebusch, and M. Tamame, manuscript in preparation.

5 J. Anderson, M. Pak, L. Phan, R. Cuesta, K. Asano, M. Tamame, and A. G. Hinnebusch, submitted for publication.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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