Translation initiation factor 2gamma mutant alters start codon selection independent of Met-tRNA binding

Selection of the AUG start codon for translation in eukaryotes is governed by codon-anticodon interactions between the initiator Met-tRNA_i^Met and the mRNA. Translation initiation factor 2 (eIF2) binds Met-tRNA_i^Met to the 40S ribosomal subunit, and previous studies identified Sui⁻ mutations in eIF2 that enhanced initiation from a noncanonical UUG codon, presumably by impairing Met-tRNA_i^Met binding. Consistently, an eIF2γ-N135D GTP-binding domain mutation impairs Met-tRNA_i^Met binding and causes a Sui⁻ phenotype. Intragenic A208V and A382V suppressor mutations restore Met-tRNA_i^Met binding affinity and cell growth; however, only A208V suppresses the Sui⁻ phenotype associated with the eIF2γ-N135D mutation. An eIF2γ-A219T mutation impairs Met-tRNA_i^Met binding but unexpectedly enhances the fidelity of initiation, suppressing the Sui⁻ phenotype associated with the eIF2γ-N135D,A382V mutant. Overexpression of eIF1, which is thought to monitor codon-anticodon interactions during translation initiation, likewise suppresses the Sui⁻ phenotype of the eIF2γ mutants. We propose that structural alterations in eIF2γ subtly alter the conformation of Met-tRNA_i^Met on the 40S subunit and thereby affect the fidelity of start codon recognition independent of Met-tRNA_i^Met binding affinity.     

Position of eukaryotic translation initiation factor eIF1A on the 40S ribosomal subunit mapped by directed hydroxyl radical probing

The universally conserved eukaryotic initiation factor (eIF), eIF1A, plays multiple roles throughout initiation: it stimulates eIF2/GTP/Met-tRNA_i^Met attachment to 40S ribosomal subunits, scanning, start codon selection and subunit joining. Its bacterial ortholog IF1 consists of an oligonucleotide/oligosaccharide-binding (OB) domain, whereas eIF1A additionally contains a helical subdomain, N-terminal tail (NTT) and C-terminal tail (CTT). The NTT and CTT both enhance ribosomal recruitment of eIF2/GTP/Met-tRNA_i^Met, but have opposite effects on the stringency of start codon selection: the CTT increases, whereas the NTT decreases it. Here, we determined the position of eIF1A on the 40S subunit by directed hydroxyl radical cleavage. eIF1A''s OB domain binds in the A site, similar to IF1, whereas the helical subdomain contacts the head, forming a bridge over the mRNA channel. The NTT and CTT both thread under Met-tRNA_i^Met reaching into the P-site. The NTT threads closer to the mRNA channel. In the proposed model, the NTT does not clash with either mRNA or Met-tRNA_i^Met, consistent with its suggested role in promoting the ‘closed’ conformation of ribosomal complexes upon start codon recognition. In contrast, eIF1A-CTT appears to interfere with the P-site tRNA-head interaction in the ‘closed’ complex and is likely ejected from the P-site upon start codon recognition.     

AIMP3/p18 Controls Translational Initiation by Mediating the Delivery of Charged Initiator tRNA to Initiation Complex

Aminoacyl-tRNA synthetase-interacting multifunctional proteins (AIMPs) are nonenzymatic scaffolding proteins that comprise multisynthetase complex (MSC) with nine aminoacyl-tRNA synthetases in higher eukaryotes. Among the three AIMPs, AIMP3/p18 is strongly anchored to methionyl-tRNA synthetase (MRS) in the MSC. MRS attaches methionine (Met) to initiator tRNA (tRNA_i^Met) and plays an important role in translation initiation. It is known that AIMP3 is dispatched to nucleus or nuclear membrane to induce DNA damage response or senescence; however, the role of AIMP3 in translation as a component of MSC and the meaning of its interaction with MRS are still unclear. Herein, we observed that AIMP3 specifically interacted with Met-tRNA_i^Metin vitro, while it showed little or reduced interaction with unacylated or lysine-charged tRNA_i^Met. In addition, AIMP3 discriminates Met-tRNA_i^Met from Met-charged elongator tRNA based on filter-binding assay. Pull‐down assay revealed that AIMP3 and MRS had noncompetitive interaction with eukaryotic initiation factor 2 (eIF2) γ subunit (eIF2γ), which is in charge of binding with Met-tRNA_i^Met for the delivery of Met-tRNA_i^Met to ribosome. AIMP3 recruited active eIF2γ to the MRS-AIMP3 complex, and the level of Met-tRNA_i^Met bound to eIF2 complex was reduced by AIMP3 knockdown resulting in reduced protein synthesis. All these results suggested the novel function of AIMP3 as a critical mediator of Met-tRNA_i^Met transfer from MRS to eIF2 complex for the accurate and efficient translation initiation.     

Contributions of aminoacyl-tRNA synthetase-interacting multifunctional protein-3 to mammalian translation initiation

Aminoacyl-tRNA synthetase-interacting multifunctional protein-3 (AIMP3) stabilizes and protects mammalian methionyl-tRNA synthetase (MRS) and eukaryotic initiation factor 2 subunit gamma (eIF2γ), factors involved in the formation and the delivery of Met-tRNA _i ^Met respectively, through the binding interactions. Due to the protections that MRS and eIF2γ are provided from the interactions with AIMP3, cellular levels of MRS and eIF2γ may be able to be maintained high enough for their canonical and/or non-canonical functions.     

Molecular Mechanism of Scanning and Start Codon Selection in Eukaryotes

Summary: The correct translation of mRNA depends critically on the ability to initiate at the right AUG codon. For most mRNAs in eukaryotic cells, this is accomplished by the scanning mechanism, wherein the small (40S) ribosomal subunit attaches to the 5′ end of the mRNA and then inspects the leader base by base for an AUG in a suitable context, using complementarity with the anticodon of methionyl initiator tRNA (Met-tRNA_i^Met) as the key means of identifying AUG. Over the past decade, a combination of yeast genetics, biochemical analysis in reconstituted systems, and structural biology has enabled great progress in deciphering the mechanism of ribosomal scanning. A robust molecular model now exists, describing the roles of initiation factors, notably eukaryotic initiation factor 1 (eIF1) and eIF1A, in stabilizing an “open” conformation of the 40S subunit with Met-tRNA_i^Met bound in a low-affinity state conducive to scanning and in triggering rearrangement into a “closed” conformation incompatible with scanning, which features Met-tRNA_i^Met more tightly bound to the “P” site and base paired with AUG. It has also emerged that multiple DEAD-box RNA helicases participate in producing a single-stranded “landing pad” for the 40S subunit and in removing the secondary structure to enable the mRNA to traverse the 40S mRNA-binding channel in the single-stranded form for base-by-base inspection in the P site.     

The β Subunit of Eukaryotic Translation Initiation Factor 2 Binds mRNA through the Lysine Repeats and a Region Comprising the C2-C2 Motif

Eukaryotic translation initiation factor 2 (eIF2) has been implicated in the selection of the AUG codon as the start site for eukaryotic translation initiation, since mutations in its three subunits in yeast that allow the recognition of a UUG codon by the anticodon of the initiator Met-tRNA^Met have been identified. All such mutations in the beta subunit of eIF2 (eIF2β) mapped to a region containing a putative zinc finger structure of the C₂-C₂ type, indicating that these sequences could be involved in RNA recognition. Another feature of eIF2β that could mediate an interaction with RNA is located in the amino-terminal sequences and is composed of three repeats of seven lysine residues which are highly conserved in other species. We show here the ability of eIF2β, purified from Escherichia coli as a fusion to glutathione S-transferase, to bind mRNA in vitro. Through a deletion analysis, mRNA binding was found to be dependent on the lysine repeats and a region encompassing the C₂-C₂ motif. Strong mRNA binding in vitro could be maintained by the presence of only one lysine or one arginine run but not one alanine run. We further show that only one run of lysine residues is sufficient for the in vivo function of eIF2β, probably through charge interaction, since its replacement by arginines did not impair cell viability, whereas substitution for alanines resulted in inviable cells. mRNA binding, but not GTP-dependent initiator Met-tRNA^Met binding, by the eIF2 complex was determined to be dependent on the presence of the lysine runs of the beta subunit.     

Enhanced eIF1 binding to the 40S ribosome impedes conformational rearrangements of the preinitiation complex and elevates initiation accuracy

In the current model of translation initiation by the scanning mechanism, eIF1 promotes an open conformation of the 40S subunit competent for rapidly loading the eIF2·GTP·Met-tRNA_i ternary complex (TC) in a metastable conformation (P_OUT) capable of sampling triplets entering the P site while blocking accommodation of Met-tRNA_i in the P_IN state and preventing completion of GTP hydrolysis (P_i release) by the TC. All of these functions should be reversed by eIF1 dissociation from the preinitiation complex (PIC) on AUG recognition. We tested this model by selecting eIF1 Ssu⁻ mutations that suppress the elevated UUG initiation and reduced rate of TC loading in vivo conferred by an eIF1 (Sui⁻) substitution that eliminates a direct contact of eIF1 with the 40S subunit. Importantly, several Ssu⁻ substitutions increase eIF1 affinity for 40S subunits in vitro, and the strongest-binding variant (D61G), predicted to eliminate ionic repulsion with 18S rRNA, both reduces the rate of eIF1 dissociation and destabilizes the P_IN state of TC binding in reconstituted PICs harboring Sui⁻ variants of eIF5 or eIF2. These findings establish that eIF1 dissociation from the 40S subunit is required for the P_IN mode of TC binding and AUG recognition and that increasing eIF1 affinity for the 40S subunit increases initiation accuracy in vivo. Our results further demonstrate that the GTPase-activating protein eIF5 and β-subunit of eIF2 promote accuracy by controlling eIF1 dissociation and the stability of TC binding to the PIC, beyond their roles in regulating GTP hydrolysis by eIF2.     

Initiation of mammalian protein synthesis: Dynamic properties of the assembly process in vitro

Binding of the Met-tRNA^Met_f·eIF-2 GTP complex to the 40 S ribosomal subunit is the first step in initiation of eukaryotic protein synthesis. The extent of binding and the stability of the complex are enhanced by initiation factors eIF-3 and eIF-4C, AUG and elevated magnesium concentration. The reversibility of reaction steps occurring during the assembly of the initiation complex is measured as the rate of Met-tRNA^Met_f exchange in the initiation complex and its intermediates. This rate progressively decreases and Met-tRNA^Met_f binding becomes irreversible upon binding of mRNA. The association of the 40 S Met-tRNA^Met_f mRNA initiation complex with the 60 S ribosomal subunit is again reversible as long as elongation does not occur.     

Translation initiation complex formation in the crenarchaeon Sulfolobus solfataricus

The function of initiation factors in and the sequence of events during translation initiation have been intensively studied in Bacteria and Eukaryotes, whereas in Archaea knowledge on these functions/processes is limited. By employing chemical probing, we show that translation initiation factor aIF1 of the model crenarchaeon Sulfolobus solfataricus binds to the same area on the ribosome as the bacterial and eukaryal orthologs. Fluorescence energy transfer assays (FRET) showed that aIF1, like its eukaryotic and bacterial orthologs, has a fidelity function in translation initiation complex formation, and that both aIF1 and aIF1A exert a synergistic effect in stimulating ribosomal association of the Met-tRNAi^Met binding factor a/eIF2. However, as in Eukaryotes their effect on a/eIF2 binding appears to be indirect. Moreover, FRET was used to analyze for the first time the sequence of events toward translation initiation complex formation in an archaeal model system. These studies suggested that a/eIF2-GTP binds first to the ribosome and then recruits Met-tRNAi^Met, which appears to comply with the operational mode of bacterial IF2, and deviates from the shuttle function of the eukaryotic counterpart eIF2. Thus, despite the resemblance of eIF2 and a/eIF2, recruitment of initiator tRNA to the ribosome is mechanistically different in Pro- and Eukaryotes.     

Protein synthesis in rabbit reticulocytes. XIII. Lack of mRNA (poly r (A)) binding activity in highly purified EIF-1.

Met-tRNA_f^Met binding factor (EIF-1) has been purified more than 100 fold over crude high salt (0.5 M KCl) ribosomal wash. The purified factor binds 2 nmoles Met-tRNA_f^Met per mg protein and shows very little poly r(A) binding activity. Crude ribosomal high salt wash possesses significant amounts of poly r(A) binding activity and also binds to other RNAs. The bulk of this unspecific RNA binding protein is separated from EIF-1 by DEAE-cellulose chromatography.     

Archaeal translation initiation factor aIF2 can substitute for eukaryotic eIF2 in ribosomal scanning during mammalian 48S complex formation

Heterotrimeric translation initiation factor (IF) a/eIF2 (archaeal/eukaryotic IF 2) is present in both Eukarya and Archaea. Despite strong structural similarity between a/eIF2 orthologs from the two domains of life, their functional relationship is obscure. Here, we show that aIF2 from Sulfolobus solfataricus can substitute for its mammalian counterpart in the reconstitution of eukaryotic 48S initiation complexes from purified components. aIF2 is able to correctly place the initiator Met-tRNA_i into the P-site of the 40S ribosomal subunit and accompany the entire set of eukaryotic translation IFs in the process of cap-dependent scanning and AUG codon selection. However, it seems to be unable to participate in the following step of ribosomal subunit joining. In accordance with this, aIF2 inhibits rather than stimulates protein synthesis in mammalian cell-free system. The ability of recombinant aIF2 protein to direct ribosomal scanning suggests that some archaeal mRNAs may utilize this mechanism during translation initiation.     

Rps5-Rps16 communication is essential for efficient translation initiation in yeast S. cerevisiae

Conserved ribosomal proteins frequently harbor additional segments in eukaryotes not found in bacteria, which could facilitate eukaryotic-specific reactions in the initiation phase of protein synthesis. Here we provide evidence showing that truncation of the N-terminal domain (NTD) of yeast Rps5 (absent in bacterial ortholog S7) impairs translation initiation, cell growth and induction of GCN4 mRNA translation in a manner suggesting incomplete assembly of 48S preinitiation complexes (PICs) at upstream AUG codons in GCN4 mRNA. Rps5 mutations evoke accumulation of factors on native 40S subunits normally released on conversion of 48S PICs to 80S initiation complexes (ICs) and this abnormality and related phenotypes are mitigated by the SUI5 variant of eIF5. Remarkably, similar effects are observed by substitution of Lys45 in the Rps5-NTD, involved in contact with Rps16, and by eliminating the last two residues of the C-terminal tail (CTT) of Rps16, believed to contact initiator tRNA base-paired to AUG in the P site. We propose that Rps5-NTD-Rps16-NTD interaction modulates Rps16-CTT association with Met-tRNA_i^Met to promote a functional 48S PIC.     

Evidence for a Negative Cooperativity between eIF5A and eEF2 on Binding to the Ribosome

eIF5A is the only protein known to contain the essential and unique amino acid residue hypusine. eIF5A functions in both translation initiation due to its stimulation of methionyl-puromycin synthesis and translation elongation, being highly required for peptide-bound formation of specific ribosome stalling sequences such as poly-proline. The functional interaction between eIF5A, tRNA, and eEF2 on the surface of the ribosome is further clarified herein. Fluorescence anisotropy assays were performed to determine the affinity of eIF5A to different ribosomal complexes and reveal its interaction exclusively and directly with the 60S ribosomal subunit in a hypusine-dependent manner (K_i^{60S-eIF5A-Hyp} = 16 nM, K_i^{60S-eIF5A-Lys} = 385 nM). A 3-fold increase in eIF5A affinity to the 80S is observed upon charged-tRNA_i^Met binding, indicating positive cooperativity between P-site tRNA binding and eIF5A binding to the ribosome. Previously identified conditional mutants of yeast eIF5A, eIF5A^Q22H/L93F and eIF5A^K56A, display a significant decrease in ribosome binding affinity. Binding affinity between ribosome and eIF5A-wild type or mutants eIF5A^K56A, but not eIF5A^Q22H/L93F, is impaired in the presence of eEF2 by 4-fold, consistent with negative cooperativity between eEF2 and eIF5A binding to the ribosome. Interestingly, high-copy eEF2 is toxic only to eIF5A^Q22H/L93F and causes translation elongation defects in this mutant. These results suggest that binding of eEF2 to the ribosome alters its conformation, resulting in a weakened affinity of eIF5A and impairment of this interplay compromises cell growth due to translation elongation defects.     

A functional interaction between methionyl-transfer RNA hydrolase and a transfer RNA binding factor

Met-tRNA_f bound at low Mg ion concentrations to rabbit reticulocyte 40 S ribosomal subunits in the presence of ApUpG and a eukaryotic tRNA binding factor serves readily as a substrate for a Met-tRNA hydrolase from rabbit reticulocytes. This hydrolysis occurs rapidly at 0 °C, appears to be specific for Met-tRNA_f, and is not inhibited by 60 S ribosomal subunits. These reactions may be responsible for the accumulation of deacylated tRNA_f^Met observed in ribosomes isolated from sodium fluoride-treated cells.     

Interaction of rat liver ribosomal subunits with homologous Met-tRNAs

Rat liver 40 S ribosomal subunits, in the presence of magnesium ions, bind homologous, resolved Met-tRNAs in the absence of added exogenous proteins. The interaction of the aminoacyl-tRNAs with the particle is dependent on the concentration of magnesium ions in the incubation. At various Mg²⁺ concentrations examined, binding of the putative initiator Met-tRNA_i to 40 S subunits is greater than that observed with Met-tRNA_m. Also, binding of Met-tRNA_i to 40 S subunits is greater than that obtained with 40 S plus 60 S particles. The initial rate of formation of the 40 S·Met-tRNA_i complex is greater at 25 °C than at 37 or 4 °C; decay of the complex, which is observed after 15 min of incubation, is greater at 37 °C but it is slower if 60 S subunits are added after the complex has been formed. If 60 S subunits are added to the incubation with 40 S subunits at the start of the reaction, binding of Met-tRNA_i is inhibited; inhibition is also obtained if elongation (binding) factor EF-1 or stripped tRNAs (particularly tRNA^Met) are present in the incubation mixture containing 40 S subunits. Acetyl-Met-tRNA_i binds to 40 S·ApUpG complex to the same extent as unacetylated Met-tRNA_i and, after addition of 60 S subunits, reacts extensively with puromycin; the addition of elongation (translocation) factor EF-2 and GTP do not affect the extent of the puromycin reaction, suggesting that the acMet-tRNA_i is bound to a site on the 40 S subunits which becomes the P site on 80 S ribosomes.     

Stress granules: RNP-containing cytoplasmic bodies arising in stress: Structure and mechanism of organization

The review considers recent data on stress granules, which are dense RNP-containing cytoplasmic bodies that arise under stress conditions, e.g., in heat shock, UV irradiation, energy depletion, and oxidative stress. There is evidence that stress granules accumulate incomplete initiation complexes containing mRNA associated with proteins, small ribosomal subunits, and some translation initiation factors, and that stress granules are formed when cells are depleted of the ternary complex (eIF2-tRNA^Met-GTP), in particular, upon eIF2A phosphorylation or a decrease in GTP. Large ribosomal subunits and the ternary complex are absent from stress granules. The structural basis of stress granules is known. It is probable, however, that RNA-binding protein TIA-1, which normally occurs in the nucleus, forms prion-like aggregates that serve as scaffolds for other components of stress granules. The cytoskeleton facilitates the accumulation of stress granule components in local cytoplasmic sites. Studies of the formation and composition of stress granules are important for a better understanding of the regulation of translation initiation in vivo and the mechanisms of the cell response to stress factors.     

β-Hairpin Loop of Eukaryotic Initiation Factor 1 (eIF1) Mediates 40 S Ribosome Binding to Regulate Initiator tRNAMet Recruitment and Accuracy of AUG Selection in Vivo

Recognition of the translation initiation codon is thought to require dissociation of eIF1 from the 40 S ribosomal subunit, enabling irreversible GTP hydrolysis (P_i release) by the eIF2·GTP·Met-tRNA_i ternary complex (TC), rearrangement of the 40 S subunit to a closed conformation incompatible with scanning, and stable binding of Met-tRNA_i to the P site. The crystal structure of a Tetrahymena 40 S·eIF1 complex revealed several basic amino acids in eIF1 contacting 18 S rRNA, and we tested the prediction that their counterparts in yeast eIF1 are required to prevent premature eIF1 dissociation from scanning ribosomes at non-AUG triplets. Supporting this idea, substituting Lys-60 in helix α1, or either Lys-37 or Arg-33 in β-hairpin loop-1, impairs binding of yeast eIF1 to 40 S·eIF1A complexes in vitro, and it confers increased initiation at UUG codons (Sui⁻ phenotype) or lethality, in a manner suppressed by overexpressing the mutant proteins or by an eIF1A mutation (17–21) known to impede eIF1 dissociation in vitro. The eIF1 Sui⁻ mutations also derepress translation of GCN4 mRNA, indicating impaired ternary complex loading, and this Gcd⁻ phenotype is likewise suppressed by eIF1 overexpression or the 17–21 mutation. These findings indicate that direct contacts of eIF1 with 18 S rRNA seen in the Tetrahymena 40 S·eIF1 complex are crucial in yeast to stabilize the open conformation of the 40 S subunit and are required for rapid TC loading and ribosomal scanning and to impede rearrangement to the closed complex at non-AUG codons. Finally, we implicate the unstructured N-terminal tail of eIF1 in blocking rearrangement to the closed conformation in the scanning preinitiation complex.     

Initiation of mammalian protein synthesis. II. The assembly of the initiation complex with purified initiation factors

The assembly of initiation complexes is studied in a protein synthesis initiation assay containing ribosomal subunits, globin [¹²⁵I]mRNA, [³H]Met-tRNA_f, seven purified initiation factors, ATP and GTP. By omitting single components from the initiation assay, specific roles of the initiation factors, ATP and GTP are demonstrated. The initiation factor eIF-2 is required for the binding of Met-tRNA_f to the 40 S ribosomal subunit. The initial Met-tRNA_f binding to the small ribosomal subunit is a stringent prerequisite for the subsequent mRNA binding. The initiation factors eIF-3, eIF-4A, eIF-4B and eIF-4C together with ATP promote the binding of mRNA to the 40 S initiation complex. The association of the 40 S initiation complex with the 60 S ribosome subunit to form an 80 S initiation complex is mediated by the initiation factor eIF-5 and requires the hydrolysis of GTP. The factor eIF-1 gives a twofold overall stimulation of initiation complex formation. A model of the sequential steps in the assembly of the 80 S initiation complex in mammalian protein synthesis is presented.     

Gene-specific translational control of the yeast GCN4 gene by phosphorylation of eukaryotic initiation factor 2

Phosphorylation of the α subunit of eukaryotic initiation factor 2 (elF-2α) is one of the best-characterized mechanisms for down-regulating total protein synthesis in mammalian cells in response to various stress conditions. Recent work indicates that regulation of the GCN4 gene of Saccharomyces cerevisiae by amino acid availability represents a gene-specific case of translational control by phosphorylation of elF-2α, Four short open reading frames in the leader of GCN4 mRNA (uORFs) restrict the flow of scanning ribosomes from the cap site to the GCN4 initiation codon. When amino acids are abundant, ribosomes translate the first uORF and reinitiate at one of the remaining uORFs in the leader, after which they dissociate from the mRNA. Under conditions of amino acid starvation, many ribosomes which have translated uORFI fail to reinitiate at uORFs 2-4 and utilize the GCN4 start codon instead. Failure to reinitiate at uORFs 2-4 in starved cells results from a reduction in the GTP-bound form of elF-2 that delivers charged initiator tRNA_i^Met to the ribosome. When the levels of elF-2·GTP·Met-tRNA_i^Met ternary complexes are low, many ribosomes will not rebind this critical initiation factor following translation of uORF1 until after scanning past uORF4, but before reaching GCN4. Phosphorylation of elF-2 by the protein kinase GCN2 decreases the concentration of elF-2·GTP·Met-tRNA_i^Met complexes by inhibiting the guanine nucleotide exchange factor for elF-2, which is the same mechanism utilized in mammalian cells to inhibit total protein synthesis by phosphorylation of elF-2.     

Identification of a Translation Initiation Factor 3 (eIF3) Core Complex, Conserved in Yeast and Mammals, That Interacts with eIF5

Only five of the nine subunits of human eukaryotic translation initiation factor 3 (eIF3) have recognizable homologs encoded in the Saccharomyces cerevisiae genome, and only two of these (Prt1p and Tif34p) were identified previously as subunits of yeast eIF3. We purified a polyhistidine-tagged form of Prt1p (His-Prt1p) by Ni²⁺ affinity and gel filtration chromatography and obtained a complex of ≈600 kDa composed of six polypeptides whose copurification was completely dependent on the polyhistidine tag on His-Prt1p. All five polypeptides associated with His-Prt1p were identified by mass spectrometry, and four were found to be the other putative homologs of human eIF3 subunits encoded in S. cerevisiae: YBR079c/Tif32p, Nip1p, Tif34p, and YDR429c/Tif35p. The fifth Prt1p-associated protein was eIF5, an initiation factor not previously known to interact with eIF3. The purified complex could rescue Met-tRNA_i^Met binding to 40S ribosomes in defective extracts from a prt1 mutant or extracts from which Nip1p had been depleted, indicating that it possesses a known biochemical activity of eIF3. These findings suggest that Tif32p, Nip1p, Prt1p, Tif34p, and Tif35p comprise an eIF3 core complex, conserved between yeast and mammals, that stably interacts with eIF5. Nip1p bound to eIF5 in yeast two-hybrid and in vitro protein binding assays. Interestingly, Sui1p also interacts with Nip1p, and both eIF5 and Sui1p have been implicated in accurate recognition of the AUG start codon. Thus, eIF5 and Sui1p may be recruited to the 40S ribosomes through physical interactions with the Nip1p subunit of eIF3.