Translation initiation factor 2 (eIF2) bound to GTP transfers the initiator methionyl tRNA to the 40S ribosomal subunit. The eIF5 stimulates GTP hydrolysis by the eIF2/GTP/Met-tRNA i Met ternary complex on base-pairing between Met-tRNA i Met and the start codon. The eIF2, eIF5, and eIF1 all have been implicated in stringent selection of AUG as the start codon. The eIF3 binds to the 40S ribosome and promotes recruitment of the ternary complex; however, physical contact between eIF3 and eIF2 has not been observed. We show that yeast eIF5 can bridge interaction in vitro between eIF3 and eIF2 by binding simultaneously to the amino terminus of eIF3 subunit NIP1 and the amino-terminal half of eIF2␤, dependent on a conserved bipartite motif in the carboxyl terminus of eIF5. Additionally, the amino terminus of NIP1 can bind concurrently to eIF5 and eIF1. These findings suggest the occurrence of an eIF3/eIF1/eIF5/eIF2 multifactor complex, which was observed in cell extracts free of 40S ribosomes and found to contain stoichiometric amounts of tRNA i Met . The multifactor complex was disrupted by the tif5-7A mutation in the bipartite motif of eIF5. Importantly, the tif5-7A mutant is temperature sensitive and displayed a substantial reduction in translation initiation at the restrictive temperature. We propose that the multifactor complex is an important intermediate in translation initiation in vivo.

Gcd10p andMet maturation. The chromatographic behavior of elongator and initiator tRNA Met on a RPC-5 column indicated that both species are altered structurally in gcd10⌬ cells, and analysis of base modifications revealed that 1-methyladenosine (m 1 A) is undetectable in gcd10⌬ tRNA. Interestingly, gcd10 and gcd14 mutations had no effect on processing or accumulation of elongator tRNA Met , which also contains m 1 A at position 58, suggesting a unique requirement for this base modification in initiator maturation.

In the initiation phase of eukaryotic translation, eIF5 stimulates the hydrolysis of GTP bound to eIF2 in the 40S ribosomal pre-initiation complex, and the resultant GDP on eIF2 is replaced with GTP by the complex nucleotide exchange factor, eIF2B. Bipartite motifs rich in aromatic and acidic residues are conserved at the C-termini of eIF5 and the catalytic (ε) subunit of eIF2B. Here we show that these bipartite motifs are important for the binding of these factors, both in vitro and in vivo, to the β subunit of their common substrate eIF2. We also find that three lysine-rich boxes in the N-terminal segment of eIF2β mediate the binding of eIF2 to both eIF5 and eIF2B. Thus, eIF5 and eIF2Bε employ the same sequence motif to facilitate interaction with the same segment of their common substrate. In agreement with this, archaea appear to lack eIF5, eIF2B and the lysine-rich binding domain for these factors in their eIF2β homolog. The eIF5 bipartite motif is also important for its interaction with the eIF3 complex through the NIP1-encoded subunit of eIF3. Thus, the bipartite motif in eIF5 appears to be multifunctional, stimulating its recruitment to the 40S preinitiation complex through interaction with eIF3 in addition to binding of its substrate eIF2.

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 Ni 2؉ affinity and gel filtration chromatography and obtained a complex of Ϸ600 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.The initiation of protein synthesis in eukaryotic cells is dependent on multiple initiation factors (eIFs) that stimulate the binding of mRNA and methionyl-initiator tRNA (tRNA i Met ) to 40S ribosomes to form the 48S preinitiation complex (39). The Met-tRNA i Met is delivered to 40S ribosomes in a ternary complex with eIF2 and GTP, whereas the binding of mRNA to ribosomes is stimulated by eIF4F, eIF4A, eIF4B (39), and the poly(A)-binding protein Pab1p (54). Joining of the 60S subunit to form an 80S initiation complex requires hydrolysis of the GTP bound to eIF2, dissociation of the ternary complex, and release of the eIF2-GDP binary complex, and eIF5 promotes these events by stimulating GTP hydrolysis on ternary complexes bound to 40S ribosomes (39).Mammalian eIF3 is a multisubunit complex that has been implicated in several aspects of 48S complex formation. The purified factor promotes dissociation of 80S ribosomes into 40S and 60S subunits, forming a complex with the 40S subunits, and stabilizes binding of the eIF2-GTP-Met-tRNA i Met ternary complex to the 40S ribosome. It also stimulates binding of mRNA to 40S subunits (9, 56), presumably through its interactions with the cap-binding initiation factor eIF4F (36, 38) or eIF4B (41). A mammalian eIF3 complex, purified by its ability to promote methionylpuromycin (Met-puromycin) synthesis by an 80S initiation complex in an assay containing purified eIF1A, eIF2, eIF5, eIF5A, and ribos...

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 twohybrid 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.In the initiation phase of eukaryotic protein synthesis, mRNA and Met-tRNA i Met 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-tRNA i Met 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-MettRNA iMet 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 co...