A genetic reversion analysis at the HIS4 locus in Saccharomyces cerevisiae has identified SUI1 as a component of the translation initiation complex which plays an important role in ribosomal recognition of the initiator codon. SUI1 is an essential protein of 12.3 kDa that is required in vivo for the initiation of protein synthesis. Here we present evidence that SUI1 is identical to the smallest subunit, p16, of eukaryotic translation initiation factor 3 (eIF-3) in S. cerevisiae. SUI1 and eIF3-p16 comigrate upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis and cross-react with anti-SUI1 and anti-eIF3 antisera. Anti-SUI1 antisera immunoprecipitate all of the subunits of eIF3, whereas antisera against the eIF3 complex and the individual PRT1 and GCD10 subunits of eIF3 immunoprecipitate SUI1. Finally, the N-terminal amino acid sequence of a truncated form of eIF3-p16 matches the sequence of SUI1. eIF3 isolated from a sui1(ts) strain at 37 degrees C lacks SUI1 and fails to exhibit eIF3 activity in the in vitro assay for methionyl-puromycin synthesis. A free form of SUI1 separate from the eIF3 complex is found in S. cerevisiae but lacks activity in the in vitro assay. The results, together with prior genetic experiments, indicate that SUI1 is essential for eIF3 activity and functions as part of eIF3 and in concert with eIF2 to promote eIF2-GTP-Met-tRNAi ternary complex recognition of the initiator codon.

Eukaryotic translation initiation factor 3 (eIF3) in the yeast Saccharomyces cerevisiae comprises about eight polypeptides and plays a central role in the binding of methionyl-tRNA i and mRNA to the 40S ribosomal subunit. The fourth largest subunit, eIF3-p39, was gel purified, and a 12-amino-acid tryptic peptide was sequenced, enabling the cloning of the TIF34 gene. TIF34 encodes a 38,753-Da protein that corresponds to eIF3-p39 in size and antigenicity. Disruption of TIF34 is lethal, and depletion of eIF3-p39 by glucose repression of TIF34 expressed from a GAL promoter results in cessation of cell growth. As eIF3-p39 levels fall, polysomes become smaller, indicating a role for eIF3-p39 in the initiation phase of protein synthesis. Unexpectedly, depletion results in degradation of all of the subunit proteins of eIF3 at a rate much faster than the normal turnover rates of these proteins. eIF3-p39 has 46% sequence identity with the p36 subunit of human eIF3. Both proteins are members of the WD-repeat family of proteins, possessing five to seven repeat elements. Taken together, the results indicate that eIF3-p39 plays an important, although not necessarily direct, role in the initiation phase of protein synthesis and suggest that it may be required for the assembly and maintenance of the eIF3 complex in eukaryotic cells.Initiation of protein synthesis is promoted by at least 10 proteins called initiation factors (reviewed in reference 14). The largest and most complex of these is eukaryotic translation initiation factor 3 (eIF3), a factor comprising at least eight subunits. eIF3 plays a central role in the initiation pathway in mammalian cells (1a, 26). It binds to 40S ribosomal subunits and is implicated in dissociating 80S ribosomes into 40S and 60S subunits (1a, 6). It prevents dissociation of the MettRNA i ⅐ eIF2 ⅐ GTP ternary complex caused by addition of RNA (7) and stabilizes ternary complex binding to 40S ribosomal subunits (1a). eIF3 is required for mRNA binding to 40S and 80S ribosomes (1a, 26), in part by binding the eIF4G subunit of the cap-binding complex, eIF4F (12, 13). Therefore, knowledge of the structure and function of eIF3 is essential for understanding the mechanism of the initiation phase of protein synthesis.To better elucidate the function of eIF3 by the application of both biochemical and genetic methods, we have been studying the factor in the yeast Saccharomyces cerevisiae. Yeast eIF3 was initially isolated and purified by a biochemical approach that used an eIF3-dependent mammalian assay for the synthesis of methionyl-puromycin (Met-PM) (17). The resulting yeast complex comprised eight subunits with apparent masses of 16,21, 29, 33, 39, 62, 90, and 135 kDa. The genes for three of the subunits had been identified previously, but it was not realized that they encode subunits of eIF3. The second-largest subunit, p90, is encoded by PRT1; the temperature-sensitive prt1-1 mutant causes destabilization of Met-tRNA i binding to 40S ribosomal subunits (8,15). GCD10, first characterized genetical...

GCN4 mRNA is translated by a reinitiation mechanism involving four short upstream open reading frames (uORFs) in its leader sequence. Decreasing the activity of eukaryotic initiation factor-2 (eIF-2) by phosphorylation inhibits general translation in yeast but stimulates GCN4 expression by allowing ribosomes to scan past the uORFs and reinitiate at GCN4 instead. GCDIO was first identified genetically as a translational repressor of GCN4. We show here that GCD10 is an essential protein of 54.6 kD that is required in vivo for the initiation of total protein synthesis. GCD10 binds RNA in vitro and we present strong biochemical evidence that it is identical to the RNA-binding subunit of yeast initiation factor-3 (eIF-3). eIF-3 is a multisubunit complex that stimulates translation initiation in vitro at several different steps. We suggest that gcdlO mutations decrease the ability of eIF-3 to stimulate binding of eIF-2/GTP/Met-tRNAi ~et ternary comPlexes to small ribosomal subunits in vivo. This would explain why mutations in eiF-3 mimic eIF-2a phosphorylation in allowing ribosomes to bypass the uORFs and reinitiate at GCN4. Our results indicate that GCN4 expression provides a sensitive in vivo assay for the function of eIF-3 in initiation complex formation.

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