Biochemical characterization of mammalian translation initiation factor 3 (eIF3) -: Molecular cloning reveals that p110 subunit is the mammalian homologue of Saccharomyces cerevisiae protein Prt1

Eukaryotic translation initiation factor 3 (eIF3), which plays an essential role in initiation of protein synthesis, was purified from rabbit reticulocyte lysates using an assay that specifically measures its ability to stimulate the binding of Met-tRNA(f), (as a Met-tRNA.eIF2. GTP ternary complex) to 40 S ribosomal subunits, Purified eIF3 consisted of six major polypeptides of molecular masses 110, 67, 42, 40, 36, and 35 kDa but lacked the 170-kDa polypeptide reported to be a constituent of other eIF3 preparations, Characterization of purified eIF3 lacking the 170-kDa polypeptide showed that the eIF3-mediated 40 S initiation complex formed in the presence of AUG codon efficiently joined 60 S ribosomal subunits in an eIF5-dependent reaction to form a functional 80 S initiation complex, eIF3, which was originally bound to the 40 S initiation complex, was released from the 40 S subunit during the subunit joining reaction, Additionally, chicken antibodies raised against rabbit reticulocyte eIF3 were used to immunochemically characterize eIF3 subunits and to isolate a 3.1-kilobase pair human cDNA that encodes the p110 subunit of mammalian eIF3, The derived amino acid sequence (calculated M-r, 95,214) shows that the p110 subunit is the mammalian homologue of Saccharomyces cerevisiae protein Prt1p, a subunit of yeast eIF3.