PURIFIED YEAST TRANSLATIONAL INITIATION-FACTOR EIF-3 IS AN RNA-BINDING PROTEIN COMPLEX THAT CONTAINS THE PRT1 PROTEIN

Eukaryotic initiation factor-3 (eIF-3) plays a pivotal role in the ini tiation phase of protein synthesis where it promotes dissociation of 8 0 S ribosomes into subunits, stabilizes methionyl-tRNA(i) binding to 4 0 S ribosomal subunits, and is required for mRNA binding. Mammalian eI F-3 is comprised of eight subunits, but no mammalian cDNA encoding the se proteins has been cloned and sequenced, nor has the corresponding f actor been characterized in yeast. Since many initiation factors are s trongly conserved between mammalian and yeast systems, we employed a m ammalian assay for initiation, the synthesis of methionyl-puromycin, t o detect eIF-3 activity in yeast subcellular fractions. Yeast eIF-3 wa s purified from the high salt wash of ribosomes by Superose 6 molecula r sieve and MonoS ion exchange chromatography. Yeast eIF-3 contains ei ght subunits with masses of 16, 21, 29, 33, 39, 62, 90, and 135 kiloda ltons all of which coelute with an apparent mass of 550 kilodaltons fr om the Superose 6 column. Immunoblotting shows that the 90-kDa subunit corresponds to the product of the PRT1 gene whose mutant form, prt1-1 , exhibits destabilization of methionyl-tRNA(i) binding to 40 S riboso mal subunits. eIF-3, and specifically the 62-kDa subunit, bind to RNA. These biochemical approaches to defining yeast eIF-3 complement genet ic methods so far used in characterizing the initiation factors and pr ovide another route to defining the yeast translational machinery.