A subcomplex of three eIF3 subunits binds eIF1 and eIF5 and stimulates ribosome binding of mRNA and tRNA(i)(Met)

Yeast translation initiation factor 3 contains five core subunits (known as TIF32, PRT1, NIP1, TIF34 and TIF35) and a less tightly associated componen t known as HCR1, We found that a stable subcomplex of His(8)-PRT1, NIP1 and TIF32 (PN2 subcomplex) could be affinity purified from a strain overexpres sing these eIF3 subunits, eIF5, eIF1 and HCR1 co-purified with this subcomp lex, but not with distinct His(8)-PRT1-TIF34-TIF35 (P45) or His(8)-PRT1-TIF 32 (P2) subcomplexes. His(8)-PRT1 and NIP1 did not form a stable binary sub complex, These results provide in vivo evidence that TIF32 bridges PRT1 and NIP1, and that eIFs 1 and 5 bind to NIP1, in native eIF3, Heat-treated prt 1-1 extracts are defective for Met-tRNA(i)(Met) binding to 40S subunits, an d we also observed defective 40S binding of mRNA, eIFs 1 and 5 and eIF3 its elf in these extracts. We could rescue 40S binding of Met-tRNA(i)(Met) and mRNA, and translation of luciferase mRNA, in a prt1-1 extract almost as wel l with purified PN2 subcomplex as with five-subunit eIF3, whereas the P45 s ubcomplex was nearly inactive. Thus, several key functions of eIF3 can be c arried out by the PRT1-TIF32-NIP1 subcomplex.