A subcomplex of three eIF3 subunits binds eIF1 and eIF5 and stimulates ribosome binding of mRNA and tRNAiMet

Abstract

Yeast translation initiation factor 3 contains five core subunits (known as TIF32, PRT1, NIP1, TIF34 and TIF35) and a less tightly associated component known as HCR1. We found that a stable subcomplex of His₈‐PRT1, NIP1 and TIF32 (PN2 subcomplex) could be affinity purified from a strain overexpressing these eIF3 subunits. eIF5, eIF1 and HCR1 co‐purified with this subcomplex, but not with distinct His₈‐PRT1–TIF34–TIF35 (P45) or His₈‐PRT1–TIF32 (P2) sub complexes. His₈‐PRT1 and NIP1 did not form a stable binary subcomplex. 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 prt1‐1 extracts are defective for Met‐tRNA_i^Met binding to 40S subunits, and we also observed defective 40S binding of mRNA, eIFs 1 and 5 and eIF3 itself 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 well with purified PN2 subcomplex as with five‐subunit eIF3, whereas the P45 subcomplex was nearly inactive. Thus, several key functions of eIF3 can be carried out by the PRT1–TIF32–NIP1 subcomplex.