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

EMBO J. 2001 Jun 1;20(11):2954-65. doi: 10.1093/emboj/20.11.2954.

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.

MeSH terms

  • Eukaryotic Initiation Factor-1 / chemistry
  • Eukaryotic Initiation Factor-1 / isolation & purification
  • Eukaryotic Initiation Factor-1 / metabolism*
  • Eukaryotic Initiation Factor-3*
  • Eukaryotic Initiation Factor-5
  • Fungal Proteins / chemistry
  • Fungal Proteins / isolation & purification
  • Fungal Proteins / metabolism*
  • Genotype
  • Kinetics
  • Models, Molecular
  • Peptide Initiation Factors / chemistry
  • Peptide Initiation Factors / isolation & purification
  • Peptide Initiation Factors / metabolism*
  • Prokaryotic Initiation Factor-3
  • Protein Biosynthesis
  • Protein Subunits
  • RNA, Messenger / chemistry
  • RNA, Messenger / isolation & purification
  • RNA, Messenger / metabolism*
  • RNA, Transfer, Met / chemistry
  • RNA, Transfer, Met / isolation & purification
  • RNA, Transfer, Met / metabolism*
  • Ribosomes / metabolism*
  • Ribosomes / ultrastructure
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins*
  • Thermodynamics

Substances

  • Eukaryotic Initiation Factor-1
  • Eukaryotic Initiation Factor-3
  • Eukaryotic Initiation Factor-5
  • Fungal Proteins
  • Peptide Initiation Factors
  • Prokaryotic Initiation Factor-3
  • Protein Subunits
  • Prt1 protein, S cerevisiae
  • RNA, Messenger
  • RNA, Transfer, Met
  • Saccharomyces cerevisiae Proteins