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
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Eukaryotic Initiation Factor-1 / chemistry
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Eukaryotic Initiation Factor-1 / isolation & purification
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Eukaryotic Initiation Factor-1 / metabolism*
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Eukaryotic Initiation Factor-3*
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Eukaryotic Initiation Factor-5
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Fungal Proteins / chemistry
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Fungal Proteins / isolation & purification
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Fungal Proteins / metabolism*
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Genotype
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Kinetics
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Models, Molecular
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Peptide Initiation Factors / chemistry
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Peptide Initiation Factors / isolation & purification
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Peptide Initiation Factors / metabolism*
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Prokaryotic Initiation Factor-3
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Protein Biosynthesis
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Protein Subunits
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RNA, Messenger / chemistry
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RNA, Messenger / isolation & purification
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RNA, Messenger / metabolism*
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RNA, Transfer, Met / chemistry
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RNA, Transfer, Met / isolation & purification
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RNA, Transfer, Met / metabolism*
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Ribosomes / metabolism*
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Ribosomes / ultrastructure
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Saccharomyces cerevisiae / genetics*
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Saccharomyces cerevisiae / metabolism*
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Saccharomyces cerevisiae Proteins*
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Thermodynamics
Substances
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Eukaryotic Initiation Factor-1
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Eukaryotic Initiation Factor-3
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Eukaryotic Initiation Factor-5
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Fungal Proteins
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Peptide Initiation Factors
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Prokaryotic Initiation Factor-3
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Protein Subunits
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Prt1 protein, S cerevisiae
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RNA, Messenger
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RNA, Transfer, Met
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Saccharomyces cerevisiae Proteins