Identification of a translation initiation factor 3 (eIF3) core complex, conserved in yeast and mammals, that interacts with eIF5.
Only five of the nine subunits of human eukaryotic translation initiation factor 3 (eIF3) have recognizable homologs encoded in the Saccharomyces cerevisiae genome, and only two of these (Prt1p and Tif34p) were identified previously as subunits of yeast eIF3. We purified a polyhistidine-tagged form of Prt1p (His-Prt1p) by Ni2+ affinity and gel filtration chromatography and obtained a complex of approximately 600 kDa composed of six polypeptides whose copurification was completely dependent on the polyhistidine tag on His-Prt1p. All five polypeptides associated with His-Prt1p were identified by mass spectrometry, and four were found to be the other putative homologs of human eIF3 subunits encoded in S. cerevisiae: YBR079c/Tif32p, Nip1p, Tif34p, and YDR429c/Tif35p. The fifth Prt1p-associated protein was eIF5, an initiation factor not previously known to interact with eIF3. The purified complex could rescue Met-tRNAiMet binding to 40S ribosomes in defective extracts from a prt1 mutant or extracts from which Nip1p had been depleted, indicating that it possesses a known biochemical activity of eIF3. These findings suggest that Tif32p, Nip1p, Prt1p, Tif34p, and Tif35p comprise an eIF3 core complex, conserved between yeast and mammals, that stably interacts with eIF5. Nip1p bound to eIF5 in yeast two-hybrid and in vitro protein binding assays. Interestingly, Sui1p also interacts with Nip1p, and both eIF5 and Sui1p have been implicated in accurate recognition of the AUG start codon. Thus, eIF5 and Sui1p may be recruited to the 40S ribosomes through physical interactions with the Nip1p subunit of eIF3.
Laboratory of Eukaryotic Gene Regulation, National Institute of Child Health and Human Development, Bethesda, Maryland 20892, USA.
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L
Phan
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Zhang
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Asano
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J
Anderson
H P
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Vornlocher
J R
JR
Greenberg
J
J
Qin
A G
AG
Hinnebusch
D000818:Animals; D000939:Epitopes; D015977:Eukaryotic Initiation Factor-1; D039621:Eukaryotic Initiation Factor-3; D039603:Eukaryotic Initiation Factor-4G; D040061:Eukaryotic Initiation Factor-5; D005656:Fungal Proteins / Q000302:isolation & purification / Q000378:metabolism; D006639:Histidine; D006801:Humans; D008322:Mammals; D013058:Mass Spectrometry; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000378:metabolism; D010448:Peptide Initiation Factors / Q000378:metabolism*; D010455:Peptides; D011233:Precipitin Tests; D012358:RNA, Transfer, Met / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000378:metabolism
D000939:Epitopes; D015977:Eukaryotic Initiation Factor-1; D039621:Eukaryotic Initiation Factor-3; D039603:Eukaryotic Initiation Factor-4G; D040061:Eukaryotic Initiation Factor-5; D005656:Fungal Proteins; C112736:Met-tRNA(i)(Met); C077915:NIP1 protein, S cerevisiae; D009687:Nuclear Proteins; D010448:Peptide Initiation Factors; D010455:Peptides; C050998:Prt1 protein, S cerevisiae; D012358:RNA, Transfer, Met; C072076:SUI1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C102947:TIF34 protein, S cerevisiae; D014157:Transcription Factors; C033223:polyhistidine; D006639:Histidine