10.1021/pr100877m.s001
Adam R. Farley
Adam R.
Farley
David W. Powell
David W.
Powell
Connie M. Weaver
Connie M.
Weaver
Jennifer L. Jennings
Jennifer L.
Jennings
Andrew J. Link
Andrew J.
Link
Assessing the Components of the eIF3 Complex and their Phosphorylation Status
American Chemical Society
2011
Phosphorylation StatusThe eukaryotic initiation factor 3
eIF 3 phosphopeptides
yeast eIF 3
translation initiation machinery
phosphorylation
casein kinase 2
Prt 1 phosphopeptides
Nip 1
Nip 1 serines
eIF 3 complexes
Tif 5 phosphopeptide
IMAC
tandem mass spectrometry
CK 2 phophorylates Nip 1
eIF 3 Complex
Nip 1 peptide
phosphorylated
LC
CK 2 consensus sequences
metal affinity chromatography
2011-04-01 00:00:00
Journal contribution
https://acs.figshare.com/articles/journal_contribution/Assessing_the_Components_of_the_eIF3_Complex_and_their_Phosphorylation_Status/2674033
The eukaryotic initiation factor 3 (eIF3) is an essential, highly conserved multiprotein complex that is a key component in the recruitment and assembly of the translation initiation machinery. To better understand the molecular function of eIF3, we examined its composition and phosphorylation status in <i>Saccharomyces cerevisiae</i>. The yeast eIF3 complex contains five core components: Rpg1, Nip1, Prt1, Tif34, and Tif35. 2-D LC−MS/MS analysis of affinity purified eIF3 complexes showed that several other initiation factors (Fun12, Tif5, Sui3, Pab1, Hcr1, and Sui1) and the casein kinase 2 complex (CK2) copurify. <i>In Vivo</i> metabolic labeling of proteins with <sup>32</sup>P revealed that Nip1 is phosphorylated. Using 2-D LC−MS/MS analysis of eIF3 complexes, we identified Prt1 phosphopeptides indicating phosphorylation at S22 and T707 and a Tif5 phosphopeptide with phosphorylation at T191. Additionally, we used immobilized metal affinity chromatography (IMAC) to enrich for eIF3 phosphopeptides and tandem mass spectrometry to identify phosphorylated residues. We found that three CK2 consensus sequences in Nip1 are phosphorylated: S98, S99, and S103. Using <i>in vitro</i> kinase assays, we showed that CK2 phophorylates Nip1 and that a synthetic Nip1 peptide containing S98, S99, and S103 competitively inhibits the reaction. Replacement of these three Nip1 serines with alanines causes a slow growth phenotype.