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Title page for ETD etd-12012011-180347

Type of Document Dissertation
Author Farley, Adam Richard
URN etd-12012011-180347
Title Assessing the Components of the eIF3 Complex and Their Phosphorylation Status
Degree PhD
Department Biochemistry
Advisory Committee
Advisor Name Title
Andrew Link Committee Chair
Bruce Carter Committee Member
David Cortez Committee Member
Katherine Friedman Committee Member
Richard Caprioli Committee Member
  • initiation
  • translation
  • mass spectrometry
  • proteomics
  • posttranslational modifications
Date of Defense 2010-08-30
Availability unrestricted



Dissertation under the direction of Professor Andrew J. Link

The eukaryotic initiation factor 3 (eIF3) is a highly conserved multi-protein complex that is an essential component in the recruitment and assembly of the translation initiation machinery. To better understand the molecular function of eIF3, I examined its composition and phosphorylation status in Saccharomyces cerevisiae. The yeast eIF3 complex contains five core components: Rpg1, Nip1, Prt1, Tif34, and Tif35. I hypothesized that for eIF3, there are unexpected and unidentified eIF3 protein-protein interactions and protein phosphorylations that regulate its function and activity in the process of translation initiation. 2-D LC-MS/MS mass spectrometry 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) co-purify with the core complex. These novel identifications expand the knowledge base for what is known about the function of eIF3 in yeast and expands its role to additional steps in protein synthesis.

In vivo metabolic labeling of proteins with 32P revealed that Nip1 is phosphorylated. Using 2-D LC-MS/MS analysis of eIF3 complexes, I identified Prt1 phosphopeptides indicating phosphorylation at S22 and T707 and a Tif5 phosphopeptide at T191. Additionally, I used immobilized metal affinity chromatography (IMAC) to enrich for eIF3 phosphopeptides and tandem mass spectrometry to identify phosphorylated residues. I found that three CK2 consensus sequences in Nip1 are phosphorylated: S98, S99, and S103. Using in vitro kinase assays, I showed that CK2 phosphorylates 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. Quantitative growth assay studies revealed that mutant strains lacking the phosphorylation have a doubling time that is increased by 33% relative to a control yeast strain. I propose that the observed phosphorylations stabilize interactions with the Prt1 protein as this region of Nip1 is suspected to be involved in Nip1-Prt1 contacts.

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