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Submitted on October 16, 2006
Revised on February 16, 2007
Accepted on February 22, 2007

Structural characterization of the human eukaryotic initiation factor 3 protein complex by mass spectrometry

Eugen Damoc, Christopher S Fraser, Min Zhou, Hortense Videler, Greg L Mayeur, John WB Hershey, Jennifer A Doudna, Carol V Robinson, and Julie A Leary

Chemistry and Molecular Cell Biology, UC Davis, Davis

Corresponding Author: nedamoc{at}ucdavis.edu

Protein synthesis in mammalian cells requires initiation factor eIF3, a ~800 kDa protein complex that plays a central role in binding of initiator methionyl-tRNA and mRNA to the 40S ribosomal subunit to form the 48S initiation complex. The eIF3 complex also prevents premature association of the 40S and 60S ribosomal subunits and interacts with other initiation factors involved in start codon selection. The molecular mechanisms by which eIF3 exerts these functions are poorly understood. Since its initial characterization in the 1970s, the exact size, composition and post-translational modifications of mammalian eIF3 have not been rigorously determined. Two powerful mass spectrometric approaches have been used in the present study to determine post-translational modifications that may regulate its activity during the translation initiation process and to characterize the molecular structure of the human eIF3 protein complex purified from HeLa cells. In the first approach, the bottom-up analysis of eIF3 allowed for the identification of a total of 13 protein components (eIF3a-m) with a sequence coverage of approximately 79%. Furthermore, 29 phosphorylation sites and several other post-translational modifications were unambiguously identified within the eIF3 complex. The second mass spectrometric approach, involving analysis of intact eIF3, allowed the detection of a complex with each of the 13 subunits present in stoichiometric amounts. Tandem mass spectrometry indicated that four eIF3 subunits (h, i, k, and m) were found to be most easily dissociated, and therefore likely to be on the periphery of the complex. It is noteworthy that none of these four subunits was found to be phosphorylated. These data raise interesting questions about the function of phosphorylation as it relates to the core subunits of the complex.


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