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Structural Characterization and Identification of Post‐Translational Modifications of Human Eukaryotic Initiation Factor 3 (eIF3) by FTICR Mass Spectrometry
Author(s) -
Damoc Eugen,
Fraser Christopher,
Doudna Jennifer,
Leary Julie
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a528-c
Subject(s) - eukaryotic small ribosomal subunit , protein subunit , eukaryotic translation , initiation factor , mass spectrometry , biology , chemistry , biochemistry , eukaryotic initiation factor , ribosomal protein , translation (biology) , ribosome , messenger rna , chromatography , rna , gene
Mammalian eIF3 is the largest initiation factor with a mass of about 600 kDa and contains at least 12 non‐identical subunits ranging in mass from 25 to 167 kDa. eIF3 is almost universally required for both cap‐dependent and cap‐independent initiation, and plays a central role in binding of initiator methionyl‐tRNA and mRNA to the 40S ribosomal subunit to form the 40S initiation complex. The molecular mechanisms by which eIF3 exert these functions are poorly understood. The overall goal of this project is to understand how the mammalian eIF3 contributes to the initiation mechanism, and how the eIF3 controls ribosomal subunit association. The present study is focused on elucidating the structure/function of eIF3 and how eIF3 activity may be modulated by covalent modifications of its subunits. To answer these questions, FTICR mass spectrometry has been used as a powerful tool for structure analysis of biopolymers which simultaneously can provide high mass resolution (>10 6 ), mass‐determination accuracy (<1 ppm), sensitivity and MS n capabilities. By nanoLC‐MS/MS analysis of the eIF3 tryptic digest using a hybrid linear ion trap‐FTICR mass spectrometer (Thermo Electron, San Jose, CA) we found that the eIF3 protein complex consists of at least 13 non‐identical subunits, the 13 th subunit being identified as GA17, a 42.5 kDa protein that contains a PCI domain like those found in eIF3a, eIF3c, and eIF3e subunits. Furthermore, five phosphorylation sites on the eIF3b, eIF3c, eIF3g and eIF3j subunits and several other PTMs (e.g. loss of N‐terminal methionine and/or N‐terminal acetylation) were identified.