title: Characterization of the mammalian initiation factor eIF2B complex as a GDP dissociation stimulator protein.
abstract: Initiation factor eIF2B mediates a key regulatory step in the initiation of mRNA translation, i.e. the regeneration of active eIF2.GTP complexes. It is composed of five subunits, alpha-epsilon. The largest of these (epsilon) displays catalytic activity in the absence of the others. The catalytic mechanism of eIF2B and the functions of the other subunits remain to be clarified. Here we show that, when present at similar concentrations to eIF2, mammalian eIF2B can mediate release of eIF2-bound GDP even in the absence of free nucleotide, indicating that it acts as a GDP dissociation stimulator protein. Consistent with this, addition of GDP to purified eIF2.eIF2B complexes causes them to dissociate. The alternative sequential mechanism would require that eIF2Bepsilon itself bind GTP. However, we show that it is the beta-subunit of eIF2B that interacts with GTP. This indicates that binding of GTP to eIF2B is not an essential element of its mechanism. eIF2B preparations that lack the alpha-subunit display reduced activity compared with the holocomplex. Supplementation of such preparations with recombinant eIF2Balpha markedly enhances activity, indicating that eIF2Balpha is required for full activity of mammalian eIF2B.
authors.affiliation: School of Life Sciences, Medical Sciences Institute/Wellcome Trust Biocentre Complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, United Kingdom.
authors.forename: D D
authors.initials: DD
authors.lastname: Williams
authors.forename: N T
authors.initials: NT
authors.lastname: Price
authors.forename: A J
authors.initials: AJ
authors.lastname: Loughlin
authors.forename: C G
authors.initials: CG
authors.lastname: Proud
mesh_terms: D001665:Binding Sites; D020717:Eukaryotic Initiation Factor-2B / Q000737:chemistry / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006801:Humans; D008956:Models, Chemical; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship
chemical_list: D020717:Eukaryotic Initiation Factor-2B; D020662:Guanine Nucleotide Exchange Factors; D006153:Guanosine Diphosphate