Characterization of the mammalian initiation factor eIF2B complex as a GDPdissociation stimulator protein

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 (E) disp lays catalytic activity in the absence of the others. The catalytic mechani sm of eIF2B and the functions of the other subunits remain to be clarified. Here we show that, when present at similar concentrations to eIF2, mammali an eIF2B can mediate release of eIF2-bound GDP even in the absence of free nucleotide, indicating that it acts as a GDP dissociation stimulator protei n. Consistent with this, addition of GDP to purified eIF2(.)eIF2B complexes causes them to dissociate. The alternative sequential mechanism would requ ire that eIF2B epsilon itself bind GTP. However, we show that it is the bet a -subunit of eIF2B that interacts with GTP, This indicates that binding of GTP to eIF2B is not an essential element of its mechanism. eIF2B preparati ons that lack the alpha -subunit display reduced activity compared with the holocomplex. Supplementation of such preparations with recombinant eIF2B a lpha markedly enhances activity, indicating that eIF2B alpha is required fo r full activity of mammalian eIF2B.