The heterotrimeric eukaryotic initiation factor 2 (eIF2) plays a critical role in the mechanics and regulation of protein synthesis. Unlike yeast and archaeal eIF2, the purified baculovirus-expressed recombinant human eIF2 subunits used in these studies reveal that the alpha- and beta-subunits interact with each other. Consistent with this observation, the beta-subunit specifically interacts with the purified eIF2B in ELISA studies and this interaction is enhanced when wt eIF2 alpha in the recombinant trimeric complex is phosphorylated or replaced by a mutant phosphomimetic eIF2 alpha (S51D). These findings together with other observations raise the possibility that the beta-subunit plays a key role in the regulation and function of mammalian eIF2 complex. PERK, an eIF2 alpha kinase, is found to interact with wt and mutants of eIF2a in which the serine 51 or 48 residue is replaced by alanine or aspartic acid thereby suggesting that the phosphorylation site in the substrate is not important for interaction. Fluorescence spectroscopic and fluorescence resonance energy transfer analyses reveal that the energy transfer occurs from PERK to eIF2 alpha. The dissociation constant of alpha-subunit-PERK complex (K-d alpha-subunit) is 0.74 mu M and the interaction is stoichiometric. (c) 2005 Elsevier Inc. All rights reserved.