Tight binding of the phosphorylated alpha subunit of initiation factor 2 (eIF2 alpha) to the regulatory subunits of guanine nucleotide exchange factor eIF2B is required for inhibition of translation initiation

Translation initiation factor 2 (eIF2) is a heterotrimeric protein that tra nsfers methionyl-initiator tRNA(Met) to the small ribosomal subunit in a te rnary complex with GTP. The eIF2 phosphorylated on serine 51 of its alpha s ubunit [eIF2(alphaP)] acts as competitive inhibitor of its guanine nucleoti de exchange factor, eIF2B, impairing formation of the ternary complex and t hereby inhibiting translation initiation. eIF2B is comprised of catalytic a nd regulatory subcomplexes harboring independent eIF2 binding sites; howeve r, it was unknown whether the ct subunit of eIF2 directly contacts any eIF2 B subunits or whether this interaction is modulated by phosphorylation. We found that recombinant eIF2 alpha (glutathione S-transferase [GST]-SUI2) bo und to the eIF2B reguiatorg subcomplex in vitro, in a manner stimulated by Ser-51 phosphorylation. Genetic data suggest that this direct interaction a lso occurred in vivo, allowing overexpressed SUI2 to compete with eIF2(alph aP) holoprotein for binding to the eIF2B regulatory subcomplex. Mutations i n SUI2 and in the eIF2B regulatory subunit GCD7 that eliminated inhibition of eIF2B by eIF2(alphaP) also impaired binding of phosphorylated GST-SUI2 t o the eIF2B regulatory subunits. These findings provide strong evidence tha t tight binding of phosphorylated SUI2 to the eIF2B regulatory subcomplex i s crucial for the inhibition of eIF2B and attendant downregulation of prote in synthesis exerted by eIF2(alphaP). We propose that this regulatory inter action prevents association of the eIF2B catalytic subcomplex with the beta and gamma subunits of eIF2 in the manner required for GDP-CTP exchange.