REGULATION OF GUANINE-NUCLEOTIDE EXCHANGE THROUGH PHOSPHORYLATION OF EUKARYOTIC INITIATION-FACTOR EIF2-ALPHA

The guanine nucleotide exchange activity of eIF2B plays a key regulato ry role in the translation initiation phase of protein synthesis. The activity is markedly inhibited when the substrate, i,e, eIF2, is phosp horylated on Ser(51) of its alpha-subunit. Genetic studies in yeast im plicate the alpha-, beta-, and delta-subunits of eLF2B in mediating th e inhibition by substrate phosphorylation. However, the mechanism invo lved in the inhibition has not been defined biochemically. In the pres ent study, we have coexpressed the five subunits of rat eIF2B in Sf9 c ells using the baculovirus system and have purified the recombinant ho loprotein to >90% homogeneity, We have also expressed and purified a f our-subunit eIF2B complex lacking the alpha-subunit, Both the five-and four-subunit forms of eIF2B exhibit similar rates of guanine nucleoti de exchange activity using unphosphorylated eIF2 as substrate. The fiv e-subunit form is inhibited by preincubation with phosphorylated eIF2 (eIFS(alpha P)) and exhibits little exchange activity when eIF2(alpha P) is used as substrate. In contrast, eIF2B lacking the cu-subunit is insensitive to inhibition by eIF2(alpha P) and is able to exchange gua nine nucleotide using eIF2(alpha P) as substrate at a faster rate comp ared with five-subunit eIF2B, Finally, a double point mutation in the S-subunit of eIF2B has been identified that results in insensitivity t o inhibition by eIF2(alpha P) and exhibits little exchange activity wh en eIFS(alpha P) is used as substrate. The results provide the first d irect biochemical evidence that the alpha- and delta-subunits of eIF2B are involved in mediating the effect of substrate phosphorylation.