EIF2 INDEPENDENTLY BINDS 2 DISTINCT EIF2B SUBCOMPLEXES THAT CATALYZE AND REGULATE GUANINE-NUCLEOTIDE EXCHANGE

eIF2B is a heteropentameric guanine-nucleotide exchange factor essenti al for protein synthesis initiation in eukaryotes. Its activity is inh ibited in response to starvation or stress by phosphorylation of the a lpha subunit of its substrate, translation initiation factor eIF2, res ulting in reduced rates of translation and cell growth. We have used a n in vitro nucleotide-exchange assay to show that wild-type yeast eIF2 B is inhibited by phosphorylated eIF2 [eIF2(alpha P)] and to character ize eIF2B regulatory mutations that render translation initiation inse nsitive to eIF2 phosphorylation in vivo. Unlike wild-type eIF2B, eIF2B complexes with mutated GCN3 or GCD7 subunits efficiently catalyzed GD P exchange using eIF2(alpha P) as a substrate. Using an affinity-bindi ng assay, we show that an eIF2B subcomplex of the GCN3, GCD7, and GCD2 subunits binds to eIF2 and has a higher affinity for eIF2(alpha P), b ut it lacks nucleotide-exchange activity. In contrast, the GCD1 and GC D6 subunits form an eIF2B subcomplex that binds equally to eIF2 and eI F2(alpha P). Remarkably, this second subcomplex has higher nucleotide- exchange activity than wild-type eIF2B that is not inhibited by eIF2(a lpha P). The identification of regulatory and catalytic eIF2B subcompl exes leads us to propose that binding of eIF2(alpha P) to the regulato ry subcomplex prevents a productive interaction with the catalytic sub complex, thereby inhibiting nucleotide exchange.