The β/Gcd7 Subunit of Eukaryotic Translation Initiation Factor 2B (eIF2B), a Guanine Nucleotide Exchange Factor, Is Crucial for Binding eIF2 In Vivo

Eukaryotic translation initiation factor 2B (eIF2B) is the guanine nucleotide exchange factor (GEF) for eukaryotic translation initiation factor 2, which stimulates formation of the eIF2-GTP-Met-tRNA(i)(Met) ternary complex (TC) in a manner inhibited by phosphorylated eIF2 [eIF2(alpha P)]. While eIF2B contains five subunits, the epsilon/Gcd6 subunit is sufficient for GEF activity in vitro. The delta/Gcd2 and beta/Gcd7 subunits function with alpha/Gcn3 in the eIF2B regulatory subcomplex that mediates tight, inhibitory binding of eIF2(alpha P)-GDP, but the essential functions of delta/Gcd2 and beta/Gcd7 are not well understood. We show that the depletion of wild-type beta/Gcd7, three lethal beta/Gcd7 amino acid substitutions, and a synthetically lethal combination of substitutions in beta/Gcd7 and eIF2 alpha all impair eIF2 binding to eIF2B without reducing epsilon/Gcd6 abundance in the native eIF2B-eIF2 holo-complex. Additionally, beta/Gcd7 mutations that impair eIF2B function display extensive allele-specific interactions with mutations in the S1 domain of eIF2 alpha (harboring the phosphorylation site), which binds to eIF2B directly. Consistent with this, beta/Gcd7 can overcome the toxicity of eIF2(alpha P) and rescue native eIF2B function when overexpressed with delta/Gcd2 or gamma/Gcd1. In aggregate, these findings provide compelling evidence that beta/Gcd7 is crucial for binding of substrate by eIF2B in vivo, beyond its dispensable regulatory role in the inhibition of eIF2B by eIF (alpha P).