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pubmed:abstractText |
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(?P)]. While eIF2B contains five subunits, the ?/Gcd6 subunit is sufficient for GEF activity in vitro. The ?/Gcd2 and ?/Gcd7 subunits function with ?/Gcn3 in the eIF2B regulatory subcomplex that mediates tight, inhibitory binding of eIF2(?P)-GDP, but the essential functions of ?/Gcd2 and ?/Gcd7 are not well understood. We show that the depletion of wild-type ?/Gcd7, three lethal ?/Gcd7 amino acid substitutions, and a synthetically lethal combination of substitutions in ?/Gcd7 and eIF2? all impair eIF2 binding to eIF2B without reducing ?/Gcd6 abundance in the native eIF2B-eIF2 holocomplex. Additionally, ?/Gcd7 mutations that impair eIF2B function display extensive allele-specific interactions with mutations in the S1 domain of eIF2? (harboring the phosphorylation site), which binds to eIF2B directly. Consistent with this, ?/Gcd7 can overcome the toxicity of eIF2(?P) and rescue native eIF2B function when overexpressed with ?/Gcd2 or ?/Gcd1. In aggregate, these findings provide compelling evidence that ?/Gcd7 is crucial for binding of substrate by eIF2B in vivo, beyond its dispensable regulatory role in the inhibition of eIF2B by eIF (?P).
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