Identification of domains and residues within the epsilon subunit of eukaryotic translation initiation factor 2B (eIF2B epsilon) required for guaninenucleotide exchange reveals a novel activation function promoted by eIF2B complex formation

Eukaryotic translation initiation factor 2B (eIF2B) is the guanine nucleoti de exchange factor for protein synthesis initiation factor 2 (eIF2). Compos ed of five subunits, it converts eIF2 from a GDP-bound form to the active e IF2-GTP complex. This is a regulatory step of translation initiation. In vi tro, eIF2B catalytic function can be provided by the largest (epsilon) subu nit alone (eIF2B epsilon). This activity is stimulated by complex formation with the other eIF2B subunits. We have analyzed the roles of different reg ions of eIF2B epsilon in catalysis, in eIF2B complex formation, and in bind ing to eIF2 by characterizing mutations in the Saccharomyces cerevisiae gen e encoding eIF2B epsilon (GCD6) that impair the essential function of eIF2B . Our analysis of nonsense mutations indicates that the C terminus of eIF2B epsilon (residues 518 to 712) is required for both catalytic activity and interaction with eIF2. In addition, missense mutations within this region i mpair the catalytic activity of eIF2B epsilon without affecting its ability to bind eIF2. Internal, in-frame deletions within the N-terminal half of e IF2B epsilon disrupt eIF2B complex formation without affecting the nucleoti de exchange activity of eIF2B epsilon alone. Finally, missense mutations id entified within this region do not affect the catalytic activity of eIF2B e psilon alone or its interactions with the other eIF2B subunits or with eIF2 . Instead, these missense mutations act indirectly by impairing the enhance ment of the rate of nucleotide exchange that results from complex formation between eIF2B epsilon and the other eIF2B subunits. This suggests that the N-terminal region of eIF2B epsilon is an activation domain that responds t o eIF2B complex formation.