Identification of Domains and Residues within the ɛ Subunit of Eukaryotic Translation Initiation Factor 2B (eIF2Bɛ) Required for Guanine Nucleotide Exchange Reveals a Novel Activation Function Promoted by eIF2B Complex Formation

Abstract

Eukaryotic translation initiation factor 2B (eIF2B) is the guanine nucleotide exchange factor for protein synthesis initiation factor 2 (eIF2). Composed of five subunits, it converts eIF2 from a GDP-bound form to the active eIF2-GTP complex. This is a regulatory step of translation initiation. In vitro, eIF2B catalytic function can be provided by the largest (epsilon) subunit alone (eIF2B). This activity is stimulated by complex formation with the other eIF2B subunits. We have analyzed the roles of different regions of eIF2B in catalysis, in eIF2B complex formation, and in binding to eIF2 by characterizing mutations in the Saccharomyces cerevisiae gene encoding eIF2B (GCD6) that impair the essential function of eIF2B. Our analysis of nonsense mutations indicates that the C terminus of eIF2B (residues 518 to 712) is required for both catalytic activity and interaction with eIF2. In addition, missense mutations within this region impair the catalytic activity of eIF2B without affecting its ability to bind eIF2. Internal, in-frame deletions within the N-terminal half of eIF2B disrupt eIF2B complex formation without affecting the nucleotide exchange activity of eIF2B alone. Finally, missense mutations identified within this region do not affect the catalytic activity of eIF2B alone or its interactions with the other eIF2B subunits or with eIF2. Instead, these missense mutations act indirectly by impairing the enhancement of the rate of nucleotide exchange that results from complex formation between eIF2B and the other eIF2B subunits. This suggests that the N-terminal region of eIF2B is an activation domain that responds to eIF2B complex formation.Eukaryotic translation initiation factor 2B (eIF2B) is a guanine nucleotide exchange factor (GEF) that converts its substrate, eIF2, from an inactive eIF2-GDP binary complex to eIF2-GTP. This active complex binds charged initiator tRNA Met (Met-tRNA i Met ) forming a ternary complex that interacts with eIF3 and the 40S ribosomal subunit. Following addition of mRNA, associated initiation factors, and the 60S ribosomal subunit, the G-protein cycle is completed by hydrolysis of eIF2-bound GTP and the release of eIF2-GDP from the ribosome (reviewed in references 14, 31, and 42). Thus, the functions of eIF2 and eIF2B are believed to be similar to those of the small GTPases and exchange factors, respectively, of the RAS superfamily (4). Recent three-dimensional structure determinations have demonstrated that while the nucleotide binding domains of the G proteins are very similar, GEF structures differ markedly from one another, each employing different amino acid motifs to drive the release of GDP (8, 41). eIF2 and eIF2B are complex proteins of three (␣ to ␥) and five (␣ to ε) nonidentical subunits, respectively. The subunit complexity of eIF2B reflects, at least in part, its novel mechanism of regulation. Four protein kinases, called PKR, HCR (HRI), PERK (PEK), and GCN2, specifically phosphorylate the seryl residue at position 51 of the ␣ subunit of eIF2 (eIF2␣) under different ...

An eIF5/eIF2 complex antagonizes guanine nucleotide exchange by eIF2B during translation initiation

“…These alleles used in Figure 5D cause significant impairment of eIF2B GEF activity and impart a slow-growth phenotype and 3AT resistance (Gomez and Pavitt, 2000) that was enhanced here by hc eIF5. Interestingly, hc eIF5 did not alter the growth rate of slow-growing strains analyzed bearing mutations in eIF2Bb (gcd2), g (gcd1), and d (gcd7) genes ( Figure 5E).…”

Identification of Residues That Underpin Interactions within the Eukaryotic Initiation Factor (eIF2) 2B Complex

“…The Asn-Phe Motif in eIF2B⑀ is Necessary for Activity in Human eIF2B-Gomez and Pavitt (5) showed that an Asn-Phe (NF) motif in yeast eIF2B⑀ (residues 249/250 in yeast, 263/264 in human) was important for facilitating the enhancement of eIF2B activity that results from the formation of holocomplexes. This motif is conserved in all available eIF2B⑀ sequences.…”

“…eIF2B⑀ contains within its C-terminal region the catalytic domain (approximately residues 527-726 in human eIF2B⑀), which mediates GDP/GTP exchange on eIF2 (5,6) (Fig. 1A).…”

Structure of the Catalytic Fragment of Translation Initiation Factor 2B and Identification of a Critically Important Catalytic Residue

“…This region encompasses residues 518 -583 of the yeast protein and is well conserved in all eIF2B⑀ proteins (12). In addition, mutation of either Thr 552 to Ile (T552I) or Ser 576 to Asn (S576N) directly impairs catalytic function without detectably impairing binding to eIF2 (14). Thus, although the eIF2B⑀ C terminus is only a small fragment of the entire ϳ275-295-kDa eIF2B complex, it contains the major functional regions required for nucleotide exchange and provides a structural model for the related C termini of eIF4G and eIF5.…”