eIF2B promotes eIF5 dissociation from eIF2•GDP to facilitate guanine nucleotide exchange for translation initiation

Abstract

Protein synthesis factor eIF2 delivers initiator tRNA to the ribosome. Two proteins regulate its G-protein cycle: eIF5 has both GTPase-accelerating protein (GAP) and GDP dissociation inhibitor (GDI) functions, and eIF2B is the guanine nucleotide exchange factor (GEF). In this study, we used protein-protein interaction and nucleotide exchange assays to monitor the kinetics of eIF2 release from the eIF2•GDP/eIF5 GDI complex and determine the effect of eIF2B on this release. We demonstrate that eIF2B has a second activity as a GDI displacement factor (GDF) that can recruit eIF2 from the eIF2•GDP/eIF5 GDI complex prior to GEF action. We found that GDF function is dependent on the eIF2Be and eIF2Bg subunits and identified a novel eIF2-eIF2Bg interaction. Furthermore, GDF and GEF activities are shown to be independent. First, eIF2B GDF is insensitive to eIF2a phosphorylation, unlike GEF. Second, we found that eIF2Bg mutations known to disrupt GCN4 translational control significantly impair GDF activity but not GEF function. Our data therefore define an additional step in the protein synthesis initiation pathway that is important for its proper control. We propose a new model to place eIF2B GDF function in the context of efficient eIF2 recycling and its regulation by eIF2 phosphorylation.

Mechanism and Regulation of Protein Synthesis in Saccharomyces cerevisiae

“…eIF2B displaces eIF5 from eIF2-GDP: Because the CTD of eIF5 and the GEF domain of eIF2Be (see Figure 3, C and D) share a common HEAT repeat structure (Boesen et al 2004;Bieniossek et al 2006) necessary for binding to eIF2b (Asano et al 1999), binding of each factor to eIF2 is mutually exclusive (Jennings and Pavitt 2010b). eIF5 must dissociate from the stable eIF2-GDP/eIF5 complex to enable eIF2B GEF action; however, eIF2B itself can displace eIF5 (Jennings et al 2013). In common with other G protein regulator nomenclature, eIF2B is a GDI displacement factor (GDF) ( Figure 7A).…”

“…Importantly, missense mutations in eIF2Bg (gcd1-G12V and gcd1-L480Q) specifically impair GDF function (release of eIF5 from eIF2-GDP) and only affect nucleotide exchange function when eIF2-GDP is prebound by eIF5 (Jennings et al 2013). As the gcd1 mutations confer slow-growth and Gcd 2 phenotypes, the GDF activity of eIF2B is critical in vivo.…”

“…activity, suggesting that these subunits cooperate in GDF function (Jennings et al 2013). Importantly, missense mutations in eIF2Bg (gcd1-G12V and gcd1-L480Q) specifically impair GDF function (release of eIF5 from eIF2-GDP) and only affect nucleotide exchange function when eIF2-GDP is prebound by eIF5 (Jennings et al 2013).…”

“…Regulation of eIF2B activity: eIF2a is phosphorylated at Ser51 by Gcn2 causing inhibition of eIF2B GEF activity (Pavitt et al 1998;Jennings et al 2013). Genetic and biochemical experiments implicate the homologous eIF2Ba, b, and d subunits in mediating translational control.…”

“…eIF2Be alone is $5-to 10-fold less effective than intact eIF2B at promoting nucleotide exchange, while eIF2Bge subcomplexes have the same activity as intact eIF2B (Gomez and Pavitt 2000;Jennings et al 2013). The GEF domain comprises the C-terminal $200 residues (eIF2Be 518-712 ) (Gomez and Pavitt 2000;Gomez et al 2002) and adopts a HEAT repeats structure with conserved residues important for GEF activity on one face (Boesen et al 2004).…”