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 eIF2Bε and eIF2Bγ subunits and identified a novel eIF2-eIF2Bγ interaction. Furthermore, GDF and GEF activities are shown to be independent. First, eIF2B GDF is insensitive to eIF2α phosphorylation, unlike GEF. Second, we found that eIF2Bγ 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.

Keywords: G-protein regulators; GDF; GDI; GEF; protein synthesis initiation.

Figure 1.

eIF5 GDI mutants restore growth to the lethal eIF2B mutant gcd6-N249K. Strains derived from GP5934 (gcd6Δ gcn2Δ tif5Δ) expressing either wild-type (WT) or mutant tif5 TRP1 plasmids as the sole source of eIF5 and a GCD6 URA3 plasmid (encoding eIF2Bɛ) were transformed with LEU2 plasmids expressing either wild-type or mutant gcd6, as indicated in the figure. Tenfold serial dilutions of liquid cultures were then plated on SD or SD + 5-fluoro-orotic acid (5-FOA) to inhibit growth of cells that had not lost the covering GCD6 URA3 plasmid, thereby making the indicated gcd6 allele the sole source of eIF2Bɛ.

Figure 2.

eIF2B acts as a GDF to effectively displace eIF2 from eIF5. (A) Scheme of GDF assay. (B,C) Assay assessing remaining complex formation between GST-eIF5 and eIF2 in the presence of increasing concentrations of either eIF2B (B) or eIF5-Flag (C). Typical data are shown (n > 3). GST-bound proteins were detected with the indicated antibodies. Input samples represent dilutions of proteins used in the assay to assist Western quantification (1.7, 0.83, and 0.33 pmol of eIF2 and eIF2B and 7, 2.9, and 0.7 pmol of eIF5). (D,E) Signal intensity quantification (Adobe Photoshop) of bound eIF2 from B, C, and Supplemental Figure S2E. (F) Mean IC₅₀ ± the standard deviation (n > 3) for each protein tested.

Figure 3.

eIF2B GDF activity is required for efficient GEF activity. Rate of [³H]GDP dissociation (K_off) from eIF2 was monitored under increasing concentrations of the eIF2B complex (A), eIF2Bγɛ (B), or eIF2Bɛ (C) with GST-eIF5 prebound to eIF2 (gray) or without (black). K_1/2 and K_max values are shown in the inset tables, derived from fitting the data to y = [(K_max × x)/(K_1/2 + x)] + c.

Figure 4.

eIF2B GDF is independent of eIF2α phosphorylation. (A) The GDF assay depicted in Figure 2A using phosphorylated eIF2, GST-eIF5, and eIF2B. Inputs represent 1.7, 0.83, and 0.33 pmol eIF2 and eIF2B and 7, 2.9, and 0.7 pmol of eIF5. (B) Quantification of A (open symbols). Unphosphorylated eIF2 quantification is shown for comparison (filled symbols), as shown in Figure 2D. (C) The top panel shows the rate of [³H]GDP dissociation (K_off) from eIF2αP (broken lines) or eIF2 (solid lines) with increasing concentrations of eIF2B either with (gray symbols) or without GST-eIF5 (black symbols). The bottom panel shows the legend with quantified K_1/2 and K_max values.

Figure 5.

eIF2Bγ mutants have a GDF activity defect. (A,B) GEF and coupled GDI–GDF–GEF assays of eIF2Bγ mutants L480Q (A) and G12V (B) are shown, as per Figure 3. (C,D) GDF assay of eIF2 displacement from eIF5 for the same mutants. (E,F) Quantification of GDF assays; mean IC₅₀ ± standard deviation (n = 3).

Figure 6.

Revised model for eIF2 ternary complex formation and its inhibition by phosphorylation of eIF2. Model showing proposed paths for eIF2 (2, two-lobed ovals), eIF5 (5, open circles), and eIF2B (2B, open five-pointed stars) integrating eIF5 GDI and eIF2B GDF and GEF activities in the recycling of eIF2 (part I), its inhibition by eIF2αP (part II), and the affect of eIF2Bγ GDF mutations (part III). Dashed gray arrows represent steps that limit eIF2 recycling.