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. 2016 Nov 16;44(20):9698-9709.
doi: 10.1093/nar/gkw657. Epub 2016 Jul 25.

eIF2β is critical for eIF5-mediated GDP-dissociation inhibitor activity and translational control

Martin D Jennings  1 , Christopher J Kershaw  1 , Christopher White  1 , Danielle Hoyle  1 , Jonathan P Richardson  1 , Joseph L Costello  1 , Ian J Donaldson  1 , Yu Zhou  1 , Graham D Pavitt  2
Affiliations

eIF2β is critical for eIF5-mediated GDP-dissociation inhibitor activity and translational control

Martin D Jennings et al. Nucleic Acids Res. .

Abstract

In protein synthesis translation factor eIF2 binds initiator tRNA to ribosomes and facilitates start codon selection. eIF2 GDP/GTP status is regulated by eIF5 (GAP and GDI functions) and eIF2B (GEF and GDF activities), while eIF2α phosphorylation in response to diverse signals is a major point of translational control. Here we characterize a growth suppressor mutation in eIF2β that prevents eIF5 GDI and alters cellular responses to reduced eIF2B activity, including control of GCN4 translation. By monitoring the binding of fluorescent nucleotides and initiator tRNA to purified eIF2 we show that the eIF2β mutation does not affect intrinsic eIF2 affinities for these ligands, neither does it interfere with eIF2 binding to 43S pre-initiation complex components. Instead we show that the eIF2β mutation prevents eIF5 GDI stabilizing nucleotide binding to eIF2, thereby altering the off-rate of GDP from eIF2•GDP/eIF5 complexes. This enables cells to grow with reduced eIF2B GEF activity but impairs activation of GCN4 targets in response to amino acid starvation. These findings provide support for the importance of eIF5 GDI activity in vivo and demonstrate that eIF2β acts in concert with eIF5 to prevent premature release of GDP from eIF2γ and thereby ensure tight control of protein synthesis initiation.

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Figures

Figure 1.
Figure 1.
An extragenic spontaneous slow-growth suppressor of eIF2Bϵ mutations. (A) sif285-1 suppresses the slow-growth of the gcd6-F250L mutation. Growth of strains GP3771-3774 on YPD. Relevant genotypes and doubling times from liquid cultures are shown. (B) Total protein synthesis rates (35S-met incorporation) ± SE (n = 3). Student's t-test indicates only F250L differs significantly from the other three strains (P < 0.05). (C and D) Polysome profile analysis for strains shown in (A) grown in YPD or following 10 min incubation in YP lacking glucose (D). Traces show A254 recorded by continuous fractionation of 15–50% sucrose gradients. (E) Replica printing of the strains shown in panel A on minimal SD ±3AT. (F) Growth on SD ±3AT media and β-galactosidase assay of HIS4-lacZ for strains shown in (A), but following transformation with a GCN2 plasmid (pAV1198). Right box summarizes the signalling pathway leading to derepression of HIS4 expression by amino acid starvation (+3AT).
Figure 2.
Figure 2.
sif285-1 is the sui3-E189K mutation in eIF2β. (A) Sanger sequencing confirms sif285-1 cells (GP3773) contain sui3-G565A mutation. (B) Growth of strains GP3771-4 transformed with plasmid vector (rows 1–4), SUI3 low copy (rows 5–8) or SUI3 high copy (rows 9–12) on SD dropout medium. (C) Growth of WT (GP7124: Flag-SUI3) and E189K (GP7125 Flag-SUI3-E189K) strains transformed with GCN2 (pAV1198) or a vector control (pAV195) on SC-histidine ±3AT media and HIS4-LacZ assays. (D) Growth of gcd6Δ sui3Δ double deletion strains (GP7216-7219) bearing the indicated alleles of each gene on YPD and SC-histidine +3AT media.
Figure 3.
Figure 3.
E189 is conserved in eIF2β. Please view the online version for a colour version of this figure. (A) Multiple sequence alignment (Clustal X) of eIF2β proteins from diverse eukaryotes showing homology around residue E189. Red highlight, identical, blue and yellow show similarity. Accession numbers for sequences are given in the ‘Materials and Methods’ section. (B) Cartoon with relative position of mutated E189 residue in Sui3/eIF2β protein, as well as the S2264Y (SUI3-2) mutation and conserved N-terminal lysine boxes described in previous studies and referred to in the text. The region with determined molecular structure is shown (blue). (C) Model of yeast eIF2 ternary complex structure showing Met–tRNAi (magenta) and GTP (black) binding to eIF2 (brown-α, green-γ and blue-β). Residues E189 (red) and S264 (light blue) are shown filled in eIF2β. The C-terminus of eIF2β is shown in light blue. It binds both initiator Met–tRNAi and Switch 1 (gold) of eIF2γ. The image was generated using UCSF Chimera software and the protein data bank coordinate file 3JAP. Only chains 1, j, k and l are shown. GTP is the analogue phosphomethylphosphonic acid guanylate ester.
Figure 4.
Figure 4.
E189K has a modest impact on eIF2–eIF2B interaction and activity. (A) Affinity (Kd) of GDP, GTP and Met–tRNAi to purified WT and mutant (β E189K) apo–eIF2 complexes measured by monitoring the fluorescence intensity of 100 nM BODIPY-FL-GDP (left), 100 nM BODIPY-FL-GTP (middle) or 20 nM BOP-N-Met–tRNAi with 1 mM GTP (right). (B) Kinetics of BODIPY-FL-GDP release from preformed eIF2 complexes in the presence of different eIF2B concentrations. K1/2 and Kmax values were determined from curve fitting y = [(Kmax × x)/(K1/2 + x)] + c. (C) Western blotting of IP of Flag-eIF2β, Flag-E189K and an untagged control from cells showing its co-association with known binding proteins. Quantification of at least three repeats using Li-Cor fluorescent secondary antibodies ±SE. Student's t-test indicates significant reduction in eIF2B–eIF2 interactions (P = 2.9 × 10−6) with E189K (marked *). Other factors are not significantly altered. Tif5 is indicated with an arrow, ‘♦’ marks a non-specific band.
Figure 5.
Figure 5.
E189K antagonizes eIF5 GDI activity. (A) Kinetics of BODIPY-FL-GDP release from preformed purified WT and mutant (β E189K) eIF2•GDP complexes (20 nM) with varying concentrations of GST-eIF5 ± SD (n = 3). Molar eIF2:GST–eIF5 protein ratios are shown. Asterisks (*) mark points with statistically significant difference to WT (P < 0.01, unpaired Student's t-test). (B) Kinetics of BODIPY-FL-GDP release from preformed eIF2 complexes (20 nM) in the presence of different eIF2B concentrations and eIF5 (20 nM). Curves shown in Figure 3B are repeated for direct comparison. Right panel shows a zoomed image focussing on eIF2B 0–12.5 nM ± SD (n = 3). Statistical significant increase in the rate of GDP release from eIF5/eIF2βE189K relative to eIF5/eIF2 WT is indicated (* = P < 0.01, † = P < 0.05, unpaired Student's t-test). (C) Growth and HIS4-LacZ expression of E189K and WT eIF2 cells (strains GP3773 and GP3771) transformed with plasmids carrying the indicated constitutively active GCN2 alleles.
Figure 6.
Figure 6.
Model for role of βE189 ensuring tight control of GDP release from eIF2. (A) eIF2 heterotrimer is shown in cartoon form based on the structure model shown in Figure 3C with eIF2γ core bound to GTP and with α and β ‘arms’ ‘grasping’ Met–tRNAi (purple). The structure of the N-terminal region of eIF2β bearing the lysine repeat ‘K-boxes’ is not known and is shown here as a white oval with black K-box stripes and is speculatively positioned over eIF2γ. βS264 is depicted as a yellow triangle adjacent to GTP bound to eIF2γ G domain and Sw1 and βE189 is shown as a magenta triangle. (B) Following GTP hydrolysis and eIF2 release from the PIC and in the absence of other ligands, eIF2β ‘arm’ position is flexible and its movement contributes to the relatively high off-rate of GDP. (C) Binding of eIF5 GDI acts as a molecular clamp constraining eIF2β and stabilizing GDP binding to eIF2γ. (D) E189K alters eIF5-eIF2 interactions so that GDP is no longer stabilized despite eIF5 interaction, shown by eIF2β ‘arm’ flexibility.
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