eIF5 has GDI activity necessary for translational control by eIF2 phosphorylation

Abstract

In protein synthesis initiation, the eukaryotic translation initiation factor (eIF) 2 (a G protein) functions in its GTP-bound state to deliver initiator methionyl-tRNA (tRNA(i)(Met)) to the small ribosomal subunit and is necessary for protein synthesis in all cells. Phosphorylation of eIF2 [eIF2(alphaP)] is critical for translational control in diverse settings including nutrient deprivation, viral infection and memory formation. eIF5 functions in start site selection as a GTPase accelerating protein (GAP) for the eIF2.GTP.tRNA(i)(Met) ternary complex within the ribosome-bound pre-initiation complex. Here we define new regulatory functions of eIF5 in the recycling of eIF2 from its inactive eIF2.GDP state between successive rounds of translation initiation. First we show that eIF5 stabilizes the binding of GDP to eIF2 and is therefore a bi-functional protein that acts as a GDP dissociation inhibitor (GDI). We find that this activity is independent of the GAP function and identify conserved residues within eIF5 that are necessary for this role. Second we show that eIF5 is a critical component of the eIF2(alphaP) regulatory complex that inhibits the activity of the guanine-nucleotide exchange factor (GEF) eIF2B. Together our studies define a new step in the translation initiation pathway, one that is critical for normal translational controls.

Figure 1. eIF5 has GDI activity

a) Scheme for GDI activity assay. b) Increasing eIF5 stabilises GDP-binding to eIF2. K_off GDP from 60 pmol eIF2 with varying concentrations of GST-eIF5 (0-240 pmol, open circles) or GST alone (filled circle). Molar eIF2:GST-eIF5 protein ratios are indicated. c) Defining regions required for GDI activity. Mean K_off GDP (60 pmol eIF2) for indicated constructs derived from reactions with GST- or FLAG-eIF5 proteins (120 pmol). Black bars represent a significant reduction in K_off GDP (P<0.0001, unpaired Student's t-test). Errors show standard deviation (n>3). 2.9 mM Mg²⁺ was used in b) and c).

Figure 2. The CTD of eIF5 is critical for interaction with eIF2

Affinity chromatography assay between eIF2 (110 pmol) and the indicated immobilized GST-eIF5 constructs. eIF2 was detected by immunoblotting using antibodies specific for a) eIF2γ or b) eIF2α. Representative blots are shown. Signal intensity was quantified (Adobe Photoshop) and the mean ± standard deviation (n=3) are shown below. c) Total protein in each sample stained with Ponceau S. Inputs (lanes 1) represent 10% of total.

Figure 3. The Linker Region of eIF5 interacts with γ subunit of eIF2

Affinity chromatography as in figure 2 between indicated immobilized GST-eIF5 constructs and total cell extracts (500 μg) expressing c-Myc-6xHis-eIF2γ from either a single copy (sc) or high copy (hc) plasmid. Immunoblots were developed with c-Myc and eIF2α antibodies. Total protein in each sample was stained with Ponceau S. Inputs (lane 1) represent 5% (blots) or 1% (stain) of total.

Figure 4. GDI activity antagonises eIF2B and affects GCN4 activation in vivo

a-c) Strains expressing single (sc) or high copy (hc) eIF5 plasmids as the source of eIF5 and co-transformed with plasmids expressing GCN2, vector alone (gcn2Δ) [a, b] or the constitutively active mutant GCN2^M788V,E1591K (GCN2^c) [c] were grown as stated. d) Left, immunoblots following FLAG-eIF5 immune precipitation of protein complexes from cells grown in nutrient sufficient conditions (SCD) and following starvation (SD+3AT). Right, quantification ± standard deviation (n=3). e) Model for recycling and regulation of eIF2, incorporating eIF5 GDI activity. Dashed grey arrows indicate steps that limit eIF2 recycling.