Plant cap-binding complexes eukaryotic initiation factors eIF4F and eIFISO4F: molecular specificity of subunit binding

Abstract

The initiation of translation in eukaryotes requires a suite of eIFs that include the cap-binding complex, eIF4F. eIF4F is comprised of the subunits eIF4G and eIF4E and often the helicase, eIF4A. The eIF4G subunit serves as an assembly point for other initiation factors, whereas eIF4E binds to the 7-methyl guanosine cap of mRNA. Plants have an isozyme form of eIF4F (eIFiso4F) with comparable subunits, eIFiso4E and eIFiso4G. Plant eIF4A is very loosely associated with the plant cap-binding complexes. The specificity of interaction of the individual subunits of the two complexes was previously unknown. To address this issue, mixed complexes (eIF4E-eIFiso4G or eIFiso4E-eIF4G) were expressed and purified from Escherichia coli for biochemical analysis. The activity of the mixed complexes in in vitro translation assays correlated with the large subunit of the respective correct complex. These results suggest that the eIF4G or eIFiso4G subunits influence translational efficiency more than the cap-binding subunits. The translation assays also showed varying responses of the mRNA templates to eIF4F or eIFiso4F, suggesting that some level of mRNA discrimination is possible. The dissociation constants for the correct complexes have K(D) values in the subnanomolar range, whereas the mixed complexes were found to have K(D) values in the ∼10 nm range. Displacement assays showed that the correct binding partner readily displaces the incorrect binding partner in a manner consistent with the difference in K(D) values. These results show molecular specificity for the formation of plant eIF4F and eIFiso4F complexes and suggest a role in mRNA discrimination during initiation of translation.

FIGURE 1.

A, domain organization for eIF4G and eIFiso4G. Plant eIF4G and eIFiso4G have similar domain organization, except eIF4G has an extended N-terminal region (19). The eIF4E binding site and HEAT domains are indicated. The HEAT domains interact with eIF4A and eIF3 as indicated. Plant eIF4G and eIFiso4G lack the third HEAT domain present in mammalian eIF4G, and yeast eIF4G only has HEAT domain 1. B, sequence of wheat eIF4G gene. The coding region is shown in capital letters, and noncoding regions and introns are in lowercase. Differences between the gene sequence and expression clone are indicated (green, silent mutants; red, amino acid changes). The N terminus of the expression clone was altered to generate an NcoI site as indicated and has a single methionine, and the second amino acid was changed to glycine. The N-terminal peptide obtained from Edmund degradation of native protein is indicated by a double underlining, and internal peptides obtained from trypsin digests of native protein are indicated by single underlining. The naturally occurring NcoI and AgeI sites used to generate the expression clone are indicated. The eIF4E binding site (dotted underlining) and HEAT domains (yellow and blue) are indicated.

FIGURE 2.

SDS-PAGE analysis of eIF4F, eIFiso4F, and mixed complexes. SDS-PAGE was carried out on a 12.5% acrylamide gel and stained with Coomassie Brilliant Blue. Each lane contains 25 pmol of the indicated complex. First lane, native eIF4F; second lane, recombinant eIF4F; third lane, eIF4G-eIFiso4E; fourth lane, eIFiso4G-eIF4E; fifth lane, recombinant eIFiso4F; sixth lane, native eIFiso4F.

FIGURE 3.

Monoclonal antibodies to wheat eIF4F. Mouse monoclonal antibodies were raised to wheat native eIF4F. The ability of individual monoclonal antibodies to react with native (n) eIF4F and recombinant (r) eIF4F was tested by Western blotting. Mouse ascites fluid (1/1000) was incubated overnight at 4 °C, and the second antibody was goat anti-mouse HRP (1/20,000, Kirkegaard-Perry). The chemiluminescent substrate was Super Signal West Pico (Thermo-Pierce).

FIGURE 4.

Comparison of recombinant and native eIF4F and eIFiso4F in translation. Each 100-μl translation reaction contained 5 pmol of capped rabbit β-hemoglobin mRNA and the indicated amounts of eIF4F or eIFiso4F complexes (■, solid line, native eIF4F; □, dashed line, recombinant eIF4F; ●, solid line, native eIFiso4F; △, dashed line, recombinant eIFiso4F) as described under “Experimental Procedures.” The amount of [¹⁴C]leucine incorporated in the absence of cap-binding complexes was 3.8 pmol. Each point represents the average of three experiments, and the error bars are indicated.

FIGURE 5.

Translation Assay of eIF4F, eIFiso4F, and mixed complexes. Each 100-μl translation reaction contained 5 pmol of the indicated mRNA (capped rabbit β-hemoglobin, capped barley α-amylase, capped AtHSP21, STNV RNA (uncapped), and capped AMV RNA 4) and the indicated amounts of recombinant eIF4F, eIFiso4F or mixed complex (●, eIF4F); (○, eIF4G-eIFiso4E); (▴, eIFiso4F); or (△, eIFiso4G-eIF4E) as described under “Experimental Procedures.” The amount of [¹⁴C]leucine incorporated in the absence of cap-binding complexes was as indicated: β-hemoglobin (β-Hb, 4.0 pmol); barley α-amylase (BαA, 4.1 pmol); AtSHSP 21 (6.6 pmol); STNV RNA (16.8 pmol); and AMV RNA 4 (5.3 pmol). Each point represents the average of three experiments, and the error bars are indicated.

FIGURE 6.

Immunoprecipitation of wheat germ extracts. Wheat germ extract (1 ml) was immunoprecipitated (IP) with rabbit antisera raised to either recombinant eIF4G or eIFiso4G bound to protein A magnetic beads (Genscript). The precipitated proteins were eluted with 50 μl of 2× SDS-PAGE sample buffer, 20 μl of which was separated on a 10–20% acrylamide gel (Bio-Rad) and transferred to nitrocellulose. Controls of recombinant wheat eIF4E (0.05 μg) and eIFiso4E (0.05 μg) were also separated on the gel. The blots were probed with either mouse monoclonal antibody for eIF4E (8F7) or rabbit polyclonal serum raised to native gel-purified eIFiso4E using a mouse or rabbit 1-h complete IP Western kit (Genscript).

FIGURE 7.

SPR binding analysis of correct and mixed complexes. eIF4E (A and D) or eIFiso4E (B and C) were tested for binding to the respective immobilized proteins eIF4G (A and B) or eIFiso4G (C and D). The running buffer contained 20 mm HEPES, 100 mm KCl, 1.0 mm DTT, 0.1 mm EDTA, 100 μm m⁷GTP, 5% glycerol, 0.01% Tween 20, and 0.1 mg/ml BSA, pH 7.6. Starting at 50 nm, a 3-fold dilution series of eIFiso4E or eIF4E were tested in triplicate. The response data were globally fit using Scrubber2 (Biologic Software Pty Ltd) to a 1:1 interaction model to extract binding constants.

FIGURE 8.

Displacement assays. To determine whether the correct cap-binding protein is able to displace a mismatched cap-binding protein in a mixed complex, the indicated amounts of eIF4E or eIFiso4E were added to 2.5 pmol of mixed complexes, eIF4F, or eIFiso4F. A and B were probed with mouse monoclonal antibody (8F7) to eIF4E (1/1000). The blots were stripped and probed with rabbit polyclonal antibody raised to native gel-purified eIFiso4E (1/1000) shown in C and D. The antibody to native eIFiso4E displays a small amount of cross-reactivity with eIFiso4G (see D, eIFiso4G-eIF4E with no eIFiso4E added).