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. 2000 Jan;20(2):468-77.
doi: 10.1128/MCB.20.2.468-477.2000.

Eukaryotic translation initiation factor 4E (eIF4E) binding site and the middle one-third of eIF4GI constitute the core domain for cap-dependent translation, and the C-terminal one-third functions as a modulatory region

S Morino  1 , H Imataka, Y V Svitkin, T V Pestova, N Sonenberg
Affiliations

Eukaryotic translation initiation factor 4E (eIF4E) binding site and the middle one-third of eIF4GI constitute the core domain for cap-dependent translation, and the C-terminal one-third functions as a modulatory region

S Morino et al. Mol Cell Biol. 2000 Jan.

Abstract

The mammalian eukaryotic initiation factor 4GI (eIF4GI) may be divided into three roughly equal regions; an amino-terminal one-third (amino acids [aa] 1 to 634), which contains the poly(A) binding protein (PABP) and eIF4E binding sites; a middle third (aa 635 to 1039), which binds eIF4A and eIF3; and a carboxy-terminal third (aa 1040 to 1560), which harbors a second eIF4A binding site and a docking sequence for the Ser/Thr kinase Mnk1. Previous reports demonstrated that the middle one-third of eIF4GI is sufficient for cap-independent translation. To delineate the eIF4GI core sequence required for cap-dependent translation, various truncated versions of eIF4GI were examined in an in vitro ribosome binding assay with beta-globin mRNA. A sequence of 540 aa encompassing aa 550 to 1090, which contains the eIF4E binding site and the middle region of eIF4GI, is the minimal sequence required for cap-dependent translation. In agreement with this, a point mutation in eIF4GI which abolished eIF4A binding in the middle region completely inhibited ribosomal binding. However, the eIF4GI C-terminal third region, which does not have a counterpart in yeast, modulates the activity of the core sequence. When the eIF4A binding site in the C-terminal region of eIF4GI was mutated, ribosome binding was decreased three- to fourfold. These data indicate that the interaction of eIF4A with the middle region of eIF4GI is necessary for translation, whereas the interaction of eIF4A with the C-terminal region plays a modulatory role.

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Figures

FIG. 1
FIG. 1
Toeprint analysis of 48S ribosomal complex formation on β-globin mRNA with recombinant eIF4GI. The components of each reaction mixture are indicated above the lanes. Formation of complex II was quantified as described in Materials and Methods, and the value for eIF4F (lane 2) was set at 100.
FIG. 2
FIG. 2
Functional analysis of eIF4GI deletion mutants. (A) Schematic representation of eIF4GI deletion mutants. PABP, eIF4E, eIF4A, eIF3, and Mnk1 binding sites are indicated. (B) Toeprint analysis of 48S ribosomal complex formation on β-globin mRNA with eIF4GI deletion mutants. The reaction components are indicated above the lanes. The value for eIF4GI(157–1560) (lane 3) was set at 100. (C) Analysis of eIF4GI deletion mutants in a reticulocyte lysate translation system. Translation was performed as described in Materials and Methods. A rabbit reticulocyte lysate treated with rhinovirus 2Apro was supplemented with recombinant proteins as indicated and programmed for translation with the capped bicistronic mRNA CAT/EMCV IRES/LUC. For quantitation of luciferase (LUC) synthesis, the value obtained for translation in untreated lysate in the absence of additional proteins (lane 1) was set at 100. For the quantitation of CAT synthesis, the value obtained for translation in the treated lysate in the presence of eIF4E alone was subtracted as background, and then the value for treated lysate translated in the presence of eIF4E and eIF4GI(157–1560) (lane 4) was set at 100. (D) Western blotting of eIF4G deletion mutants. Recombinant protein preparations (∼1 μg) containing the same amount of eIF4GI according to Coomassie blue staining were subjected to SDS-PAGE (10% gel) and analyzed by Western blotting with anti-Xpress antibody (to detect the epitope located between the His tag and eIF4G coding sequence) or with anti-FLAG antibody.
FIG. 3
FIG. 3
Protein sequence alignment of human eIF4GI (13, 32), eIF4GII (7), and p97 (11). Conserved amino acids are boxed. Amino acids mutated to alanine are highlighted. The eIF4E binding site (18) and the rhinovirus 2Apro cleavage site (15) are also indicated.
FIG. 4
FIG. 4
Minimal essential region for eIF4A and eIF3 binding in the middle fragment of eIF4GI and point mutation for eIF4A binding. (A) N-terminal boundary. N-terminally truncated middle region fragments of eIF4GI were expressed as C-terminally FLAG-tagged proteins in HeLa cells, using the vaccinia virus system as described in Materials and Methods. Cell extract (1 mg) was immunoprecipitated (IP) with anti-FLAG antibody, and immunoprecipitates were resolved by SDS-PAGE (10% gel) for Western blotting with an anti-FLAG (upper panel), anti-hPrt1 (middle panel), or anti-eIF4A (lower panel) antibody. HeLa cell extract (40 μg of protein) was used as a control for the Western blotting in lane 1. IgG, immunoglobulin G. (B) C-terminal boundary. C-terminally truncated middle region fragments of eIF4GI were expressed in HeLa cells as N-terminally HA-tagged proteins. Cell extracts were immunoprecipitated with anti-HA antibody for Western blotting with an anti-HA (upper panel), anti-hPrt1 (middle panel), or anti-eIF4A (lower panel) antibody. HeLa cell extract (40 μg of protein) was used as a control for the Western blotting in lane 1. (C) Minimal essential region for eIF4A and eIF3 binding. HA-eIF4GI(672–1065), HA-eIF4GI(672–970), or HA-eIF4GI(702–970) was expressed in HeLa cells and processed as for panel B. (D) Coimmunoprecipitation of point mutants of the eIF4GI middle region with eIF4A and eIF3. HA-eIF4GI(613–1090) wild type (WT) or HA-eIF4GI(613–1090) point mutants were expressed in HeLa cells and processed as for panel. (E) Recombinant eIF4GI(157–1090) Y776A does not bind eIF4A. Four micrograms of FLAG-eIF4A immobilized on anti-FLAG resin was incubated with 5 μg of His-eIF4GI(157–1090) wild type or His-eIF4GI(157–1090) Y776A on ice for 10 min. After washing, bound proteins were solubilized with SDS sample buffer and subjected to SDS-PAGE followed by Western blotting with an anti-FLAG (middle panel) or anti-His (lower panel) antibody. Twenty percent of the input His-tagged protein was resolved by SDS-PAGE followed by Western blotting with anti-His antibody (upper panel).
FIG. 5
FIG. 5
Demarcation of the C-terminal eIF4A-binding domain of eIF4GI and point mutants. (A) Coimmunoprecipitation of deletion mutants of the eIF4GI C-terminal domain with eIF4A. GST-CAT or GST-eIF4GI deletion mutants were coexpressed with HA-eIF4A in HeLa cells, using the vaccinia virus system as described in Materials and Methods. One-twentieth of the cell extract was used for Western blotting with anti-GST antibody (upper panel). The remaining extract was immunoprecipitated (IP) with anti-HA antibody, and immunoprecipitates were resolved by SDS-PAGE (15% gel) followed by Western blotting with anti-HA (middle panel) or anti-GST (lower panel) antibody. (B) Coimmunoprecipitation of point mutants of the eIF4GI C-terminal domain with eIF4A. HA-eIF4GI(1040–1560) wild type (WT) or HA-eIF4GI(1040–1560) point mutants were expressed in HeLa cells. The cell extract was immunoprecipitated with anti-HA antibody, and immunoprecipitates were resolved by SDS-PAGE (10% gel) followed by Western blotting with an anti-HA (upper panel) or anti-eIF4A (lower panel) antibody. HeLa cell extract (40 μg of protein) was used as a control for Western blotting in lane 1. (C) Recombinant eIF4GI(1040–1560) FVR1239AAA does not bind eIF4A in vitro. Four micrograms of FLAG-eIF4A immobilized on anti-FLAG resin was incubated with 5 μg of His-eIF4GI(1040–1560) or His-eIF4GI(1040–1560) FVR1239AAA on ice for 10 min. After washing, bound proteins were solubilized for SDS-PAGE followed by Western blotting with an anti-FLAG (middle panel) or anti-His (lower panel) antibody. Twenty percent of the input His-tagged protein was subjected to SDS-PAGE followed by Western blotting with anti-His antibody (upper panel).
FIG. 6
FIG. 6
Functional analysis of eIF4GI point mutants. (A) Western blotting of eIF4GI point mutants. One microgram of each recombinant protein preparation was subjected to SDS-PAGE (10% gel) and analyzed by Western blotting with anti-Xpress antibody to detect the epitope located between the His-tag and eIF4GI coding sequence. WT, wild type. (B) toeprinting analysis of 48S ribosomal complex formation on β-globin mRNA with eIF4GI point mutants (2 μg of each). The components of the reaction mixtures are indicated above the lanes. The value for eIF4GI(157–1560) (lane 3) was set at 100. (C) Dose-dependent analysis of eIF4GI FVR1239AAA in a ribosomal binding assay. Increasing amounts of wild-type eIF4GI and eIF4GI FVR1239AAA were used in toeprinting analysis. The value for wild-type eIF4GI (3 μg) was set at 100. (D) Binding of translation factors to eIF4GI point mutants in vitro. The components indicated above the lanes in panel B were mixed and incubated as for toeprinting analysis. The mixture was then immunoprecipitated with anti-eIF4GI(1–329), and immunoprecipitates were analyzed by Western blotting with anti-Xpress for eIF4G, anti-hPrt1 for eIF3, anti-eIF4A, or anti-His for eIF4E. (E) Analysis of eIF4GI point mutants in a rabbit reticulocyte lysate translation system. Translation was performed as described in Materials and Methods. Translation products were analyzed as for Fig. 2C. LUC, luciferase. (F) Dose-response analysis of eIF4GI FVR1239AAA in a rabbit reticulocyte lysate treated with 2Apro.
FIG. 7
FIG. 7
Demarcation of the Mnk1 binding site in the C-terminal region of eIF4GI. (A) N-terminal boundary. GST, GST-CAT, or GST-eIF4GI deletion mutants were coexpressed with FLAG-Mnk1 in HeLa cells. One-fortieth of the cell extract was subjected to SDS-PAGE for Western blotting with anti-FLAG antibody to confirm the expression of FLAG-Mnk1 (upper panel). The remaining extract was mixed with glutathione-Sepharose beads. Bound proteins eluted with reduced glutathione were subjected to Western blotting with an anti-GST (middle panel) or anti-FLAG (lower panel) antibody. (B) C-terminal boundary. GST, GST-CAT, or GST-eIF4GI deletion mutants were coexpressed with FLAG-Mnk1 in HeLa cells. One-fortieth of the cell extract was subjected to SDS-PAGE for Western blotting with anti-GST antibody to confirm the expression of GST fusion proteins (upper panel). The remaining extract was immunoprecipitated with anti-FLAG antibody, and immunoprecipitates were subjected to Western blotting with anti-FLAG (middle panel) or anti-GST (lower panel) antibody. IgG, immunoglobulin G.
FIG. 8
FIG. 8
Model of eIF4GI functional domains. Previous studies have mapped the eIF4E (18) and PABP (13) binding sites to the N-terminal third of eIF4G. The middle third region was shown to bind eIF4A and eIF3 (12), while the C-terminal third region was shown to bind eIF4A (12, 14) and Mnk1 (26). See Discussion section for explanations of models.
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