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Figure 1 Alignment of amino acid sequences of the N-terminal region of human eIF4GI and eIF4GII. The pattern-induced multi-sequence alignment program (Smith and Smith, 1992) was used to align amino acid sequences of the N-terminal region of eIF4GI (Yan et al., 1992; Imataka and Sonenberg, 1997; this study) and eIF4GII (Gradi et al., 1998). Identical amino acids are boxed and conservative substitutions are shaded. The first methionine of the original eIF4GI (Yan et al., 1992) is indicated by an arrow. Numbers above amino acids represent positions of truncations used in the text. A schematic representation of the binding sites of eIF4E (Mader et al., 1995), eIF4A and eIF3 (Lamphear et al., 1995; Imataka and Sonenberg, 1997) is shown above the alignment.
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 | Figure 2 Co-immunoprecipitation of eIF4GI and eIF4GII with PABP. (A) HeLa cells infected with vTF7-3 were transfected with vector (pcDNA3-HA) alone (lane 1) or the vector expressing an HA-tagged protein as indicated in each lane (lanes 2–5). Proteins were immunoprecipitated with anti-HA antibody and immunoprecipitates were resolved by SDS–10% PAGE. Western blotting was performed with anti-PABP (upper panel), anti-eIF4A (middle panel) or anti-HA antibody (lower panel). HeLa cell extract (40  g of protein) was loaded to the left of lane 1. (B) Extracts from uninfected and non-transfected HeLa cells were incubated with pre-immune serum (lane 2) or anti-PABP serum (lane 3). Following precipitation with protein G–Sepharose, bound proteins were resolved by SDS–8% PAGE. Western blotting was performed with anti-eIF4GI (left upper panel), anti-eIF4GII (right upper panel) or anti-PABP antibody (lower panels). One-fiftieth of the extracts used for immunoprecipitation was loaded in lane 1.
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Figure 3 Localization of the PABP-binding site in human eIF4GI. (A) N-terminal deletions. HeLa cells infected with vTF7-3 were co-transfected with pcDNA3-PABP-HA and pcDNA3-GST-eIF4GI fragments indicated in each lane (lanes 1–5) or pcDNA3-GST-CAT (lane 6). One-tenth of the cell extract was used for Western blotting with anti-GST antibody (upper panel). The remaining extract was used for immunoprecipitation with anti-HA antibody. Immunoprecipitates were resolved by SDS–10% PAGE. Western blotting was performed with anti-GST (middle panel) or anti-HA antibody (lower panel). (B) N-terminal boundary. Experiments were as in (A), with plasmids expressing GST fusion proteins indicated in each lane. (C) C-terminal boundary. Experiments were as in (A), with plasmids expressing GST fusion proteins indicated in each lane. (D) eIF4GI(132–160) is the PABP-binding site. HeLa cells infected with vTF7-3 were co-transfected with pcDNA3-FLAG-PABP and pcDNA3-GST-eIF4GI fragments indicated in each lane (lanes 1–3) or pcDNA3-GST (lane 4). One-tenth of the cell extract was used for Western blotting with anti-FLAG antibody (upper panel), and the remaining extract was mixed with glutathione–Sepharose beads. Bound proteins were eluted with reduced glutathione, and were resolved by SDS–12.5% PAGE. Western blotting was performed with anti-FLAG (middle panel) or anti-GST antibody (lower panel). The amino acid sequence of the PABP-binding site in human eIF4GI and the corresponding region of human eIF4GII are aligned at the bottom of the figure. Identical amino acids are boxed and conservative substitutions are shaded. (E) eIF4GI(132–160) binds endogenous PABP. GST (lane 1) or GST–eIF4GI(132–160) (lane 2) was expressed as in (D), but without co-expression of FLAG-PABP. The cell extract was mixed with glutathione–Sepharose beads. Bound proteins eluted with reduced glutathione were subjected to Western blotting with anti-PABP (upper panel) or anti-GST antibody (lower panel). HeLa cell extract (40 g protein) was loaded to the left of lane 1.
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 | Figure 4 Localization of the eIF4GI-binding site in PABP. (A) Schematic representation of PABP mutants examined in (B) and (C). (B) eIF4GI binds the N-terminal region (RRMs 1–4) of PABP. HeLa cells infected with vTF7-3 were co-transfected with pcDNA3-HA-eIF4G(1–329) and pcDNA3-FLAG-PABP(RRMs 1–4) (lane 1), -PABP(C) (lane 2) or -eIF4E (lane 3). One-twentieth of the cell extract was used for Western blotting with anti-FLAG antibody (upper panel). The remaining extract was used for immunoprecipitation with anti-HA antibody. Immunoprecipitates were resolved by SDS–10% PAGE. Western blotting was performed with anti-FLAG (middle panel) or anti-HA antibody (lower panel). (C) eIF4GI binds RRMs 1–2. Experiments were as in (A), with plasmids expressing FLAG-tagged proteins indicated in each lane. (D) RRMs 1–2 binds eIF4GI(132–160). GST (lane 1) or GST–eIF4GI(132–160) (lane 2) was co-expressed with FLAG-RRMs 1–2 as in Figure 3D. One-tenth of the cell extract was used for Western blotting with anti-FLAG antibody (upper panel), and the remaining extract was mixed with glutathione–Sepharose beads. Bound proteins eluted with reduced glutathione were subjected to Western blotting with anti-FLAG (middle panel) or anti-GST antibody (lower panel).
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Figure 5 Functional analysis of the N-terminal region of eIF4GI. (A) In vitro binding of the N-terminal sequence of eIF4GI to PABP. PABP(RRMs 1–4)-His was incubated with GST (lane 1), GST–eIF4GI(1–204) (lane 2) or GST–eIF4GI(1–204:mut) (lane 3) immobilized on glutathione–Sepharose beads. Bound proteins were eluted with reduced glutathione, and resolved by SDS–12.5% PAGE. Western blotting was performed with anti-GST (upper panel) or anti-His (lower panel) antibody. One-fifth of the input PABP(RRMs 1–4)-His was loaded to the left of lane 1. (B) Effects of the N-terminal region on poly(A)-dependent translation. Buffer (lanes 1 and 5) (1 l), GST (lanes 2 and 6), GST–eIF4G(1–204) (lanes 3, 8, 9 and 10) or GST–eIF4G(1–204:mut) (lanes 4 and 7) (2, 4 or 6 g) (1 l) was added to a rabbit reticulocyte lysate (10 l). After incubation on ice 30 min, the lysate was programmed with capLUC (lanes 1–4) or capLUCpA RNA (lanes 5–10) (1 l, 60 ng). The translation reaction mixture was incubated at 30°C for 30 min. Luciferase activity was measured using a luminometer. The luciferase activity of the lysate programmed with capLUC in the presence of buffer alone (lane 1) was set at 100%. Error bars denote the standard error of four independent experiments.
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