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First published online 9 January 2007
doi: 10.1242/jcs.03339

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After fertilization of sea urchin eggs, eIF4G is post-translationally modified and associated with the cap-binding protein eIF4E

Nathalie Oulhen^*, Patrick Salaün^*,, Bertrand Cosson, Patrick Cormier and Julia Morales

Université Pierre et Marie Curie-Paris6, UMR 7150, Equipe Cycle Cellulaire et Développement and CNRS, UMR 7150, Station Biologique de Roscoff, 29682 Roscoff CEDEX, France

View larger version (44K): [in this window] [in a new window]   Fig. 1. Characterization of sea urchin eIF4G. (A) Schematic diagram of SgIF4G protein deduced from the cDNA. The SgIF4G conserved domains MIF4G (middle domain of eIF4G: predicted eIF4A- and eIF3-binding domain), MA3 (domain present in DAP-5, eIF4G and MA-3), eIF5C (domain at the C-termini of GCD6, eIF-2B , eIF5 and eIF4G) and amino acid sequence corresponding to the eIF4E- and PABP-binding sites are indicated. The last 1005 residues of the predicted protein scored 40% with human eIF4GI. The sequence corresponding to the peptide used to generate an SgIF4G-GST fusion protein is indicated by a grey line. Numbers indicate the positions of amino acids in the protein sequence deduced from the cDNA sequence. (B) Protein alignment of the eIF4E-binding domain of sea urchin eIF4G, and human eIF4GI and eIF4GII. Identical and conserved amino acid residues are on black and grey background, respectively. The common eIF4E recognition motif is indicated above (where x is any amino acid and is an aliphatic residue, usually L, M or F). (C) Protein alignment of the PABP-binding region of SgIF4G and human eIF4GI and eIF4GII. Residues suggested to be important for the interaction with PABP in human eIF4GI are indicated above (Imataka and Sonenberg, 1997; Wakiyama et al., 2000). (D) A single ortholog of SgIF4G is expressed in unfertilized eggs of sea urchin. Northern blot derived from gel loaded with 1 or 8 µg poly(A+) mRNAs from unfertilized sea urchin eggs was assessed for the presence of SgIF4G transcript as described in Materials and Methods. (E) The SgIF4G fusion protein (GST-SgIF4G) interacts with a mouse eIF4E-fusion protein (GST-mIF4E). After incubation, the fusion proteins were affinity-purified using an m7GTP column (lanes 1-5), analysed by immunoblotting and detected by chemifluorescence using an anti-GST antibody as described in Materials and Methods. Affinity-purified proteins were compared with the GST-fusion proteins loaded separately (lanes 6-8). The positions of the respective GST proteins are indicated with arrows on the right side of the panel.

View larger version (39K): [in this window] [in a new window]   Fig. 2. Sea urchin eIF4G is modified following fertilization and interacts with eIF4E in vivo. (A) Specificity of SgIF4G antibodies. Supernatants obtained from low-level speed centrifugation of egg homogenates were prepared prior to (lanes 1, 3, 5 and 7) or following (lanes 2, 4, 6 and 8) fertilization. Identical amounts of extract proteins (10 µg) were resolved by 7.5% SDS-PAGE and subjected to western blot analyses using anti-SgIF4G antibody (lanes 1 and 2), corresponding pre-immune serum (lanes 3 and 4), anti-SgIF4G antibody pre-incubated with 1 µg recombinant GST-SgIF4G (lanes 5 and 6) or anti-SgIF4G antibody pre-incubated with 1 µg of GST (lanes 7 and 8); anti-actin antibody was used as loading control. (B) The multiple isoforms of SgIF4G associate with the GST-tagged mIF4E. Proteins extracted from unfertilized eggs or eggs 60 minutes post fertilization were incubated with GST alone or GST-tagged mIF4E proteins bound to glutathione-Sepharose beads as described in Materials and Methods. Precipitates were analysed by western blotting using anti-SgIF4G (top panels) and anti-GST (bottom panels) antibodies. Input represents 20% of total protein used for pull-down. (C) SgIF4G co-purifies with sea urchin eIF4E. Total-extract proteins (left panels) or affinity-purified proteins using an m7GTP column (right panels) prior to or 60 minutes post-fertilization were subjected to western blot analyses using anti-SgIF4G (top panels), anti-actin (middle panels) or anti-eIF4E (bottom) antibodies. (D) Sea urchin eIF4E coimmunoprecipitates with SgIF4G after fertilization. Proteins extracted from unfertilized eggs or eggs 60 minutes post fertilization were immunoprecipitated with SgIF4G, resolved by SDS-PAGE and analysed by western blotting using anti-rabbit eIF4E (lower panel) and anti-SgIF4G antibodies (upper panels). The arrow indicates IgG recognized by secondary antibodies. Protein A Sepharose beads with anti-SgIF4G but without proteins and protein A Sepharose beads with proteins but without anti-SgIF4G were used as controls.

View larger version (39K): [in this window] [in a new window]   Fig. 3. The increase in the association of SgIF4G and eIF4E following fertilization correlates with the dissociation of eIF4E and 4E-BP, and occurs after modifications of SgIF4G protein. (A) SgIF4G associates with eIF4E when 4E-BP dissociates from eIF4E. m7GTP-column affinity-purified egg proteins extracted prior to or at the indicated times after fertilization were resolved by 15% SDS-PAGE and subjected to western blotting using anti-4E-BP2 (bottom), anti-eIF4E (middle) and anti-SgIF4G (top) antibodies. (B) SgIF4G modifications occur rapidly following fertilization. Supernatants obtained from low-speed centrifugation of egg homogenates prepared prior to or at the indicated times following fertilization, were resolved by 7.5% SDS-PAGE and subjected to western blotting using anti-SgIF4G (top). Anti-actin was used as loading control (bottom).

View larger version (41K): [in this window] [in a new window]   Fig. 4. SgIF4G-eIF4E association following fertilization, but not SgIF4G modification, is sensitive to rapamycin treatment. (A) SgIF4G-eIF4E association is inhibited by rapamycin. Affinity-purified protein (using an m7GTP column) obtained prior to or at the indicated times after fertilization from untreated (left) or 20 µM rapamycin-treated (right) eggs were subjected to western blot analyses using anti-SgIF4G (top), anti-human 4E-BP2 (bottom) and anti-eIF4E (middle) antibodies. (B) SgIF4G modification is not affected by rapamycin. The total amount of SgIF4G from untreated (left) or rapamycin-treated (20 µM) eggs (right), obtained prior to and at the indicated times after fertilization, was analysed by western blotting using anti-SgIF4G antibody (top). Anti-actin was used as loading control (bottom).

View larger version (29K): [in this window] [in a new window]   Fig. 5. A peptide based on the eIF4E-binding sequence of sea urchin eIF4G inhibits formation of the SgIF4G-eIF4E complex and affects the first mitotic division of sea urchin embryos. (A) Incubation of a peptide based on the eIF4E-binding site of sea urchin eIF4G with recombinant eIF4E and SgIF4G fusion proteins inhibits their interaction. The fusion proteins, untreated (lane 1) or incubated with the SgIF4G peptide corresponding to the binding site on eIF4E (wild type) used at 50 µM (lane 2) or 20 µM (lane 3) or a scrambled peptide used at 50 µM (lane 4) or 20 µM (lane 5) were affinity-purified using an m7GTP column and were analysed by immunoblotting as described in Materials and Methods using an anti-GST antibody. Affinity-purified proteins were compared with the GST-fusion proteins loaded separately (lanes 6 and 7). The positions of the respective GST-fusion proteins are indicated by arrows on the right side of the panel. (B) A peptide based on the eIF4E-binding of sea urchin eIF4G affects the first mitotic division of sea urchin embryos. SgIF4G peptide corresponding to the binding site on eIF4E (wild type) or a scrambled peptide was introduced by microinjection at a final intracellular concentration of 20 µM into unfertilized eggs. The control corresponds to non-injected eggs. Cleavage was assessed under a light microscope at 150 minutes after fertilization. An average of 100 unfertilized eggs were injected for each compound in each experiment and error bars represent the standard deviation (± s.d.) of three independent experiments. Significance was assessed using Fisher's F-test and Student's t-test. *P<0.005, significant difference between eggs microinjected with the SgIF4G wild-type peptide, microinjected with the scrambled peptide and non-injected eggs.

View larger version (18K): [in this window] [in a new window]   Fig. 6. Microinjection of m7GDP inhibits the first mitotic division of sea urchin embryos. m7GDP was introduced by microinjection at a final intracellular concentration of 1 mM into unfertilized eggs. The control corresponds to non-injected eggs and buffer corresponds to eggs injected with the microinjection buffer. Cleavage was scored by observation under a light microscope at 150 minutes post-fertilization. An average of 60 unfertilized eggs were injected for each compound in each experiment, and error bars represent the standard deviation (± s.d.) of three independent experiments. Significance was assessed using Fisher's F-test and Student's t-test. *P<0.005, significant difference between eggs microinjected with m7GDP, microinjected with buffer and non-injected eggs.