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First published online 5 May 2004
doi: 10.1242/jcs.01106

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Expression of fragments of translation initiation factor eIF4GI reveals a nuclear localisation signal within the N-terminal apoptotic cleavage fragment N-FAG

School of Life Sciences, University of Sussex, Falmer, Brighton, BN1 9QG, UK

View larger version (42K): [in a new window]   Fig. 2. Exogenously expressed myc-tagged isoforms of eIF4GI are localised to the cytoplasm. (A) Schematic representation of eIF4GI showing sites of alternative translation initiation and binding sites of other components of the translation initiation machinery (Bradley et al., 2002; Byrd et al., 2002; Morley, 2001). Sites of cleavage by caspase-3 and the picornaviral L and 2A proteases are indicated. Black lines indicate individual sequences used for expression in this figure. (B) Total cell lysates were prepared from HeLa cells transfected with plasmids containing eIF4GI cDNAs and resolved by SDS-PAGE. Expressed proteins were identified by immunoblotting using the 9E10 monoclonal antibody to the c-myc epitope; molecular mass markers are shown on the left. (C) Anti-myc monoclonal antibody (9E10) followed by goat anti-mouse IgG conjugated to FITC (green) was used to visualise the localisation of the tagged isoforms of eIF4G expressed in HeLa cells, as indicated. Actin was visualised with phalloidin-TRITC (red) and nuclei with DAPI (blue). Scale bars: 20 µm.

View larger version (34K): [in a new window]   Fig. 1. eIF4GI, eIF4E and PABP are predominantly cytoplasmic and do not directly co-localise with the actin cytoskeleton. (A) HeLa cells seeded on glass coverslips were fixed in 4% paraformaldehyde for 15 min, rinsed with PBS three times and permeabilised in PBS containing 0.8% Triton-X-100 (v/v) for 8 minutes. Rabbit antisera recognising the C terminus of eIF4GI, the N terminus of eIF4GI, eIF4E and PABP1, followed by a secondary antibody of goat anti-rabbit IgG conjugated to rhodamine, were used to visualise the localisation of the endogenous proteins within these cells (pseudocoloured in red). Actin was visualised with phalloidin-FITC (pseudocoloured green) and nuclei with DAPI (pseudocoloured blue). Scale bars: 20 µm. (B) Cells were cultured and processed as in A. The localisation of the endogenous proteins within these cells was also visualised using combinations of rabbit and mouse antisera and secondary antibodies of goat anti-rabbit IgG conjugated to rhodamine (red) or goat anti-mouse IgG conjugated to FITC (green). Left panels: rabbit anti-eIF4GI and mouse anti-eIF4E; centre panels: rabbit anti-eIF4GI and mouse anti-PABP1; right panels: rabbit anti-eIF4E and mouse anti-PABP1. White bars represent 20 µm.

View larger version (62K): [in a new window]   Fig. 3. All isoforms of exogenous myc-tagged N-FAG are localised to the nucleus. (A) Schematic representation of eIF4GI outlining the individual N-terminal sequences used for expression in this study. (B) HeLa cells were transfected with plasmids encoding myc-tagged isoforms of the N-terminal domains of eIF4GI. Sixteen hours after transfection, total cell lysates were prepared and proteins resolved by SDS-PAGE. eIF4GI and its N-terminal cleavage fragments were identified by immunoblotting using anti-myc antiserum; molecular mass markers are shown on the left. Owing to differences in expression levels and to aid visualisation, extracts from cells transfected with N-FAGdc, N-FAGb, N-FAGa and Nta were all diluted 1:2 with sample buffer; despite this, expression of Ntf is only detectable at extremely long exposures (data not shown). (C) Anti-myc antibody followed by goat anti-mouse IgG conjugated to FITC (green) was used to visualise the localisation of the tagged isoforms of the N-terminal domain of eIF4GI expressed in HeLa cells, as indicated. Actin was visualised with phalloidin-TRITC (red) and nuclei with DAPI (blue). Scale bars: 20 µm.

View larger version (59K): [in a new window]   Fig. 4. Expressed M-FAG remains cytoplasmic while expressed Ct/p100 causes morphological defects and induces apoptosis. (A) Schematic representation of eIF4GI outlining the individual sequences used for expression in this study. (B) HeLa cells were transfected with plasmids encoding myc-tagged isoforms of the C-terminal domains of eIF4GI. Sixteen hours after transfection, total cell lysates were prepared and proteins resolved by SDS-PAGE. The fragments were identified by immunoblotting using anti-myc antiserum; molecular mass markers are shown on the left. Owing to differences in expression levels and to aid visualisation, extracts from cells expressing C-FAG were diluted 1:10 prior to analysis. (C) Anti-myc antibody followed by goat anti-mouse IgG conjugated to FITC (green) was used to visualise the localisation of the tagged isoforms of the C-terminal domains of eIF4GI expressed in HeLa cells, as indicated. Actin was visualised with phalloidin-TRITC (red) and nuclei with DAPI (blue). Scale bars: 20 µm.

View larger version (55K): [in a new window]   Fig. 5. Mutation of a putative NLS in N-FAG changes its localisation from nuclear to cytoplasmic. (A) Schematic representation of eIF4GI showing the basic region that may act as a nuclear localisation signal and the individual sequences used in this study. (B) HeLa cells were transfected with plasmids encoding the wild-type or 513KRRRK517-513AAAAA517 mutated myc-tagged eIF4GI sequences indicated. 16 hours after transfection, total cell lysates were prepared and proteins resolved by SDS-PAGE. eIF4GI and its N-terminal cleavage fragments were visualised by immunoblotting using anti-myc antiserum; molecular mass markers are shown on the left. Expression of eIF4GIfAAAAA is only detectable at extremely long exposures (data not shown). (C) HeLa cells were probed with anti-myc antibody followed by goat anti-mouse IgG conjugated to FITC (green) to visualise the localisation of wild-type N-terminal apoptotic cleavage fragments of eIF4GI, or those in which a basic sequence was mutated to alanines, as indicated. Actin was visualised with phalloidin-TRITC (red) and nuclei with DAPI (blue). Scale bars: 20 µm.

View larger version (56K): [in a new window]   Fig. 6. The eIF4GI nuclear localisation signal is sufficient to direct a change in the subcellular localisation of eGFP. (A) Schematic representation of eIF4GI outlining the individual sequences used for expression in this figure. (B) Total cell extracts from HeLa cells expressing eGFP fusion proteins were resolved by SDS-PAGE. Expressed proteins were visualised by immunoblotting using a monoclonal antibody raised against eGFP; molecular mass markers are shown on the left. (C) HeLa cells expressing eGFP fusion proteins were grown on glass coverslips and fixed with paraformaldehyde. eGFP was visualised directly using the appropriate fluorescence filters. Scale bars: 20 µm.