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First published online 19 March 2009
doi: 10.1242/jcs.040154

Journal of Cell Science 122, 1100-1110 (2009)
Published by The Company of Biologists 2009
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The nuclear pore component Nup358 promotes transportin-dependent nuclear import

Saskia Hutten1,2, Sarah Wälde1, Christiane Spillner1, Joachim Hauber3 and Ralph H. Kehlenbach1,*

1 Department of Biochemistry I, Faculty of Medicine, Georg-August-University of Göttingen, Humboldtallee 23, 37073, Göttingen, Germany
2 Wellcome Trust Centre for Gene Regulation and Expression, MSI/WTB Complex, University of Dundee, Dundee DD1 5EH, UK
3 Heinrich-Pette-Institute for Experimental Virology and Immunology, Martinistrasse 52, 20251 Hamburg, Germany


Figure 1
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  		Fig. 1. HIV-1 Rev is preferentially imported by transportin and importin-9 in vitro. (A) Digitonin-permeabilised cells were incubated with His-YFP-Rev as an import substrate and with equimolar concentrations of import receptors in the presence of 2 µM wild-type Ran at 4°C or 23°C, as indicated. trn, transportin. (B) Nuclear-import reactions were performed with reduced concentrations of either transportin or importin-9 (fourfold excess of His-YFP-Rev), as indicated, in the presence of either wild-type Ran (RanWT), RanQ69L or WGA at 23°C. (C) GST-Rev was immobilised on beads and incubated with transportin or importin-9 in the absence or presence of RanQ69L-GTP. Bound proteins were analysed by SDS-PAGE followed by immunoblotting using anti-His antibodies. The input (inp.) corresponds to 10% of the import receptor used for the binding reaction. The faster-migrating band in the input lane for importin-9 is probably a degradation product that also interacts with GST-Rev in the absence but not in the presence of RanGTP. Scale bars: 10 µm.

 

Figure 2
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  		Fig. 2. Transportin is the major import receptor for HIV-1 Rev in vivo. (A) HeLa cells were co-transfected with reporter constructs coding for GFP2-M9core, GFP2-cNLS or HA-Rev, as indicated, either with an empty vector (–M9M) or with a plasmid coding for Myc-MBP-M9M (+M9M) at a ratio of 1:7. Cells were stained for DNA and MBP-M9M (M9M), as indicated. The reporter proteins were either detected directly (GFP2-M9 core, GFP2-cNLS; top and middle panels) or by direct immunofluorescence (bottom panel). Cells were analysed by fluorescence microscopy. (B) HeLa cells were transfected with Rev-GFP either alone or in the presence of Myc-MBP-M9M and either empty vector, HA-transportin (HA-trn), HA–importin-9 (HA-imp 9) or HA–importin-β (HA-imp β), as indicated, at a ratio of 1:1:12. Cells were stained for MBP-M9M, import receptors and DNA, as indicated, and analysed by fluorescence microscopy. (C) GST-M9M was immobilised on beads an incubated with either transportin or importin-9 in the absence or presence of RanQ69L-GTP. Bound proteins were analysed by SDS-PAGE and immunoblotting using an anti-His antibody. The input (inp.) corresponds to 10% of the import receptors used in the binding reaction. (D) Mock-treated cells or those treated with an siRNA against transportin were transiently transfected with HA-Rev and NES (Rev aa 68-90)-GFP2-M9core, stained for HIV-1 Rev and analysed by fluorescence microscopy. Scale bars: 10 µm.

 

Figure 3
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  		Fig. 3. Nup358 is required for the nuclear localisation of HIV-1 Rev in vivo. (A) Mock-treated or Nup358-depleted HeLa cells were transfected with a plasmid coding for HA-Rev and stained for DNA, HIV-1 Rev and Nup358, as indicated. (B) The mean distribution of the cells in the three categories (N>C, N=C or N<C) for HA-Rev in mock-treated or Nup358-depleted cells is shown. Bars indicate the standard deviation from the mean of six independent experiments with >100 cells analysed for each condition and experiment. (C) HeLa cells were co-transfected with a plasmid coding for untagged HIV-1 Rev and HA-Nup358 (aa 2595-2881) at a ratio of 1:7, and stained for DNA, HIV-1 Rev, RanGAP and HA-Nup358. Scale bars: 10 µm.

 

Figure 4
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  		Fig. 4. Depletion of Nup358 inhibits nuclear import of HIV-1 Rev. Mock-treated or Nup358-depleted HeLa cells were transfected with a construct coding for (A) HA-Rev or (B) Rev-GR-GFP (RGG) and either fixed before (–LMB) or after (+LMB) treatment with 10 nM LMB for 3 hours. Cells were stained for Nup358 (A,B) and HIV-1 Rev (A), and were analysed by fluorescence microscopy. Note that, in A, a rabbit anti-Nup358 antibody was used for indirect immunofluorescence, whereas a goat anti-Nup358 antibody was used in B. The RGG-protein in B was detected directly. Scale bars: 10 µm.

 

Figure 5
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  		Fig. 5. Transportin is a rate-limiting factor for the nuclear import of HIV-1 Rev in Nup358-depleted cells. (A) Mock-treated or Nup358-depleted cells were co-transfected with plasmids coding for untagged HIV-1 Rev and either empty vector, HA-transportin, HA–importin-9 or HA–importin-β, as indicated, at a ratio of between 1:4 and 1:5. Cells were stained for DNA, Nup358, HIV-1 Rev and HA-tagged import receptors, and were analysed by fluorescence microscopy. Scale bar: 10 µm. (B) A quantification of cells showing a clear nuclear localisation of HIV-1 Rev (N>C) in the absence or presence of HA-tagged import receptors. Error bars indicate the standard deviation from the mean of three independent experiments with >100 cells analysed in each single experiment.

 

Figure 6
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  		Fig. 6. Nup358 promotes transportin-mediated import. Mock-treated or Nup358-depleted cells were transfected with a construct coding for either GR2-GFP2-M9core (A,B) or GR2-GFP-hnRNP M (C,D) and either fixed before (–dex) or after (+dex) the addition of dexamethasone for 10 minutes at 37°C. Cells were stained for Nup358 and analysed by fluorescence microscopy. (B,D) A quantification of the subcellular localisation of the reporter protein (N>C, N=C and N<C) in mock-treated or Nup358-depleted cells. Error bars indicate the standard deviation from the mean of three independent experiments with >100 cells analysed in each single experiment. Scale bars: 10 µm.

 

Figure 7
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  		Fig. 7. Depletion of Nup358 leads to a reduced import kinetic of a transportin-dependent substrate. Mock- or Nup358-depleted cells were transfected with a construct coding for GR2-GFP2-M9core. After the addition of dexamethasone (t=0), the rate of nuclear import was measured for 15 minutes. (A) The graph shows the ratio of nuclear to total fluorescence over time. Error bars indicate the standard deviation from the mean of 65 (mock-treated) or 74 (Nup358-depleted) cells. (B) An example for mock-treated and Nup358-depleted cells before (t=0) or 15 minutes after (t=890) the addition of dexamethasone is shown. Efficient depletion of Nup358 was confirmed in a parallel experiment by immunofluorescence (data not shown). The experiment was repeated twice with very similar results.

 

Figure 8
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  		Fig. 8. The model depicts the role for Nup358 in the coordination of the recycling of HIV-1 Rev and transportin, as well as the formation of new import complexes containing HIV-1 Rev at the NPC. (1) Export from the nucleus results in the binding of the export complex consisting of HIV-1 Rev, viral RNA, RanGTP and CRM1 to its cytoplasmic docking site, Nup214 (Hutten and Kehlenbach, 2006Go). In parallel, transportin in complex with RanGTP is re-exported into the cytoplasm and targeted to Nup358. (2) The concerted action of Nup358-associated RanGAP and the Ran-binding domains of Nup358 promote hydrolysis of RanGTP to RanGDP, leading to the dissociation of HIV-1 Rev and of the viral RNA from the export complex, and the release of transportin for a new round of nuclear import. (3) The close proximity of these reactions at Nup358 allows the formation of new import complexes containing HIV-1 Rev and transportin at the nuclear pore. Additionally, Nup358 might serve as a recycling station for CRM1 (Hutten and Kehlenbach, 2006Go) on its way back into the nucleus. (4) The high concentration of RanGTP in the nucleus leads to the dissociation of HIV-1 Rev from transportin and the formation of new export complexes containing viral RNA, HIV-1 Rev, CRM1 and RanGTP.

 

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© The Company of Biologists Ltd 2009