Volume 110, Issue 3, Pages 349-360 (August 2002) Npap60/Nup50 Is a Tri-Stable Switch that Stimulates Importin-α:β-Mediated Nuclear Protein Import  Mark E. Lindsay, Kendra Plafker, Alicia E. Smith, Bruce E. Clurman, Ian G. Macara  Cell  Volume 110, Issue 3, Pages 349-360 (August 2002) DOI: 10.1016/S0092-8674(02)00836-X

Figure 1 Npap60 Is an Importin-α:β Binding Partner (A) Domain alignment of RanBP3 with the related Npap60 protein. Importin-α binding motif (IABM), FG repeats, and Ran binding domain (RBD) are indicated. (B) Npap60 binds importin-α:β from HeLa cytosol. Cytosol was exposed to GST or GST-Npap60 on beads ± NLS peptide (40 μM). Cytosol (100 μg) and one half of GST and GST-Npap60 bound fractions were stained with Coomassie (CBB) (Asterisk indicates GST-Npap60 and a GST-Npap60 breakdown product). Immunoblotting (IB) was on 1/20 of total fractions. (C) Yeast two-hybrid interaction assay. S. cerevisiae HF7c(MATa) expressing GAL4DBD-Crm1 or -importin-β were mated to W303(MATα) yeast expressing VP16-fusions as shown. (D) Coimmunoprecipitation of Npap60 and importin-β. Cytosol was precipitated with mAbs to importin-β (anti-imp-β) or hemaglutinin (control). (E) The Npap60:importin-β interaction is direct and specific. GST-tagged Npap60, Npap60 F domain (aa 214–319), RanBP3, or RanBP3 F domain (aa 182–292) (2 μg) were immobilized on beads in the presence of His6-tagged Crm1 or importin-β (1 μM). Proteins were stained with Coomassie or immunoblotted for His6. Cell 2002 110, 349-360DOI: (10.1016/S0092-8674(02)00836-X)

Figure 2 Npap60 Shuttles between the Nucleus and the Cytoplasmic Side of the Nuclear Pore Complex (A) Npap60 is a nucleocytoplasmic shuttling protein. GSN2 cells (labeled “D” for donor) and Rat1 cells (“A” for acceptor) were coseeded. Cells were fused and incubated for 2 hr with cycloheximide. GSN2 nuclei contain a nonshuttling GFP-Stv-NLS fusion protein. Unfused cells are labeled “U”. Inset shows specificity of the anti-Npap60 antibody for human protein. (B and C) Npap60 is accessible to the cytoplasmic side of the nuclear envelope. HeLa or NIH3T3 cells were fixed with 2% formaldehyde/PBS (FA) before permeabilization with either 0.1% Triton X-100 (TX-100) or 0.08% digitonin (Dig), or were fixed after permeabilization with 0.003% digitonin. Hela cells were stained with anti-human Npap60 and anti-lamin A/C. NIH3T3 cells were stained with anti-mouse Npap60 and anti-lamin A/C. (D) Colocalization of Npap60 and nucleoporins. HeLa cells were treated with 0.003% digitonin, fixed with 2% formaldehyde, and then stained with anti-human Npap60 (green) and mAb414 (red). Colocalization is shown by yellow in the merged image. Cell 2002 110, 349-360DOI: (10.1016/S0092-8674(02)00836-X)

Figure 3 The N Terminus of Npap60 Interacts with the C Terminus of Importin-α (A) GST-tagged importin-α3 (400 nM) was exposed to His6-Npap60 or RanBP3 (2 μM) ± NLS peptide (70 μM). Proteins were stained with Coomassie (CBB) or immunoblotted (IB) for His6-Npap60 and RanBP3. (B) Npap60 binds to the C terminus of importin-α. 35S-labeled Npap60 was incubated with GST-tagged C-terminal tails of importin-α4 (aa 349–521) and (aa 396–521) on beads. Bound proteins were visualized by fluorography. Two-hybrid mating assays were performed as described in Figure 1C. (C) Schematic representation of importin-α. Binding sites of importin-β (Görlich et al., 1996a), NLSs (Conti et al., 1998; Fontes et al., 2000), CAS (Herold et al., 1998), and Npap60 are indicated. Residue numbering is from importin-α4. (D) Npap60 binds several importin-α isoforms. In vitro binding assay in (B) was repeated with C termini of importin-α1, -4, and -5. (E) The Npap60:importin-α interaction is direct and requires a polybasic motif. GST-GFP-Npap60 (aa 1–109) (GGN109) or a mutant in which the KRR motif has been mutated to AAA (KRR) (1.2 μM) were exposed to His6-tagged (500 nM) importin-α, importin-β, or both proteins. Bound proteins were detected with anti-His6 antibodies. Cell 2002 110, 349-360DOI: (10.1016/S0092-8674(02)00836-X)

Figure 4 Importin-α Can Bind and Import both Npap60 and a Monopartite NLS Simultaneously (A) SV40 NLS and Npap60 IABM peptide beads were exposed to 400 nM of importin-α, GST-importin-α(396–521), or GST. Bound proteins were detected with anti-His6 or anti-GST antibodies. (B) Importin-α can bind NLS and the N terminus of Npap60 simultaneously. NLS beads were exposed to His6-tagged importin-α (500 nM), importin-β (500 nM), GGN109 (1.2 μM), or combinations of these factors. Bound proteins were detected with anti-His6 or anti-GFP antibodies. As a control for importin-α dimerization, the beads were exposed to importin-α, importin-β, and GGNLS (1.2 μM). (C) The N terminus of Npap60 is imported by importin-α:β. GGN109 (2 μM) was applied to digitonin-permeabilized cells plus 2 μM importin-α, importin-β, energy, and Ran. Hexokinase-glucose was added to deplete ATP (-energy). Quantitation of import is shown in arbitrary units ± SEM (n >80 cells/sample). (D) Import of (2 μM) GGN109 or GGNLS were performed ± NLS peptide (100 μM). Quantification of import was done as in 4C. Cell 2002 110, 349-360DOI: (10.1016/S0092-8674(02)00836-X)

Figure 5 Npap60 Potentiates Nuclear Protein Import at Limiting Concentrations of Transport Factors (A) Npap60, importin-β, and importin-α form a complex that binds NLS-cargo. Importin-α (200 nM), importin-β (1 μM), Npap60 (1 μM), or combinations of these factors were exposed to NLS peptide beads or to control beads (c). Bound proteins were identified by Coomassie staining. (B) Npap60 stabilizes the importin-α:β heterodimer. GST-importin-β was exposed to increasing concentrations of importin-α1 ± Npap60. Bound importin-α was detected by immunoblotting. Percent maximal binding was calculated by densitometry. (C) Npap60 specifically stimulates importin-α:β-mediated nuclear import. (a-h) Importin-α (imp-α1), importin-β (imp-β), or transportin (Tp) were applied to digitonin-permeabilized cells ± Npap60 (1 μM) plus Ran, energy and GGNLS, or GGM9 (1 μM). Quantification was done as in 4C. (i-k) Export of endogenous importin-α was monitored in digitonin-permeabilized nuclei exposed to Ran alone (12 μM), Ran plus Npap60 (1.5 μM), or Ran plus CAS (1.5 μM). Quantification of export is in arbitrary fluorescence units ± SEM (n >25 cells/sample). Cell 2002 110, 349-360DOI: (10.1016/S0092-8674(02)00836-X)

Figure 6 Npap60 Uses a Modular Structure to Interact with Importin-α and RanGTP. (A) GST-tagged Npap60, or GST-tagged N, F, or R domains of Npap60 were exposed to His6-importin-β (1 μM) ± importin-α. Bound proteins were immunoblotted with anti-importin-α antibodies. (B) As in (A), ± RanQ69L (5 μM). Bound proteins were immunoblotted with anti-Ran antibodies. (C) As in (A), ± His6-FF5 fragment of Nsp1p (10 μM). (D) GST-tagged Npap60 was exposed to importin-β and the FF5 fragment of Nsp1p (10 μM) ± importin-α or Ran Q69L. Asterisks mark full-length GST-fusions. Schematic representations of interactions are shown on the right. Components are labeled: importin-β (β), RanGTP (RT), importin-α (α), Npap60 (NFR), N domain (N), F domain (F), R domain (R), and FF5 fragment of Nsp1p (FF5). Cell 2002 110, 349-360DOI: (10.1016/S0092-8674(02)00836-X)

Figure 7 Disassembly of Npap60 Complexes (A) RanGTP is insufficient to remove Npap60:importin-α:β complex from NLS. Factors were exposed to control (C) or NLS beads as in Figure 5A with the addition of RanQ69L (6 μM). Proteins were immunoblotted with anti-His6. (B) CAS and RanGTP remove Npap60:importin-α:β complex from NLS. NLS beads were mixed 1:1 with glutathione-Sepharose beads loaded with 1 μg GST per sample. Importin-β, importin-α, and Npap60 were added to beads as in (A), with addition of RanQ69L (6 μM), CAS (2 μM), or both proteins. (C) CAS and RanGTP remove importin-α from Npap60:importin-α:β complex. Importin-β (1 μM), importin-α (200 nM), RanQ69L (6 μM), or CAS (2 μM) were added to immobilized GST-Npap60. Bound proteins were visualized by Coomassie staining. Asterisk marks GST-Npap60 breakdown product. (D) Binding of importin-α to the N terminus of Npap60 is competitive with binding of CAS. Importin-α (200 nM), CAS (2 μM), or RanQ69L (6 μM) were added to immobilized GST-N domain. Bound proteins were visualized by Coomassie. (E) Npap60 potentiates importin-α and RanBP1-mediated stimulation of RanGAP hydrolysis. GST-RanGAP (30 nM) ± RanBP1 (1 μM) were added to preassembled complexes of Ran[γ32P-GTP] (3 nM), importin-β (16 nM) ± Npap60 (1 μM), and with increasing amounts of importin-α. Protein-bound [γ32P]-GTP was detected by filter binding to nitrocellulose and scintillation counting. (F) Npap60 potentiates importin-α and RanBP1-mediated release of RanGTP from importin-β. GST-importin-β was immobilized on beads and loaded stoichiometrically with Ran[α32P-GTP]. After washing to remove unbound RanGTP, beads were split into two pools and Npap60 was preincubated with one pool for 20 min. At t = 0, bead pools were split in three and (100 nM) importin-α RanBP1, or both proteins were added and beads were incubated at 25°C. Bead aliquots were taken at indicated times, and after washing, loss of importin-β bound RanGTP was monitored by scintillation counting. (G) Model of Npap60-assisted importin-α:β-mediated nuclear protein import cycle. Npap60 (NFR), importin-β (β); importin-α (α); RanGDP (RD); and RanGTP (RT). Cell 2002 110, 349-360DOI: (10.1016/S0092-8674(02)00836-X)