J Cell Biol. 1998 Apr 6;141(1):31-49.

Major binding sites for the nuclear import receptor are the internal nucleoporin Nup153 and the adjacent nuclear filament protein Tpr.

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Department of Biology, University of California at San Diego, La Jolla, California 92093, USA.

Abstract

A major question in nuclear import concerns the identity of the nucleoporin(s) that interact with the nuclear localization sequences (NLS) receptor and its cargo as they traverse the nuclear pore. Ligand blotting and solution binding studies of isolated proteins have attempted to gain clues to the identities of these nucleoporins, but the studies have from necessity probed binding events far from an in vivo context. Here we have asked what binding events occur in the more physiological context of a Xenopus egg extract, which contains nuclear pore subcomplexes in an assembly competent state. We have then assessed our conclusions in the context of assembled nuclear pores themselves. We have used immunoprecipitation to identify physiologically relevant complexes of nucleoporins and importin subunits. In parallel, we have demonstrated that it is possible to obtain immunofluorescence localization of nucleoporins to subregions of the nuclear pore and its associated structures. By immunoprecipitation, we find the nucleoporin Nup153 and the pore-associated filament protein Tpr, previously shown to reside at distinct sites on the intranuclear side of assembled pores, are each in stable subcomplexes with importin alpha and beta in Xenopus egg extracts. Importin subunits are not in stable complexes with nucleoporins Nup62, Nup93, Nup98, or Nup214/CAN, either in egg extracts or in extracts of assembled nuclear pores. In characterizing the Nup153 complex, we find that Nup153 can bind to a complete import complex containing importin alpha, beta, and an NLS substrate, consistent with an involvement of this nucleoporin in a terminal step of nuclear import. Importin beta binds directly to Nup153 and in vitro can do so at multiple sites in the Nup153 FXFG repeat region. Tpr, which has no FXFG repeats, binds to importin beta and to importin alpha/beta heterodimers, but only to those that do not carry an NLS substrate. That the complex of Tpr with importin beta is fundamentally different from that of Nup153 is additionally demonstrated by the finding that recombinant beta or beta45-462 fragment freely exchanges with the endogenous importin beta/Nup153 complex, but cannot displace endogenous importin beta from a Tpr complex. However, the GTP analogue GMP-PNP is able to disassemble both Nup153- and Tpr-importin beta complexes. Importantly, analysis of extracts of isolated nuclei indicates that Nup153- and Tpr-importin beta complexes exist in assembled nuclear pores. Thus, Nup153 and Tpr are major physiological binding sites for importin beta. Models for the roles of these interactions are discussed.

PMID:: 9531546; [PubMed - indexed for MEDLINE]
PMCID:: PMC2132719

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Figure 3

Distinct immunofluorescent localization of Nup153, Nup214, and Tpr. Anti-Nup153 (380) antibody was covalently linked to fluorescein, whereas anti-Tpr antibody was directly linked to rhodamine or fluorescein independently, using isothiocyanate derivatives (RITC and FITC, respectively) as described in Materials and Methods. For immunostaining and colocalization, anti-Nup214 was first used to immunostain formaldehyde fixed XL177 cells. After incubation of the cells with goat anti–rabbit-RITC antibody and subsequent blocking of this antibody with excess IgG, cells were then stained with FITC–anti-Tpr antibody, as described in Materials and Methods (a–e). The anti-Tpr stain is punctate and lies more to the nuclear interior than the anti–Nup214 stain. To compare localization of Nup153 and Tpr (f–i), XL177 cells were costained with FITC–anti-Nup153 (380) antibody and RITC–anti-Tpr antibody. Nup153 and Tpr show a more overlapping localization, although a large amount of Tpr staining still lies more intranuclearly than that of Nup153. A low magnification of the colocalization is shown in c and h, a 4× higher magnification view in panels immediately above and below (b, d, g, i), and an 8× higher magnified view lies above and below these (a, e, f). The nuclei shown contain internal tunnels of nuclear envelope, with magnified views of the tunnel shown in d, e, and i. Magnified views of the circumferential nuclear envelope are shown in a, b, f, and g. Note that the Nup214 staining (a–e) is always closer to the cytoplasm, whereas the Tpr staining (a–i) is always more proximal to the nuclear interior. j shows mAb414 (Oregon Green) and anti-Tpr (RITC) staining of a XL177 tissue culture cell, showing a segment of the nuclear envelope. Bar, ∼1 μm.

Major Binding Sites for the Nuclear Import Receptor Are the Internal Nucleoporin Nup153 and the Adjacent Nuclear Filament Protein Tpr

J Cell Biol. J Cell Biol;141(1):31-49.

Figure 5

Stable independent complexes of Nup153 and Tpr with importin β exist. (A) Coimmunoprecipitation of a ∼97-kD band with Nup153 and with Tpr. Anti-Nup153 (380) and preimmune antibodies were bound to protein A–Sepharose (lane 1 and 2, respectively) and used for immunoprecipitation from 20 μl soluble Xenopus egg extract, as described in Materials and Methods. The immunoprecipitates were boiled with sample buffer, separated using SDS-PAGE (8%), and silver stained. In addition to Nup153, a prominent protein band of ∼97 kD immunoprecipitated with anti-Nup153 (lane 2). The markers for this gel are 193, 112, 86, and 70 kD. A separate experiment was performed with anti-Tpr antiserum; a band of ∼97 kD was observed to precipitate with Tpr (lane 3). The markers for this gel are 205, 116, and 97.4 kD. (B) Immunoprecipitations from soluble Xenopus egg extract were performed using anti-153 (380), anti-Tpr, or rabbit IgG antibodies coupled to protein A–Sepharose. Before antibody addition, the extract was diluted (1:100) in either ELBS buffer (lanes 1–3), or RL buffer (lanes 4 and 8). In other incubations Xenopus egg extract was preincubated with GMP-PNP (lanes 6 and 10) or AMP-PNP (lanes 5 and 9) before dilution with buffer RL and immunoprecipitation, as above. The immunoprecipitated proteins and an aliquot of soluble Xenopus egg extract (lane 7) were analyzed by gel electrophoresis and transfer to PVDF. The blot on the left was cut and probed by immunoblotting with anti-Tpr (top), anti-Nup153 (381; middle) or a mix of anti–importin α and β antibodies (bottom). The blot on the right was probed with a mix of anti-Nup153, importin β, and importin α antibodies. Importin α and β immunoprecipitate with anti-Nup153 (lane 1) and anti-Tpr (lane 2) antisera. The binding of importin β to Nup153 and Tpr is stable in both ELBS (lanes 1 and 2) and RL (lanes 4, 5, 8, and 9) buffers; however, the binding to importin α is disrupted in the more stringent RL buffer (lanes 4, 5, 8, and 9). GMP-PNP disrupts the complex of importin β with Tpr (lane 6) and Nup153 (lane 10). The differences in the amounts of TPR in Fig. 5, lanes 4–6 are due to inefficient transfer of the very large Tpr protein (∼270 kD) from the 8% gel used to simultaneously view importin α; reelectrophoresis of the samples revealed only small differences in the amounts of Tpr per lane (data not shown).

Major Binding Sites for the Nuclear Import Receptor Are the Internal Nucleoporin Nup153 and the Adjacent Nuclear Filament Protein Tpr

J Cell Biol. J Cell Biol;141(1):31-49.

Figure 4

Importin β is in a complex with Nup153, but not Nup62, Nup93, Nup98, or Nup214. (A) Immunoprecipitations were conducted from Xenopus egg extract diluted in buffer RL using anti-Nup153 (361), protein A–purified 361 preimmune, anti-Nup93, and anti-Xenopus Nup98 antibodies. The antibody used for immunoprecipitation is shown at the top of each lane. The supernatants of the immunoprecipitates (lanes 1–4) were immunoblotted with a mixture of anti-Nup93, anti-Xenopus Nup98, and anti-rat Nup153 antibodies to demonstrate that each nucleoporin had been quantitatively immunoprecipitated. Lane 1 is depleted of Nup93, lane 2 is depleted of Nup98, and lane 3 is depleted of the great majority of Nup153. The proteins of the pellets immunoprecipitated from 2.5 μl Xenopus egg extract (lanes 5–8) were immunoblotted with anti–importin β antiserum. Soluble Xenopus egg extract (0.05 μl) is shown (lane 9) to indicate where importin β migrates. Importin β coimmunoprecipitates with Nup153 (lane 7), but not with Nup93 (lane 5) or Nup98 (lane 6). (B) Immunoprecipitation from soluble Xenopus egg extract diluted in buffer RL were performed using anti-Nup153 (380) and preimmune antisera bound to protein A–Sepharose. Immunoprecipitated protein was eluted with glycine and separated on an 8% SDS-PAGE gel along with aliquots of Xenopus egg extract (lanes 1, 4, and 7). Proteins were transferred to PVDF and analyzed by immunoblotting with the following antibodies: anti–importin β (lanes 1–3), anti-Xenopus Nup98 (lanes 4–6), anti-Nup93 (lanes 7–9), and anti-Nup153 (lanes 10 and 11). Note that importin β is detected in Nup153 immunoprecipitates and Xenopus egg extract (lane 1 and 3), but not in a preimmune immunoprecipitate (lane 2). Also note that Nup98 and Nup93 are detected in Xenopus egg extract (lanes 4 and 7), but not in preimmune or Nup153 immunoprecipitates (lanes 5, 6, 8, and 9). (C) Immunoprecipitates of Nup153, Nup214, and Tpr were separated, transferred, and probed with anti–importin β, anti-Tpr, and mAb414 to detect Nup214, Nup153, and Nup62. An asterisk marks a breakdown product of Tpr. Importin β immunoprecipitates with Nup153 (lane 1) and Tpr (lane 3), but not with Nup214 (lane 2). (D) Immunoprecipitates of Nup153, Nup62, and control IgG were separated, transferred, and probed with anti–importin β, and mAb414 to detect Nup62. Full-length recombinant importin β immunoprecipitates with Nup153 (lane 2), but not with Nup62 (lane 4) or nonspecific IgG (lane 6).

Major Binding Sites for the Nuclear Import Receptor Are the Internal Nucleoporin Nup153 and the Adjacent Nuclear Filament Protein Tpr

J Cell Biol. J Cell Biol;141(1):31-49.

Figure 6

Nup153, but not Tpr, binds to importin α/β heterodimer in the NLS-bound form. (A) Anti-HSA antibody coupled to protein A–Sepharose was added to Xenopus egg extract (diluted 1:100 in buffer ELBS) containing 2.4 μg HSA (lane 2) or 1.9 μg NLS-HSA (lane 3). The proteins that bound to the NLS-HSA/anti-HSA/ Protein A beads were eluted and immunoblotted with a mix of anti–importin α and β and anti-Nup153 (381) antibodies. The proteins present in 0.2 μl of soluble Xenopus egg extract are shown in lane 1 for comparison. Importin α, importin β, and Nup153 all bind to an NLS-HSA affinity column (lane 3), but not to an HSA column (lane 2). (B) GMP-PNP disrupts Nup153 bound to importin α/β– NLS complexes. NLS-HSA/anti-HSA Sepharose beads were added to Xenopus egg extract pretreated with GMP-PNP, AMP-PNP, or no addition (lanes 2–4, respectively). Before bead addition, extracts were diluted with buffer ELBS and supplemented with 1.9 μg of NLS-HSA per aliquot. The NLS-HSA bead mixtures were incubated in extract for 2 h. Then bound proteins were eluted, electrophoresed, and immunoblotted with mAb414 (lanes 1–4). Soluble Xenopus egg extract (0.2 μl) was immunoblotted in parallel (lane 1) to indicate the nucleoporins recognized in the total extract by mAb 414 (Nup358, Nup214, Nup153, and Nup62). Much of the Nup358 and Nup153, but only a trace of Nup62, bind to the NLS-HSA affinity column (lanes 3 and 4). GMP-PNP disrupts the majority of this binding (lane 2), whereas AMP-PNP does not (lane 3). (C) Tpr does not bind to importin α/β–NLS complexes. The experiment in (A) was repeated, and the bound proteins were immunoblotted with a mix of anti-Tpr, anti-Nup153 (381), anti–importin α and β (lanes 5–8). Nup153 and importin α and β bind to the NLS-HSA beads (lane 6), but Tpr does not (lane 6). GMP-PNP releases the importin β and Nup153 from the beads (lane 7), whereas AMP-PNP does not (lane 8). (An asterisk denotes a breakdown product of Tpr.)

Major Binding Sites for the Nuclear Import Receptor Are the Internal Nucleoporin Nup153 and the Adjacent Nuclear Filament Protein Tpr

J Cell Biol. J Cell Biol;141(1):31-49.

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Major binding sites for the nuclear import receptor are the internal nucleoporin Nup153 and the adjacent nuclear filament protein Tpr.

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