Importin beta regulates the seeding of chromatin with initiation sites for nuclear pore assembly

Abstract

The nuclear envelope of higher eukaryotic cells reforms at the exit from mitosis, in concert with the assembly of nuclear pore complexes (NPCs). The first step in postmitotic NPC assembly involves the "seeding" of chromatin with ELYS and the Nup107-160 complex. Subsequent steps in the assembly process are poorly understood and different mechanistic models have been proposed to explain the formation of the full supramolecular structure. Here, we show that the initial step of chromatin seeding is negatively regulated by importin beta. Direct imaging of the chromatin attachment sites reveals single sites situated predominantly on the highest substructures of chromatin surface and lacking any sign of annular structures or oligomerized pre-NPCs. Surprisingly, the inhibition by importin beta is only partially reversed by RanGTP. Importin beta forms a high-molecular-weight complex with both ELYS and the Nup107-160 complex in cytosol. We suggest that initiation sites for NPC assembly contain single copies of chromatin-bound ELYS/Nup107-160 and that the lateral oligomerization of these subunits depends on the recruitment of membrane components. We predict that additional regulators, besides importin beta and Ran, may be involved in coordinating the initial seeding of chromatin with subsequent steps in the NPC assembly pathway.

Figure 1.

Importin β inhibits the binding of ELYS and Nup107-160 to chromatin. Anchored chromatin-binding assays were conducted with membrane-free Xenopus egg cytosol and processed for indirect immunofluorescence (A) or immunoblot analysis (B and C), as described in Materials and Methods. (A) Cytosol was preincubated with 20 μM ovalbumin (control), or 20 μM human importin β (hImp β), before addition on to coverslips with poly-lysine–tethered chromatin templates. Chromatin binding was carried out for 30 min at room temperature. DNA was stained with Hoechst 33258, and binding was visualized by staining with affinity-purified antibodies directed against ELYS and Nup107. Scale bar, 10 μm. (B) Immunoblot analysis of the chromatin-bound fraction from reactions performed on chromatin-coated coverslips. Membrane-free cytosol was preincubated with different amounts of histidine-tagged full-length human importin β (hImp β) or untagged full-length Xenopus importin β (XImp β) and then added on to chromatin-coated coverslips. The blot was probed for the endogenous proteins: ELYS, Nup160, Nup133, Nup107, Nup85, Nup43, and histone H3. ELYS and Nup107-160 complex members were absent from reactions containing an excess of recombinant importin β. (C) The inhibitory effect of importin β occurs through interactions in cytosol. Immunoblot analysis was performed as in B, with 20 μM human importin β added to the reactions in lanes 1–3 and 20 μM ovalbumin added to the control in lane 4. In lanes 1 and 4, the recombinant proteins were preincubated with cytosol before the addition onto chromatin-coated coverslips. In lane 2, importin β was preincubated with chromatin and washed once, and cytosol was subsequently added. In lane 3, cytosol was added to the chromatin-coated coverslip, followed by a wash and a subsequent incubation with importin β. Only the preincubation of importin β in cytosol (lane 1) resulted in the inhibition of binding.

Figure 2.

Direct visualization of ELYS/Nup107-160 attachment sites on chromatin by FESEM. Chromatin-coated silicon chips were incubated in egg cytosol, fixed, and processed for immunogold labeling. (A) In-lens image showing the surface topology of chromatin, and (B) the corresponding backscatter detector image revealing the position of gold particles after labeling with anti-Nup107. (C) Anti-ELYS and (D) anti-Nup133, immunolabeling with pseudocolored gold particle positions superimposed from the backscatter images. Note that the ELYS/Nup107-160 attachment sites occur predominantly on the elevated substructures of chromatin. (E) Immunogold labeling with anti-histone H3 is preferentially localized to lower substructures. Scale bars, 100 nm.

Figure 3.

Immunodepletion of ELYS inhibits chromatin seeding. (A) Membrane-free cytosol was treated by two rounds of incubation with immobilized preimmune (mock depletion) or anti-ELYS antibodies. The amount of ELYS in mock-depleted (mock) and ELYS-depleted (ΔELYS) cytosol were compared by immunoblotting. Varying amounts of untreated cytosol were loaded for comparison. Nup107 was not codepleted with ELYS. (B) Mock-depleted and ELYS-depleted cytosol were incubated with chromatin-coated silicon chips and processed for FESEM with anti-ELYS and gold-conjugated secondary antibodies as in Figure 2. Pseudocolored gold particle positions were superimposed from the backscatter images. Scale bar, 100 nm.

Figure 4.

RanGTP is not sufficient to counteract the negative effect of importin β. Anchored chromatin-binding assays were analyzed by indirect immunofluorescence with anti-ELYS antibody as in Figure 1. Recombinant proteins were preincubated with cytosol before the addition onto chromatin-coated coverslips. (A) Representative images from binding reactions containing cytosol supplemented with 20 μM ovalbumin (control), 20 μM Xenopus importin β (XImp β), or 20 μM Xenopus importin β + 40 μM RanQ69L-GTP (XImp β + RanGTP). Scale bar, 10 μm. (B) Quantitative analysis summarizing three separate experiments of the type shown in A. Anti-ELYS immunofluorescent signal intensity was measured exclusively from the chromatin surface on 12 randomly chosen, nonoverlapping templates in each category. Normalized fluorescence intensity is shown after the subtraction of nonspecific staining, measured on identical coverslips for which the primary antibody was omitted from the procedure. Error bar, SD.

Figure 5.

Two distinct subpopulations of the Nup107-160 complex in egg cytosol differ in ELYS content. Xenopus egg cytosol was subfractionated on a DEAE Affi-Gel Blue column followed by a Q-Sepharose column. The second column was eluted with a linear 0.1–1 M NaCl gradient. The column elution profile is shown by OD₂₈₀ absorbance (in blue). Fractions 26–34, from the region containing ELYS and Nup107-160 complex members, were analyzed by immunoblotting as shown in the bottom panel. The blot was probed with antibodies to ELYS and Nup107-160 complex members. Only fractions 29–31, eluted at 200–230 mM NaCl, contained significant amounts of ELYS, whereas Nup107-160 complex members peaked over the whole 200–260 mM NaCl range (fractions 29–34).

Figure 6.

Only a subfraction of the endogenous Nup107-160 complex in egg extract is associated with ELYS and capable of seeding chromatin. (A) Fractions of equal volume, eluted from the Q-Sepharose column at 225 mM NaCl (fraction A, enriched in ELYS) and 250 mM NaCl (fraction B, lacking ELYS) were desalted, concentrated, and analyzed by immunoblotting. The two fractions contain comparable amounts of Nup133, Nup107, and Nup43, but only fraction A contains ELYS. (B) Fractions A and B were tested in the immunofluorescence binding assay, as in Figure 1. Chromatin binding was visualized by staining with antibodies directed against ELYS and Nup107. Only fraction A was able to bind to chromatin. Scale bar, 10 μm.

Figure 7.

Importin β is associated with the ELYS+ subpopulation of the Nup107-160 complex. Fraction A (enriched in ELYS) and fraction B (lacking ELYS) eluted from a Q-Sepharose column as in Figure 6 were desalted and used for immunoprecipitation with affinity-purified anti-Nup107 or control rabbit IgG. Nup160, Nup133, and Nup43 were coimmunoprecipitated by anti-Nup107 from both fractions, whereas ELYS and importin β were only significantly pulled out from fraction A. Note that importin β is a very abundant protein in egg cytosol and is present throughout the entire elution profile of the Q-Sepharose column.

Figure 8.

Importin β forms a high-molecular-weight complex with both ELYS and Nup107-160. (A) ELYS-enriched fractions eluted from a Q-Sepharose column, as in Figure 5, were pooled and loaded on a Superose-6 column. One-half of the sample was preincubated with 10 μM Xenopus importin β before gel filtration (+XImp β rows). Samples eluted from these two gel filtration runs (−/+β) were analyzed by immunoblotting. Note that the addition of excess importin β does not shift ELYS to lower molecular weight fractions. (B) Two Superose-6 gel filtration runs (−/+ importin β) were performed as in A. Fractions 5–8 eluted from each column were pooled together and subjected to immunoprecipitation with anti-ELYS or anti-Nup107 antibodies. Immunoblotting confirms the existence of a high-molecular-weight complex containing ELYS, Nup107-160, and importin β.

Figure 9.

A RanGTP affinity column pulls out ELYS and the Nup107-160 complex from cytosol. (A) ELYS-enriched fractions eluted from a Q-Sepharose column were pooled and loaded on an affinity column of immobilized RanQ69L-GTP. Samples were analyzed by immunoblotting. Equivalent samples of the starting material and the flowthrough fraction of the RanQ69L-GTP column were loaded in the first two lanes. ELYS, Nup107-160, and importin β were retained on the affinity column. The column was washed with four column volumes and sequentially eluted with buffer containing 250 mM, 450 mM, and 1 M KCl. Samples from consecutive eluted fractions were loaded for each step. Only ELYS and Nup107-160 complex members were eluted in the first step, whereas importin β remained bound to the column. (B) Fractions eluted from a Q-Sepharose column were probed with antibodies directed against four different transport receptors of the importin β superfamily and compared with a sample of complete cytosol (0.3 μl). Importin β was present in all of these fractions, CRM 1 and exportin-t were not detected, whereas transportin peaked in fractions 24–28, showing minimal overlap with ELYS/Nup107-160.