Nup53 domain interaction analysis using GST-pulldown assays. (A–C) Purified recombinant GST, GST-Nup53, or various GST-Nup53 truncations immobilized on glutathione-Sepharose 4B beads were incubated with extracts from purified rat liver NEs (+) or buffers alone (−). After binding and washing, bead-bound proteins were eluted, separated by SDS-PAGE, and detected with either silver staining (SS) or Western blotting (WB) by using antibodies against the indicated Nups. Positions of GST-fusions (white asterisks), relevant Nups (black asterisks and name), and mass markers are indicated in kilodaltons.

Depletion of Nup53 from cytosol blocks formation of a closed NE. (A) Amount of xNup53 in mock-depleted (Mock) and Nup53-depleted (ΔNup53) cytosolic extracts (equal volumes) were compared by SDS-PAGE and western blotting by using α-Nup53 antibodies. Varying amounts of untreated cytosol were loaded for comparison. A cross-reacting band indicated with an asterisk serves as a loading control. (B) For add-back experiments, amounts of recombinant proteins corresponding to approximately endogenous levels of Xenopus Nup53 (extract) were used. Purified full-length Nup53^1-326 and truncation fragments (Nup53^1-300, Nup53^1-203, Nup53^204-326, and Nup53^167-300) were analyzed by SDS-PAGE and Western blotting by using anti-Nup53 antibodies. Mass markers are indicated in kilodaltons. (C) Equal amounts of mock-depleted (Mock) or Nup53-depleted (ΔNup53) cytosolic extracts were analyzed by SDS-PAGE and Western blotting by using anti-Nup155, anti-Nup93, and anti-Nup205 antibodies. (D) Nuclei assembled in vitro in Mock, ΔNup53 extracts, or ΔNup53 extracts after addition of full-length Nup53. Membranes are visualized by using the membrane dye DilC₁₈ (red). Chromatin was stained with 4,6-diamidino-2-phenylindole (DAPI) (blue). Bar, 10 μm. (E) Quantitation of closed NE formation of experiments performed as in D. More than 100 randomly chosen chromatin substrates per reaction were counted. Average of three independent experiments are shown, error bars represent the SD over the three experiments.

Characterization of in vitro-assembled nuclei. (A) Nuclei assembled in vitro in Mock, ΔNup53 extracts, or ΔNup53 extracts after addition of full-length Nup53. Nuclear import reaction was performed using fluorescein isothiocyanate (FITC)-labeled BSA-NLS 20–30 min before fixation. Membranes are visualized by using the membrane dye DilC₁₈ (red). FITC BSA-NLS (green). Chromatin was stained with DAPI (blue). Bar, 10 μm. (B) Nuclei assembled in vitro in Mock, ΔNup53 extracts, or ΔNup53 extracts after addition of GST-Nup53. Localization of the recombinant protein was analyzed by immunofluorescence using α-GST (red). Chromatin was stained with DAPI (blue).

A Nup53 fragment that interacts with Nup155 is sufficient to restore NE formation. (A) Nuclei assembled in vitro in Mock, ΔNup53 extracts, or ΔNup53 extracts after addition of full-length Nup53, and the Nup53^167-300 fragment. Maximum intensity projections of three-dimensional stack are shown. Membranes are visualized by using the membrane dye DilC₁₈ (red). Chromatin was stained with DAPI (blue). (B) Nuclei assembled as described in A were analyzed by immunofluorescence by using Nup155 antibodies (red). Chromatin was stained with DAPI (blue). (C) Nuclei assembled as in A and B were analyzed by immunofluorescence using Nup93 antibodies (red). Chromatin was stained with DAPI (blue). Bar, 10 μm.

Summary of human Nup53 truncation characterization. Compilation of Nup53 truncation interaction, localization, and depletion phenotype rescue data. NH₂- and COOH-terminal human Nup53 truncations were constructed based on regions that are well conserved among orthologues with considerations of predicted secondary structure. Helical stretches from amino acid residues 181–193, 216–226, and 314–326 are represented by light gray bars. Interaction data were obtained either by silver staining, Western blot, or both. Interactions detected (+) or not detected (−) between indicated Nups and Nup53 truncations are shown. Localization was scored as either nuclear rim (NR), nuclear (N), or cytoplasmic (C). The ability of the various Nup53 truncations to rescue the Nup53 depletion phenotype was monitored by DilC₁₈ NE membrane staining.

In vivo localization of enhanced green fluorescent protein (EGFP)–Nup53 truncations. (A and B) Plasmids encoding either full-length Nup53 or the indicated truncations fused to EGFP were transfected into HeLa cells. Cellular distribution of the indicated GFP chimeras (GFP) and merged images of the GFP fusions and nuclear DNA was detected by staining with the DNA binding dye Hoechst (GFP-DNA) was determined by fluorescence microscopy following 36 h of transient expression. The transfected cells growing on coverslips were processed for fluorescence microscopy either by formaldehyde fixation followed by Triton X-100 permeabilization (F/T) or Triton X-100 permeabilization before formaldehyde fixation (T/F). Bar, 10 μm.

Depletion of Nup53 from cytosol blocks NPC assembly. (A) Transmission electron microscopy of nuclear assembly reactions of Mock, ΔNup53 extracts, or ΔNup53 extracts after addition of full-length Nup53. Black bar, 2 μm; white bar, 210 nm. (B) Nuclear assembly reactions of Mock, ΔNup53 extracts, or ΔNup53 extracts after addition of full-length Nup53. Reactions were analyzed by immunofluorescence against mAb414 (green). Chromatin was stained with DAPI (blue). Bar, 10 μm.

Nup53 fragments that interact with NDC1 or Nup93 alone are not sufficient to restore NE formation. (A) Nuclei assembled in vitro in Mock, ΔNup53 extracts, or ΔNup53 extracts after addition of Nup53^1-300, Nup53^1-203, and Nup53^204-326. Membranes are visualized by using the membrane dye DilC₁₈ (red). Chromatin was stained with DAPI (blue). Bar, 10 μm. (B) Quantitation of closed NE formation of experiments performed as described in A, including the truncation fragment Nup53^167-300. More than 100 randomly chosen chromatin substrates per reaction were counted. Average of three independent experiments is shown. Error bars represent the SD over the three experiments.