Identification of Ndc1-interacting proteins and characterization of the Ndc1 subcomplex. (A) Affinity purification from yeast expressing TAP-tagged Ndc1 (NDC1-TAP) or untagged controls. Gel areas were excised and subjected to LC-MS/MS. Lines mark borders of areas subjected to MS analysis. (B) Profile of an Ndc1 affinity purification performed in low ionic strength conditions. (C) Comparison of purifications performed from cells expressing TAP-tagged versions of Pom34 (POM34-TAP), Pom152 (POM152-TAP), or untagged controls. (D) Comparison of Ndc1-TAP affinity purifications from wild-type (wt), pom34Δ, pom152Δ, or untagged control cells. For A–D, purified proteins were eluted, separated by SDS-PAGE, and visualized by SYPRO-Ruby. (E) Western blot analysis of the levels of myc-tagged Pom152 or myc-tagged Pom34 in wild-type, pom34Δ, or pom152Δ cells expressing Ndc1-TAP. Ndc1-TAP and Xpo1/Crm1 levels are used as loading controls. (F) Western blot analysis of Ndc1 purifications from cells expressing Ndc1-TAP together with either 13×myc-tagged Pom152_full-length, Pom152_170–1337, or Pom152_1–301. The scheme depicts the membrane topology of Pom152. Xpo1 was included as a control. sup, supernatant. (G) Comparison of Ndc1 purifications from cells expressing full-length Pom152-13×myc or its truncations. Proteins were visualized with SYPRO-Ruby. (H) Distribution of full-length Pom152 and its truncations tagged with 13×myc analyzed by immunofluorescence. Nuclei were stained with DAPI. (I) Model summarizing the interactions between Ndc1, Pom152, and Pom34. Solid arrows indicate direct protein interactions. The dashed arrow displays the functional requirement of Pom34 for the Ndc1 subcomplex integrity. Migration rate references for all gel image panels are shown in kilodaltons. Bar, 5 µm.

The interactions between Nup53, Nup59, Nup157, and Nup170. (A) Coomassie brilliant blue–stained gel showing samples of purified proteins. (B) SYPRO-Ruby–stained gel showing samples of the load (left) or eluates from TAP-Nup170, TAP-Nup157, or TAP beads after incubation with different mixtures of E. coli extract, 6×His-MBP-Nup53, 6×His-MBP-Nup59, or 6×His-MBP. Migration rate references for all gel image panels are shown in kilodaltons.

Roles of Nup53 and Nup59 in NPC organization and function. (A and B) pom152Δ pom34Δ GAL-3×HA-NUP59 (A) or pom152Δ pom34Δ nup53Δ MET3-3×HA-NUP59 (B) cells expressing GFP-tagged Nup82, Nup133, Nup188, or Nup2 or nuclear import reporters Npl3-GFP (B) or Npl3(S411A)-GFP (A) together with the ER reporter dsRed-HDEL were analyzed by fluorescence microscopy in both permissive and nonpermissive conditions. Gal, galactose medium; dex, dextrose-containing medium; −met, medium lacking methionine; +10× met, medium containing 10× methionine. Bar, 5 µm.

Transmission EM analysis of NPC morphology in conditional mutants affecting Ndc1-interacting nucleoporins. (A) NPCs (arrowheads) in pom152Δ pom34Δ GAL-3×HA-NUP59 cells in permissive conditions (i) did not display morphological abnormalities. (ii) After incubation in nonpermissive conditions, a large fraction of dilated pores often displaying fuzzy electron-dense material could be detected (asterisks). (iii) A similar phenotype was observed in pom152Δ pom34Δ nup53Δ MET3-3×HA-NUP59 cells in permissive conditions. After incubation in nonpermissive conditions, these cells displayed not only dilated pores (iv) but also large openings in NE (v, arrows). In nup53Δ nup59Δ GAL-3×HA-POM34 cells, no obvious NPC size abnormalities could be detected both in permissive (vi) and nonpermissive conditions (vii). n, nucleus. Bars: (i–iv, vi, and vii) 100 nm; (v) 500 nm. (B, i–iii) Comparison of pore size distributions in pom152Δ pom34Δ GAL-3×HA-NUP59 cells (i), pom152Δ pom34Δ nup53Δ MET3-3×HA-NUP59 cells (ii), and nup53Δ nup59Δ GAL-3×HA-POM34 cells (iii) incubated in permissive and nonpermissive conditions. (iv) Comparison of pore numbers per section in nup53Δ nup59Δ GAL-3×HA-POM34 in either permissive or nonpermissive conditions. Gal, galactose medium; dex, dextrose-containing medium; −met, medium lacking methionine; 10× met, medium containing 10× methionine. Error bars indicate SEM.

Summary model of the Ndc1 interaction network. Solid arrows depict direct protein interactions. The dashed arrow displays the functional requirement of Pom34 for the Ndc1 subcomplex integrity.

The interactions of Ndc1 with Nup53 and Nup59. (A) Purified proteins separated by SDS-PAGE and stained with Coomassie brilliant blue. (B and C) SYPRO-Ruby–stained gels showing input (load) or eluates from S beads with immobilized Ndc1-TAP or S beads alone incubated with E. coli extract (bact. extract) or E. coli extract premixed with either GST-Nup53 or GST-Nup59. (D, top) Purified proteins stained with Coomassie brilliant blue. (bottom) SYPRO-Ruby–stained gel showing eluates from Ndc1-TAP beads after incubation with variants of Nup53 and Nup59. (E) Summary of interactions between truncations of Nup53 or Nup59 and Ndc1. (F) Binding of GST-Nup59 to Ndc1-TAP beads after preincubation with 10-fold excess of GST or 10-fold access of GST-Nup53. (G) Comparison of GST-Nup59 binding with Ndc1-TAP beads after preincubation with an excess of GST-Nup53 truncations or GST alone (left), or the reciprocal experiment with GST-Nup53 and an excess of GST-Nup59 truncations or GST alone (right). (H) Model summarizing the interactions between Ndc1 and Nup53 or Nup59. Solid arrows indicate direct protein interactions. Dashed arrow reflects the competition observed between Nup53 and Nup59 for Ndc1 binding. Migration rate references for all gel image panels are shown in kilodaltons.

The role of Pom152, Pom34, Nup53, and Nup59 in targeting of Ndc1 to NE. (A) Localization of Ndc1 was compared in nup53Δ pom34Δ pom152Δ MET3-3×HA-NUP59 (i), nup53Δ MET3-3×HA-NUP59 (ii), pom34Δ pom152Δ GAL-3×HA-NUP59 (iii), and GAL-3×HA-NUP59 (iv) cells by fluorescence microscopy (via the yeast EGFP tag in i and ii) or by indirect immunofluorescence (via 13×myc tag in iii and iv) in permissive (galactose medium [gal] or medium lacking methionine [−met]) and nonpermissive conditions (dextrose-containing medium [dex] or medium containing 10× methionine [+10× met]). For immunofluorescence, cells were also stained with DAPI to label DNA. (B) Similar analysis of Ndc1–yeast EGFP localization in nup53Δ nup59Δ GAL-3×HA-POM34 (i), GAL-3×HA-POM34 (ii), nup53Δ nup59Δ GAL-3×HA-POM152 (iii), and GAL-3×HA-POM152 (iv). Bars, 5 µm.

Roles of Pom152 and Pom34 in NPC organization and function. (A and B) Fluorescence analysis as described in Fig. 5 in nup53Δ nup59Δ cells conditionally expressing Pom34 (A) or Pom152 (B). Gal, galactose medium; dex, dextrose-containing medium. Bar, 5 µm.

Dendra assay to analyze distribution of nucleoporins in nup53Δ nup59Δ GAL-3×HA-POM34 cells. (A) Representation of the use of Nup-Dendra fusions to track the localization of newly synthesized and preexisting nucleoporins. Initially, Dendra displays green fluorescence but can be irreversibly photoconverted into a red fluorophore by a short pulse of UV light. After conversion, newly made nucleoporins are detected in the green channel, whereas red fluorescence can be used to localize Nups that were synthesized before the photoconversion. (B) Distribution of red and green Nup133-2×Dendra variants before (left), immediately after (middle), and 30 min after photoconversion (right). (C) Time course to visualize the Nup82-Dendra distribution in nup53Δ nup59Δ GAL-3×HA-POM34 cells after repression of POM34 expression without photoconversion. (D) Distribution of newly made and preexisting Nup82-Dendra in nup53Δ nup59Δ GAL-3×HA-POM34 cells. Cells were incubated for 4 h in nonpermissive conditions, photoconverted (left), and the same field of cells was reimaged after an additional 6-h incubation period (right). (E) Cells were incubated in dextrose medium (dex) for 9 h to induce Nup82 mislocalization, placed in galactose medium (gal), and photoconverted (left). After a 3-h incubation, the same cells were imaged again to detect the distribution of preexisting and newly synthesized Nup82-Dendra. Bars, 5 µm.