Ubiquitylation of nucleoporins. Ni-purified 6His-ubiquitin (Ub)–conjugated forms of genomically HA-tagged Nups were extracted from cells transformed (+) or not transformed (−) with a plasmid encoding 6His-ubiquitin under control of the CUP1 promoter. Cell lysates and Ni-purified material were examined by Western blotting with an anti-HA antibody. Ubiquitin expression and efficiency of purification were controlled using an anti-6His antibody (not depicted). Localization of Nups as previously determined (Rout et al., 2000) is indicated on the right (N, nuclear side of the NPC; C, cytoplasmic side of the NPC). (A) Analysis of ubiquitin-conjugated forms of genomically HA-tagged FG-Nups. (B) Analysis of ubiquitin-conjugated forms of genomically HA-tagged transmembrane Nups. (C) Summary of the ubiquitylation status of Nups. Molecular masses are given in kilodaltons.

Ubiquitylation of Nup159 and its role in Dyn2 localization at the NPC. (A) Schematic representation of Nup159 molecular organization (DID, dynein-interacting domain). (B) Ni-purified 6His-ubiquitin (Ub)–conjugated forms of Nup159-HA were extracted from cells transformed (+) or not transformed (−) with a plasmid encoding 6His-ubiquitin under control of the CUP1 promoter. Cell lysates (top) and Ni-purified material (middle) were examined by Western blotting with an anti-HA antibody. A portion of the Ni-purified material also was examined by Western blotting on a separate membrane using an anti-6His antibody to show purification efficiency (bottom). (B and C) Analysis of ubiquitin-conjugated forms of Nup159-HA was performed in wt (Nup159-HA) and nup159K897R-HA cells (B) and in wt and cdc34ts strains (C). The white line indicates that intervening lanes have been spliced out. Molecular markers are given in kilodaltons. (D) Localization of genomically tagged Nup60-GFP and Nup159-mCherry (left) or Nup60-GFP and nup159K897R-mCherry was monitored by SIM. Centroid distances of ∼90 ± 20 nm, along the line axis indicated in the magnified areas, were estimated between the two fluorescent signals at the NPC (n = 20 for each cell types). Bars, 1 µm. (E) Localization of genomically tagged Dyn2-GFP was analyzed in Nup159-HA and nup159K897R-HA cells as well as in cdc34ts cells at 23°C and after a shift to 37°C. NPC localization corresponds to the rimlike staining. The magnified areas (boxes) are shown on the right. Bars, 5 µm.

Ubiquitylation of Nup159 facilitates nuclear migration to the daughter cells during mitosis. (A) Ubiquitylation of Nup159 does not affect cell cycle progression. Nup159-HA (black) and nup159K897R-HA (red) cells were synchronized with α-factor and then released for the indicated period of time before fixation and staining with SYTOX green and analyzed by flow cytometry. Numbers on the bottoms show fluorescence intensity (arbitrary units). (B) Ubiquitylation of Nup159 is not cell cycle dependent. Ni-purified 6His-ubiquitin (Ub)–conjugated forms of Nup159-HA were extracted from unsynchronized (O/N, overnight) cells or from cells treated with α-factor for 3 h before release for indicated periods of time. Cell lysates (top) and Ni-purified material (middle) were examined by Western blotting with an anti-HA antibody. (bottom) Ubiquitin expression and efficiency of purification were controlled using an anti-6His antibody. Molecular markers are given in kilodaltons. Numbers at the top are in minutes. (C) Cells were treated with α-factor for 3 h, released for 90 min, fixed, and stained with Hoechst before epifluorescence and interferential contrast analysis. Dotted lines show the yeast cell contours. Arrows indicate daughter cells. (D) Segregation of nuclei observed in A was analyzed in >100 wt and mutant cells per experiment, and results were obtained from five independent experiments. Error bars show standard deviations. ***, P < 0.05. (E) Localization of microtubules was analyzed by fluorescence microscopy in Nup159-HA and nup159K897R-HA cells using Tub1-GFP. DIC, differential interference contrast. Bars, 5 µm.

Ubiquitylation of Nup159 is in the dynein pathway not the Kar9 pathway. (A) Serial dilutions of the corresponding strains were spotted onto YPD plates and grown at the indicated temperature. (B) kar9ΔNup159-HA and kar9Δnup159K897R-HA cells grown at 30°C were fixed and stained with Hoechst before epifluorescence and interferential contrast analysis. Arrows indicate some defecting cells. DIC, differential interference contrast. Bar, 5 µm.