Depletion of Nsp1p causes asymmetric NPC segregation. (A) PMET3-HA-NSP1 cells were incubated with methionine for the indicated times to repress NSP1 expression. Cell lysates were analyzed by Western blotting using anti-HA and anti-Gsp1p (load control) antibodies. (B) PMET3-NSP1 NUP188-GFP SUR4-mCherry cells were grown in the absence (−Met) or presence (+Met, 3 h) of methionine. Early anaphase cells were identified, and images were acquired every 2 min as they progressed through mitosis using an epifluorescence microscope. Arrowheads indicate the position of the bud neck. Arrows highlight accumulated Nup188-GFP signal close to the bud neck in Nsp1p-depleted cells. Bar, 5 µm. (C) Cells expressing NUP188-GFP in WT and the indicated PMET3-NUP backgrounds were grown as described in A. Before (+Met 0 h) and after (+Met 4 h) Nup depletion, telophase cells (n = 23–33 per condition) were imaged using a confocal microscope, and the daughter-to-mother ratio (Idaughter/Imother) of the Nup188-GFP signal was determined. Error bars express standard deviation. (D) Cells expressing the indicated Nup-GFP fusions in the PMET3-NSP1 background were analyzed by epifluorescence microscopy before (−Met) and after depletion of Nsp1p (+Met, 6 h). Arrowheads identify mother (blue) and daughter (red) nuclei. Bar, 5 µm.
