Nup82p-depleted NPCs are selectively excluded from the daughter NE during mitosis. (A) Western analysis using anti-GFP, anti-Nup53p, or anti-Gsp1p (load control) antibodies of P_MET3-NUP82-GFP₃ and NUP82-GFP₃ cell lysates after growth in methionine-containing media for the indicated times. (B) A P_MET3-NUP82-GFP₃ NUP188-mCherry strain was grown for the indicated times in medium containing methionine to repress NUP82-GFP₃ expression. Nup82-GFP₃ and Nup188-mCherry were visualized using an epifluorescence microscope. Selected telophase cells are outlined. Two sets of Nup82-GFP₃ images are shown. Fixed exposure times reveal decreasing intensities of Nup82-GFP₃ across the time course, whereas Nup188-mCherry levels are unaffected. Adjusted exposures allow enhanced visualization of Nup82-GFP₃ foci and reveal their symmetrical distribution between mother (blue arrowheads) and daughter (red arrowheads) NEs. In contrast, Nup188-mCherry reveals greater numbers of NPCs retained in the mother, including those with no detectable Nup82-GFP₃ (yellow arrowheads). Bar, 5 µm. (C) Cells grown as described in B were examined by confocal fluorescence microscopy at 2 and 6 h after methionine addition. Z stacks of telophase cells were used to determine the I_daughter/I_mother signal intensity ratios of Nup82-GFP₃ and Nup188-mCherry. Error bars represent standard deviation. (D) A schematic model based on data presented in B and C is shown. NPCs depleted of Nup82p and compromised for Nsp1p subcomplex function are crossed with a red X. A diffusion barrier (brick wall) is proposed to prevent these damaged NPCs from entering the daughter cell. In contrast, NPCs containing a functional Nsp1p subcomplex can traverse the barrier and enter the daughter. These two events result in asymmetric NPC segregation during mitosis.