Nup60 is not required for the retention of Mlp1 at the nuclear periphery, explaining differences in the meiotic dynamics of basket nucleoporins. (A) Schematic depicting a hypothesis for two distinct roles of Nup60 in nuclear basket organization: (1) a role in recruitment (for both Mlp1 and Nup2), tested by monitoring nucleoporin localization in a nup60Δ background or upon constitutive Nup60 depletion using the NUP60-AID allele and (2) a role in retention (for Nup2, but not Mlp1), tested by monitoring nucleoporin localization upon transient Nup60 depletion using the NUP60-AID allele. (B) Immunoblot of Nup60-3V5-IAA17 (Nup60-AID) levels in strains with Nup2-GFP or Mlp1-GFP, in the absence or presence of PCUP1-osTIR1-F74G. (Nup2-GFP: −osTIR1 = UB32240 and +osTIR1 = UB32238; Mlp1-GFP: −osTIR1 = UB32248 and +osTIR1 = UB32246). Hxk2 was used as a loading control. Vegetative depletion was induced with 50 μM CuSO4 and 100 μM 5-Ph-IAA immediately upon inoculation of a logarithmic YPD culture at 0.25 OD/ml and continued for 10 h. For meiotic depletion, cells were maintained in prophase I arrest (ndt80Δ), and depletion was induced by adding 50 μM CuSO4 at 4.5 h in SPM and 100 μM 5-Ph-IAA at 5 h in SPM. Background subtracted Nup60 protein levels were divided by Hxk2 levels to control for loading and then normalized to the -osTIR1 strain at 4.5 h in SPM. (C) Nup2-GFP localization in NUP60 (UB15305) and nup60Δ (UB31600) cells from a saturated YPD culture. (D) Nup2-GFP localization in -osTIR1 (UB32240) or +osTIR1 (UB32238) cells from a saturated YPD culture after constitutive Nup60 depletion. (E) Nup2-GFP localization in −osTIR1 (UB32240) or +osTIR1 (UB32238) cells before and after Nup60 depletion during prophase I arrest. (F) Mlp1-GFP localization in NUP60 (UB14648) and nup60Δ (UB31262) cells from a saturated YPD culture. (G) Mlp1-GFP localization in −osTIR1 (UB32248) or +osTIR1 (UB32246) cells from a saturated YPD culture after constitutive Nup60 depletion. (H) Mlp1-GFP localization in −osTIR1 (UB32248) or +osTIR1 (UB32246) cells before and after Nup60 depletion during prophase I arrest. For E and H, the white arrowheads in the “Fire LUT” images denote nuclei at the time point when Nup60-AID was largely depleted in +osTIR1 strains. Scale bars, 2 μm. Source data are available for this figure: SourceData F9.
Figure 9.

Nup60 is not required for the retention of Mlp1 at the nuclear periphery, explaining differences in the meiotic dynamics of basket nucleoporins. (A) Schematic depicting a hypothesis for two distinct roles of Nup60 in nuclear basket organization: (1) a role in recruitment (for both Mlp1 and Nup2), tested by monitoring nucleoporin localization in a nup60Δ background or upon constitutive Nup60 depletion using the NUP60-AID allele and (2) a role in retention (for Nup2, but not Mlp1), tested by monitoring nucleoporin localization upon transient Nup60 depletion using the NUP60-AID allele. (B) Immunoblot of Nup60-3V5-IAA17 (Nup60-AID) levels in strains with Nup2-GFP or Mlp1-GFP, in the absence or presence of PCUP1-osTIR1-F74G. (Nup2-GFP: −osTIR1 = UB32240 and +osTIR1 = UB32238; Mlp1-GFP: −osTIR1 = UB32248 and +osTIR1 = UB32246). Hxk2 was used as a loading control. Vegetative depletion was induced with 50 μM CuSO4 and 100 μM 5-Ph-IAA immediately upon inoculation of a logarithmic YPD culture at 0.25 OD/ml and continued for 10 h. For meiotic depletion, cells were maintained in prophase I arrest (ndt80Δ), and depletion was induced by adding 50 μM CuSO4 at 4.5 h in SPM and 100 μM 5-Ph-IAA at 5 h in SPM. Background subtracted Nup60 protein levels were divided by Hxk2 levels to control for loading and then normalized to the -osTIR1 strain at 4.5 h in SPM. (C) Nup2-GFP localization in NUP60 (UB15305) and nup60Δ (UB31600) cells from a saturated YPD culture. (D) Nup2-GFP localization in -osTIR1 (UB32240) or +osTIR1 (UB32238) cells from a saturated YPD culture after constitutive Nup60 depletion. (E) Nup2-GFP localization in −osTIR1 (UB32240) or +osTIR1 (UB32238) cells before and after Nup60 depletion during prophase I arrest. (F) Mlp1-GFP localization in NUP60 (UB14648) and nup60Δ (UB31262) cells from a saturated YPD culture. (G) Mlp1-GFP localization in −osTIR1 (UB32248) or +osTIR1 (UB32246) cells from a saturated YPD culture after constitutive Nup60 depletion. (H) Mlp1-GFP localization in −osTIR1 (UB32248) or +osTIR1 (UB32246) cells before and after Nup60 depletion during prophase I arrest. For E and H, the white arrowheads in the “Fire LUT” images denote nuclei at the time point when Nup60-AID was largely depleted in +osTIR1 strains. Scale bars, 2 μm. Source data are available for this figure: SourceData F9.

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