Identification of Nup60 phosphosites at the interface with the NPC core that mediate Cdc5-dependent detachment. (A) N-terminal and C-terminal Nup60 phosphopeptides that exhibit meiotic upregulation (data from Wettstein et al., in preparation). Each line represents an individual phosphopeptide originating from Nup60 protein, measured in 1-h intervals across the entire meiotic cell division program, as well as in prophase I arrested cells (ndt80Δ, 8 h in SPM*) and metaphase I arrested cells (cdc20-mn, 10 h in SPM*). Log2 fold change (log2(FC)) relative to mean expression over all samples of each phosphopeptide is plotted on the y-axis. The average measurement of triplicates is plotted. (B) Schematic of Nup60 depicting the position of phosphosites relative to known structural features. The phosphomutants generated are indicated by colored boxes. AH = amphipathic helix, HR = helical region. (C) Visualization of the N-terminal (orange) and C-terminal phosphosites (green) of Nup60 on a cryo-EM structure of the NPC (Kim et al., 2018) visualized using Mol* (Sehnal et al., 2021). (D) Montages of cells containing different NUP60-GFP alleles and HTB1-mCherry in a prophase I arrest (ndt80Δ) before (5 h in SPM) and after (6 h in SPM) induction of PCUP1-CDC5-3xFLAG-10xHis. The following alleles were tested: NUP60-GFP (UB29129), NUP60-S89A-GFP (UB29560), NUP60-Nterm3A-GFP (UB29636), NUP60-Nterm5A-GFP (UB29638), NUP60-Cterm4A-GFP (UB29562), and NUP60-9A-GFP (UB29564). Induction was performed at 5 h in SPM medium with 50 μM CuSO4. (E) Quantification of Nup60 detachment for the experiment depicted in D. Individual DI values were normalized to the average DI for Nup60-GFP cells at the pre-induction time point (5 h in SPM). Asterisks indicate statistical significance calculated using Dunn’s test for multiple comparisons when each allele was compared with wild type for a given time point (see Table S5 for P values). Sample sizes (n) are the number of cells quantified for each strain; for Nup60-GFP and Nup609A-GFP, cells from two independent replicates were pooled. (F) Immunoblot for different Nup60-GFP alleles and Cdc5-3xFLAG-10xHis before (5 h in SPM) or after (6 h in SPM) copper induction during prophase I arrest (ndt80Δ), corresponding to the images in D. Hxk2 was used as a loading control. (G) Immunoblot for Nup60-GFP (UB29253) or Nup609A-GFP (UB30438) in cdc20-mn background. Hxk2 was used as a loading control. For F and G, the brackets to the left of the blots denote apparent phosphoshifts. For each montage, normalized DI values are indicated when calculated. Scale bars, 2 μm. Source data are available for this figure: SourceData F5.
Figure 5.

Identification of Nup60 phosphosites at the interface with the NPC core that mediate Cdc5-dependent detachment. (A) N-terminal and C-terminal Nup60 phosphopeptides that exhibit meiotic upregulation (data from Wettstein et al., in preparation). Each line represents an individual phosphopeptide originating from Nup60 protein, measured in 1-h intervals across the entire meiotic cell division program, as well as in prophase I arrested cells (ndt80Δ, 8 h in SPM*) and metaphase I arrested cells (cdc20-mn, 10 h in SPM*). Log2 fold change (log2(FC)) relative to mean expression over all samples of each phosphopeptide is plotted on the y-axis. The average measurement of triplicates is plotted. (B) Schematic of Nup60 depicting the position of phosphosites relative to known structural features. The phosphomutants generated are indicated by colored boxes. AH = amphipathic helix, HR = helical region. (C) Visualization of the N-terminal (orange) and C-terminal phosphosites (green) of Nup60 on a cryo-EM structure of the NPC (Kim et al., 2018) visualized using Mol* (Sehnal et al., 2021). (D) Montages of cells containing different NUP60-GFP alleles and HTB1-mCherry in a prophase I arrest (ndt80Δ) before (5 h in SPM) and after (6 h in SPM) induction of PCUP1-CDC5-3xFLAG-10xHis. The following alleles were tested: NUP60-GFP (UB29129), NUP60-S89A-GFP (UB29560), NUP60-Nterm3A-GFP (UB29636), NUP60-Nterm5A-GFP (UB29638), NUP60-Cterm4A-GFP (UB29562), and NUP60-9A-GFP (UB29564). Induction was performed at 5 h in SPM medium with 50 μM CuSO4. (E) Quantification of Nup60 detachment for the experiment depicted in D. Individual DI values were normalized to the average DI for Nup60-GFP cells at the pre-induction time point (5 h in SPM). Asterisks indicate statistical significance calculated using Dunn’s test for multiple comparisons when each allele was compared with wild type for a given time point (see Table S5 for P values). Sample sizes (n) are the number of cells quantified for each strain; for Nup60-GFP and Nup609A-GFP, cells from two independent replicates were pooled. (F) Immunoblot for different Nup60-GFP alleles and Cdc5-3xFLAG-10xHis before (5 h in SPM) or after (6 h in SPM) copper induction during prophase I arrest (ndt80Δ), corresponding to the images in D. Hxk2 was used as a loading control. (G) Immunoblot for Nup60-GFP (UB29253) or Nup609A-GFP (UB30438) in cdc20-mn background. Hxk2 was used as a loading control. For F and G, the brackets to the left of the blots denote apparent phosphoshifts. For each montage, normalized DI values are indicated when calculated. Scale bars, 2 μm. Source data are available for this figure: SourceData F5.

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