SWATH-MS proteomics identifies Nup60 as a target of Cdc5-dependent phosphorylation. (A) Schematic illustration of the experimental setup and flowthrough used for the proteomics screen. Strains that arrest in prophase I (ndt80∆) carrying either P_GAL1-CDC5-GFP (YML3993) or P_GAL1-CDC5^KD-GFP (YML3994) were induced to enter meiosis by transfer to SPM* and, after 7 h in SPM*, treated with 2 µM β-estradiol to initiate Cdc5 or Cdc5^KD expression. Samples for protein analyses, immunoblot, or SWATH-MS proteomics were collected at the indicated time points after transfer to SPM*. The experiment was performed in biological triplicates. (B) Volcano plot depicting the differential phosphorylation of peptides in cells ectopically expressing Cdc5 or Cdc5^KD, as described in A. For peptides of interest, the protein name and the phosphorylation site are indicated. The log₂ fold change (log₂(FC)) is plotted on the x-axis and the P value corrected by false discovery rate (−log₁₀(adj. P value)) is plotted on the y-axis. Phosphopeptides with adjusted P values >0.01 are represented by light gray dots below the dashed line. Phosphopeptides with adjusted P values ≤0.01 are represented by dark gray dots and are putative Cdc5 targets. Within this category, phosphopeptides marked in dark blue belong to previously reported Cdc5 targets, and phosphopeptides marked in light blue belong to novel Cdc5 targets further validated in this study. All data points are included in Table S6. (C) Volcano plot depicting the differential phosphorylation of nucleoporin peptides in cells ectopically expressing Cdc5 or Cdc5^KD. The data were plotted as in B, but with phosphopeptides colored according to the subcomplex that the nucleoporin belongs to. (D) Examples of phosphopeptides in Slk19, Shp1, Swi6, and Nup60 that either do not change (gray lines) or change (red lines) in abundance upon Cdc5 expression. Phosphopeptide abundance (the average of the measurements from three biological replicates) is plotted upon either CDC5^KD induction (left plot) or CDC5 induction (right plot). In samples where a peptide could not be detected, data were imputed for that time point and used for plotting and statistical analysis (e.g., see the Slk19 S707 plot upon CDC5^KD induction). Note: In the case of Swi6, the peptide containing phosphorylated S176 is downregulated upon Cdc5 expression. Such downregulation may be a consequence of concurrent phosphorylation of a second/multiple residue(s) in the same peptide, not detected in this study. Therefore, the Cdc5 target site(s) would not be S176, but the additional site(s) in the same peptide. The same effect may be relevant for other disenriched peptides in B and C. (E) Immunoblots of Nup60-9myc and Cdc5 protein in a meiotic time course (YML6662). Samples were collected in 2-h intervals and cover the full meiotic cell division program. (F) Immunoblots for Nup60-9myc and Cdc5 protein from either cdc20-mn (YML6665) or cdc20-mn cdc5-mn (YML6664) cells during metaphase I arrest. (G) Immunoblots for Nup60-9myc and Cdc5 protein (YML12234) before (0–6 h in SPM*) or after (8 h in SPM*) treatment (either addition of copper or not) during prophase I arrest (ndt80Δ). Cdc5 was under control of the CUP1 promoter (P_CUP1-CDC5). (H) Immunoblots for Nup60-9myc (YML6665) or Nup60^S89A-9myc (YML7956) in cdc20-mn strains induced to enter meiosis and arrest in metaphase I. For E–H, Crm1 was used as a loading control and the brackets to the left of the blots denote apparent phosphoshifts. For all immunoblots in this article, the values to the right of the blots indicate molecular weight in kilodaltons (kD), assessed using a ladder. Source data are available for this figure: SourceData F4.