Mitotic recruitment of Siz2 to the NE directs SUMOylation events. (A, D, E, and G) α-factor arrest-release assays were performed as in Fig. 1. Cell lysates were analyzed by Western blotting to detect SUMO conjugates (A and G), Siz2-V5₃ (D), and siz2^S522A-V5₃ (phosphorylation site mutant—Phos site; E), as well as Clb2 and the Gsp1 load control in the indicated strain background. In A, the position of Scs2-SUMO is indicated by a red arrowhead. Blue arrowheads point to three other prominent mitotic SUMO conjugates. In D, the dot highlights the position of mitotically phosphorylated Siz2-V5₃. Molecular mass markers are shown in kD. (B and H) Anti-SUMO immunofluorescence analysis of WT and siz2Δ (B) as well as siz2^S522A (H) cells was performed as described in Fig. 1. DAPI staining identifies nuclear position. Imaging and quantification of the nuclear distribution of SUMO in mitotic cells (n = 25) were performed as in Fig. 1 and Fig. 2. Note, quantification of lines scans shown in panel B were obtained at the same time as data shown in Fig. 2 B, and the WT data are also shown here for comparison. (C and F) Epifluorescence images of cells producing GFP-Siz2 (C) or GFP-siz2^S522A (F) along with the NE/ER marker Sur4-mCherry. Cell cycle stage of the highlighted cell (arrows) is indicated in C. Mitotic cells are shown in F. Imaging and quantification of the nuclear distribution of GFP-Siz2 (C) or GFP-siz2^S522A (F) compared with Sur4-mCherry were examined using scans along a 2.1-µm line (see C, red lines), as described in the Fig. 1 legend, for cells (n = 25) in mitosis (F) or interphase and mitosis (C) as indicated. Note, line scans in C show enrichment of Siz2-GFP at the NE with Sur4-mCherry in mitotic cells. Error bars represent SD. Bar, 2 µm. Phos, phosphorylation.