INM association of Scs2-Siz2 directs mitosis-specific SUMOylation events. (A) Scs2 localization was assessed using the split-superfolder GFP system. Epifluorescence images of WT cells containing GFP_1–10–Scs2 and plasmid-encoded GFP₁₁-mCherry-Hxk1(cytoplasmic) or GFP₁₁-mCherry-Pus1 (nuclear). Localization of the reporter (mCherry) and assembled GFP_1–10⋅GFP₁₁ (GFP) in representative G1-, S-, and M-phase cells is shown. Bar, 2 µm. Dot represents association of the GFP fragments. (Band D) The indicated strains were assessed for Siz2-V5₃ phosphorylation and their mitotic SUMO conjugate profile by Western blot analysis as described in Fig. 1 and Fig. 2. Dots highlight the position of mitotically phosphorylated Siz2-V5₃. GFP-Siz2 and Sur4-mCherry imaging and quantification of the nuclear distribution were performed on mitotic cells of the specified strains as described in Fig. 3. Note, quantification of line scans shown in panels B and D was obtained at the same time as data shown in Fig. 3 C, and the WT data are also shown here for comparison. The scs2^K84D/L86D mutations lie in the MSP domain (MSP) and the siz2^A569 mutation within the FFAT-like motif (FFAT). Error bars represent SD. Bar, 2 µm. In D, the position of Scs2-SUMO is indicated by a red arrowhead, and blue arrowheads point to three other prominent mitotic SUMO conjugates. Molecular mass markers are shown in kD. (C) Binding of Scs2-TAP to either Siz2-V5₃ or siz2^S522A-V5₃ and binding of scs2^K84D/L86D-TAP to Siz2-V5₃ was assessed. Scs2-TAP or scs2^K84D/L86D-TAP was affinity-purified from cells (IP) producing Siz2 derivatives, and bound proteins were eluted using a Mg²⁺ step gradient. Equivalent portions of the indicated fractions were analyzed by Western blotting (WB) to assess levels of the V5- and TAP-tagged fusions. Note, all Load, Elution, or Bound fractions shown in panel C were derived from the same Western blot.