Constitutive association of Siz2 with the NE induces SUMOylation and increases nuclear surface area. (A) WT cells producing Siz2-GFP_1-10 and a plasmid-encoded GFP₁₁-mCherry-TM (GFP₁₁^{(nucleo/cyto)}) or mCherry-TM-GFP₁₁ (GFP₁₁^{(NE/ER lumen)}) fusion protein were examined by epifluorescence microscopy. The membrane-integrated GFP₁₁ fusion proteins allow for the visualization of NE/ER morphology by mCherry fluorescence. Formation of GFP_1-10-GFP₁₁ dimers was visualized by GFP fluorescence. Arrowheads point to GFP fluorescence at the NE in several cells. Size bar, 2 µm. (B) WT cells or cells producing Siz2-GFP_1-10 and GFP₁₁-mCherry-TM (GFP₁₁^{(nucleo/cyto)}) were arrested in G1-phase using α-factor, released from arrest, and then analyzed every 10 min by Western blotting to detect SUMO conjugates, Clb2, and the Gsp1 load control. Clb2 levels peak in mitosis. Note, Western blot images of samples from the Siz2-GFP_1-10/GFP₁₁ strain were derived from the same blot. (C and H) SUMO conjugate profiles of the indicated strains were assessed by Western blotting of cell lysates derived from asynchronous cultures. Images shown were derived from the same Western blot. (D)siz2Δ mutant cells containing plasmid-encoded GFP₁₁-mCherry-TM (GFP₁₁^{(nucleo/cyto)}) and a plasmid expressing the siz2^RING-GFP_1-10 mutant from a copper-inducible promoter were examined by epifluorescence microscopy 4 h after 0.5 mM Cu²⁺ addition. GFP and mCherry fluorescence was visualized as described in A. Arrowheads point to GFP fluorescence at the NE in several cells. Size bars, 2 µm. (E) Cultures of cells producing Siz2-GFP_1-10 and plasmid-encoded GFP₁₁-mCherry-TM (GFP₁₁^{(nucleo/cyto)}) and siz2Δ mutant cells producing plasmid-encoded siz2^RING-GFP_1-10 and GFP₁₁-mCherry-TM (GFP₁₁^{(nucleo/cyto)}) (described in D) were incubated in the presence or absence of 0.5 mM Cu²⁺ for 4 h. SUMO conjugates and the GFP_1-10 fusions were detected by Western blotting. Note, following induction, the CUP1pr-siz2^RING-GFP_1-10 gene produced levels of siz2^RING-GFP_1-10 similar to Siz2-GFP_1-10. (F and I) Nuclear surface areas of WT (F) and scs2^K180R mutant (I) cells expressing the specified constructs at the indicated cell cycle stage were determined using Pus1-GFP as described in Fig. 1 A. (G) Pus1-GFP was introduced into the two strains described in E and nuclear surface areas of cells were examined after Cu²⁺ induction. Note, arrowheads on Western blots in B, C, E, and H point to SUMOylated Scs2 (red) and/or three prominent Siz2-dependent INM SUMO conjugates (blue; Ptak et al., 2021). Mass markers are shown in kilodaltons. Error bars for all graphs shown are SD, and p values were determined using a two-tailed Student’s t test for the indicated sample pairs. *p ≤ 0.05, **p ≤ 0.01. Source data are available for this figure: SourceData F2.