Figure S1. Siz2 localization and PTM.... | Rockefeller University Press

Figure S1.

Siz2 localization and PTM.(A, B, and G) α-factor arrest-release assays were performed as in Fig. 1. Shown are WT cells and cells producing HA3-Scs2 or Scs2-V53 (A), siz1Δ cells (B), and Siz2-V53–, siz2S527A-V53–, and siz2S674A-V53–producing cells (G). Cell lysates were analyzed by Western blotting using anti-SUMO, Clb2, and Gsp1 (load control) antibodies, as well as a V5 antibody in G, as indicated. Only the 0-and 60-min time points were analyzed in A. (C) Cell lysates derived from asynchronous cultures of WT, siz1Δ, siz2Δ, and mms211–184–V53 (Mms21 derivative deficient in SUMO E3 ligase activity) cells were assessed by Western blotting using an anti-SUMO antibody to assess SUMO conjugate profiles. Gsp1 is a loading control. In A–C and G, red arrowheads point to SUMOylated Scs2 or SUMOylated tagged Scs2. Blue arrowheads point to other prominent mitotic SUMO conjugates. In G, dots highlight the position of mitotically phosphorylated Siz2-V53. Molecular mass markers are shown in kD. (D) Anti-SUMO immunofluorescence analysis of siz1Δ and mms211–184–V53 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. (E) Shown are epifluorescence images of representative G1/S- and M-phase WT cells producing GFP-Siz2 and nucleolar Nop56-mCherry. Merged images show that GFP-Siz2 is largely excluded from the nucleolus. (F) WT mitotic cell lysates were isolated at 60 min after release from α-factor arrest (see Fig. 1 B). Proteins were extracted and solubilized in buffer lacking (-PPase) or containing protein phosphatase (+PPase). Samples were analyzed by Western blotting using anti-V5 and Gsp1 (loading control) antibodies. Mobility of modified Siz2-V53 is increased by phosphatase treatment. Vertical line indicates that all bands observed consist of Siz2-V53. (G) Imaging and quantification of the nuclear distribution of GFP-Siz2, GFP-siz2S527A, and GFP-siz2S674A compared with Sur4-mCherry were examined as described in Fig. 3 for cells (n = 25) in mitosis. Note, line scans show enrichment of Siz2-GFP, GFP-siz2S527A, and GFP-siz2S674A at the NE with Sur4-mCherry in these cells. Quantification of lines scans shown in G were obtained at the same time as data shown in Fig. 3 C, and the WT data are also shown here for comparison. Error bars represent SD. Bar, 2 µm. Phos, phosphorylation.

Siz2 localization and PTM. (A, B, and G) α-factor arrest-release assays were performed as in Fig. 1. Shown are WT cells and cells producing HA₃-Scs2 or Scs2-V5₃ (A), siz1Δ cells (B), and Siz2-V5₃–, siz2^S527A-V5₃–, and siz2^S674A-V5₃–producing cells (G). Cell lysates were analyzed by Western blotting using anti-SUMO, Clb2, and Gsp1 (load control) antibodies, as well as a V5 antibody in G, as indicated. Only the 0-and 60-min time points were analyzed in A. (C) Cell lysates derived from asynchronous cultures of WT, siz1Δ, siz2Δ, and mms21¹^–¹⁸⁴–V5₃ (Mms21 derivative deficient in SUMO E3 ligase activity) cells were assessed by Western blotting using an anti-SUMO antibody to assess SUMO conjugate profiles. Gsp1 is a loading control. In A–C and G, red arrowheads point to SUMOylated Scs2 or SUMOylated tagged Scs2. Blue arrowheads point to other prominent mitotic SUMO conjugates. In G, dots highlight the position of mitotically phosphorylated Siz2-V5₃. Molecular mass markers are shown in kD. (D) Anti-SUMO immunofluorescence analysis of siz1Δ and mms21¹^–¹⁸⁴–V5₃ 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. (E) Shown are epifluorescence images of representative G1/S- and M-phase WT cells producing GFP-Siz2 and nucleolar Nop56-mCherry. Merged images show that GFP-Siz2 is largely excluded from the nucleolus. (F) WT mitotic cell lysates were isolated at 60 min after release from α-factor arrest (see Fig. 1 B). Proteins were extracted and solubilized in buffer lacking (-PPase) or containing protein phosphatase (+PPase). Samples were analyzed by Western blotting using anti-V5 and Gsp1 (loading control) antibodies. Mobility of modified Siz2-V5₃ is increased by phosphatase treatment. Vertical line indicates that all bands observed consist of Siz2-V5₃. (G) Imaging and quantification of the nuclear distribution of GFP-Siz2, GFP-siz2^S527A, and GFP-siz2^S674A compared with Sur4-mCherry were examined as described in Fig. 3 for cells (n = 25) in mitosis. Note, line scans show enrichment of Siz2-GFP, GFP-siz2^S527A, and GFP-siz2^S674A at the NE with Sur4-mCherry in these cells. Quantification of lines scans shown in G were obtained at the same time as data shown in Fig. 3 C, and the WT data are also shown here for comparison. Error bars represent SD. Bar, 2 µm. Phos, phosphorylation.