FIG. 4.

Smc6 (Rad18) and Nse3 are sumoylated in an Nse2-dependent manner in vitro, and Smc6 (Rad18) is sumoylated in vivo. (A to D) ³⁵S-labeled proteins were tested with in vitro sumoylation assays using 0.05 μg of Hus5/μl and 0 or 0.2 μg of Nse2 or Pli1/μl, as indicated. (A) Smc6 (Rad18); (B) Smc5 (Spr18); (C) Nse1; (D) Nse3. (E) Smc6 (Rad18) is sumoylated in vivo. Ni²⁺ pull-down assays were performed with cell extracts from cells transformed with pREP42MH (empty vector) (lanes 1, 3, 5, and 7) or pREP42MH-Rad18 (Smc6) (lanes 2, 4, 6, and 8). TCA, total cell extract controls. Western analysis was conducted with anti-Myc or anti-Pmt3 antisera as indicated. (F) Sumoylation of Smc6 (Rad18) expressed at wild-type levels increases after exposure to MMS. Lysates (containing 50 mg of total protein) prepared from a Myc-tagged Smc6 (Rad18) strain and an untagged control with or without exposure to MMS (0.01%) were incubated overnight at 4°C with an anti-Myc antibody that had been previously cross-linked to protein G-Sepharose beads. The beads were washed extensively, and bound proteins were eluted by incubation with 100 mM glycine, pH 2.3, separated by SDS-PAGE, and analyzed by Western blotting (WB) with anti-Myc and anti-Pmt3 antibodies, as indicated. Lanes 1, 2, 5, 7, and 8, Myc-tagged Rad18 (Smc6) strain; lanes 3, 4, 6, 9, and 10, wild type (untagged Rad18 [Smc6]). Lanes 5 and 6 are the same as lanes 7 and 9, but with longer exposure times. *, modified forms.