FIG. 5.

Mutation of C195 and H197 results in a loss of sumoylating activity in vitro and in vivo. (A) Nse2.SA protein is unable to form SUMO chains in vitro. An in vitro sumoylation assay was performed in the absence of an added target protein, with 0.05 μg of Hus5/μl. The products were detected by Western analysis with anti-Pmt3 antisera. The arrow indicates the junction of stacking and separating gels. (B) Nse2.SA does not facilitate sumoylation of Smc6 (Rad18) in vitro. An in vitro sumoylation assay was performed with ³⁵S-labeled Smc6 (Rad18) and 0.05 μg of Hus5/μl. The products were detected with a phosphorimager. (C) Nse2.SA is not sumoylated in vitro. An in vitro sumoylation assay was performed with an ³⁵S-labeled wild-type or mutant (Nse2.SA) Nse2 protein. The products were detected as described for panel B. (D) Western analysis of total cell extracts probed with anti-Pmt3 antisera (top) or anti-tubulin antisera (bottom). Lane 1, wild type (sp.011); lane2, rad31.d; lane 3, hus5.17; lane 4, hus5.62; lane 5, pli1.d; lane 6, nse2.SA; lane 7, nse2.CH. (E) nse2.SA cells have reduced levels of sumoylated Smc6 (Rad18). Extracts of wild-type (nse2.CH) (lanes 3 and 4) and mutant (nse2.SA) (lanes 1 and 2) strains harboring either pREP41MH-Rad18 (Smc6) (lanes 1 and 3) or the empty pREP41MH vector (lanes 2 and 4) were bound to nickel beads under denaturing conditions, and the bound proteins were analyzed by immunoblotting with either anti-Myc or anti-Pmt3 antibodies as indicated. *, modified forms.