Figure 6

The Ulp1(3)^(C580S) truncation binds SUMO and SUMO-modified proteins. (A) and (B) Immobilized Ulp1(3)^(C580S) was analyzed for its ability to affinity-purify Smt3 from yeast whole-cell extracts (WCEs). WCEs containing FLAG-tagged Smt3 (YOK 428) (left) or GFP-Smt3 (YOK 1857) (right) (input) were prepared under nondenaturing conditions and incubated with immobilized maltose-binding protein (MBP)-Ulp1(3)^(C580S) (3^(C580S)), MBP-Ulp1(3)^(C580S) lacking the small ubiquitin-like modifier (SUMO)-binding surface (3^(C580S)ΔSBS) or unbound resin (amylose). After washing and elution, bound Smt3 and Smt3 conjugates were detected using either anti-Flag or anti-GFP antibody. (C) Immobilized Ulp1(3)^(C580S) was analyzed for its ability to affinity-purify Cdc11 from yeast WCEs. WCE containing GFP-Smt3 (YOK 1857) was prepared under nondenaturing conditions and incubated with immobilized MBP-Ulp1(3)^(C580S), MBP-Ulp1(3)^(C580S) lacking the SUMO-binding surface (3^(C580S)ΔSBS) or unbound resin (amylose). After washing and dilution, bound Cdc11 was detected using an anti-Cdc11 antibody (Santa Cruz Biotechnology). (D) WCEs from logarithmically growing yeast cells expressing GFP-tagged Ulp1(3), Ulp1(3)^(C580S) and Ulp1(3)^(C580S)ΔSBS (YOK 1839, YOK 1907, YOK 1903) (input) were prepared under nondenaturing conditions. Extracts were then incubated with SUMO2 immobilized on agarose beads (Boston Biochem). After washing and elution with sample buffer, bound proteins were detected using an anti-GFP antibody. (E) SUMO2 chains (Boston Biochem) were incubated with resin-bound MBP-Ulp1(3)^(C580S) or unbound resin (amylose). After washing and elution with sample buffer, bound proteins were detected using an anti-SUMO2 antibody. SUMO2 chains loading control (input). Concentrations of immobilized MBP-Ulp1(3)^(C580S) and MBP-Ulp1(3)^(C580S) lacking the SUMO-binding surface (3^(C580S)ΔSBS) were confirmed by Coomassie staining of eluted proteins and quantitation on an Agilent 2100 Bioanalyzer (Agilent Technologies).