MBP-Ulp1(3)(C580S) can serve as a SUMO2 binding platform for SUMO-targeted ubiquitin ligase-mediated substrate ubiquitination. (A) SUMO2 chains (Boston Biochem) were incubated with resin-bound maltose-binding protein (MBP)-Ulp1(3)(C580S). The complex of MBP-Ulp1(3)(C580S) with small ubiquitin-like modifier 2 (SUMO2) chains was then eluted and added to an in vitro ubiquitination reaction with the SUMO-targeted ubiquitin ligase (STUbL) RNF4, an E3 ubiquitin ligase. Proteins in the STUbL reactions were separated by SDS-PAGE and assessed by Western blot analysis with an anti-SUMO2 antibody. Arrows indicate modified SUMO2 chains. Lane 1: No SUMO chains; lane 2: no RNF4; lane 3: no Ulp1(3)(C580S); lane 4: all reagents. (B) Proposed model for using MBP-Ulp1(3)(C580S) as a SUMO2-binding platform for substrate ubiquitination. SUMO2, ubiquitin and RNF4 are indicated by spheres labeled S, spheres labeled Ub and the gray oval labeled RNF4, respectively.
Elmore et al. BMC Biology 2011 9:74 doi:10.1186/1741-7007-9-74