Figure 7
From: Sumo-dependent substrate targeting of the SUMO protease Ulp1
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.