Human RNF111 binds to poly-SUMOylated proteins via an N-terminal SIM region. (A) Schematic of human RNF111/Arkadia. The RING domain, two putative NLSs (Episkopou et al., 2001), and three SUMO-interacting motifs (SIMs; top), conserved in higher vertebrates (bottom), are shown. Core hydrophobic SIM residues are highlighted in green. (B) Amino acid substitutions (highlighted in red) in the RNF111 SIM region to disrupt its SUMO-binding ability (*SIM). (C) S-FLAG-Strep–tagged RNF111 (SFS-RNF111) proteins expressed in U2OS cells were purified on Strep-Tactin Sepharose, incubated with purified SUMO2 or poly-SUMO2 (3–8), and washed extensively. Bound complexes were immunoblotted with the SUMO2 antibody. WCE, whole-cell extract. (D) HeLa cells stably expressing FLAG-SUMO isoforms were transfected with Strep-HA-RNF111 plasmids as indicated. Whole-cell extracts were subjected to Strep-Tactin pull-down and immunoblotting with the FLAG antibody. (E) Plasmon surface resonance analysis of poly-SUMO2 binding kinetics of RNF111 fragments spanning the SIMs. Data shown are from a single representative experiment out of three repeats. MM, molecular mass.