FIGURE 4.

hDREF is a SUMO E3 ligase. A, ³⁵S-labeled full-length Mi2α was synthesized by cell-free coupled in vitro transcription/translation and subjected to in vitro SUMOylation reaction. The reaction mixture contained GST-hDREF, GST-PIAS1, or GST-Pc2 as an E3 enzyme in addition to GST-SUMO-1, GST-Uba2, GST-Aos1, and GST-Ubc9 and ATP, as described under “Experimental Procedures.” Proteins were separated on an SDS-polyacrylamide gel, and ³⁵S-Mi2α polypeptide was detected by autoradiography. The data are representative of three independent experiments with similar results. B, Coomassie Brilliant Blue (CBB) staining of purified recombinant proteins used in A and C. C, in vitro SUMOylation using a recombinant His-Mi2α(1617–2000). In vitro SUMOylation in the reaction mixture containing GST-SUMO-1, GST-Aos1 (E1), GST-Uba2 (E1), GST-Ubc9 (E2), GST-hDREF, and His-Mi2α(1617–2000), as indicated, was performed. Proteins were separated on an SDS-polyacrylamide gel, and His-Mi2α polypeptide was detected by immunoblotting analysis (IB) using anti-His antibody. The data are representative of three independent experiments with similar results.