Multiple methods are used to identify sumoylation of E1 and E2 enzymes.

(A) In vitro sumoylation using E1, E2 and acetylated SUMO, which cannot form poly-SUMO chains, reveals higher molecular weight sumoylated species of Uba2, Aos1 and Ubc9. Ulp1 removal of SUMO allowed the identification of unmodified lysine as sumoylation sites. (B) Peptides that led to the identification of sumoylation sites of E1 and E2 enzymes are listed. Positions of the unmodified lysine (sumoylation site) are shown in parenthesis while all labeled lysine are acetylated. The periods flanking each peptide indicate the site of trypsin cleavage. (C) Comparison between WT Smt3 and Smt3-I96R as substrate for in vitro sumoylation by Aos1-Uba2 and Ubc9-K153R. Both WT Smt3 and Smt3-I96R are acetylated to prevent poly-SUMO formation. (D) Sumoylated peptides identified for Aos1 and Uba2 using EQIGG and GG remnants with the position of lysine indicated in parenthesis. * The first methionine was removed from Aos1, which is fused with N-terminal 6xHIS tag for bacterial expression.