(A) Upon extended reaction times under conditions that favor substrate sumoylation (or SUMO chain formation), SCE C94S and SCE C94K, but not SCE C94A, form an SCE–SUMO adduct that is stable under reducing conditions. (B) WT SCE (top), but not SCE C94S (bottom), can bind SUMO in a thioester linkage under conditions favorable for the reaction of WT SCE (left part, non-reducing). The reaction proceeds quickly. Reducing condition (sample boiling with DTT) destroys the linkage (right part, reducing). (C) The SCE–SUMO adduct with SCE C94S, but not that with SCE C94K, is sensitive to alkali treatment, indicating an oxyester linkage between the two proteins. The asterisk indicates a contaminating band of the SUMO preparation.