Sumoylation of Human Translationally Controlled Tumor Protein Is Important for Its Nuclear Transport

Figure 1

TCTP can be sumoylated in vitro. GST Pull-Down assay (a). Proteins were resolved on a 12% SDS-PAGE and transferred to a nitrocellulose membrane and probed with anti-HIS antibody. Lane 1: 5 μg of rTCTP, Lane 2, shows the amount of TCTP that bound to the GST-Ubc9 enzyme, and Lane 3 shows the amount of TCTP that bound to GST alone control. These results show that TCTP strongly binds to Ubc9 enzyme. In vitro sumoylation assay (b). Proteins were resolved on 12% SDS-PAGE and transferred to nitrocellulose membrane and probed with anti-His or anti-TCTP antibody to detect TCTP. Note the high molecular weight product (TCTP-SUMO-1) in Lane 4, where the incubation mixture contained TCTP, UBA2/AOS1, and Ubc9/SUMO. Deletion of any one of these products from the reaction mixture resulted in the absence of this high molecular weight band (Lanes 1, 2, 3, and 5). Mutation studies (c), The lysine residue (aa 164) in the TCTP was mutated to arginine. When such mutated TCTP was subjected to in vitro sumoylation, SUMO-1 failed to bind to mutated TCTP, whereas wild-type TCTP could be sumoylated under the same conditions.