FIGURE 2.

Lys-509 is the major SUMO acceptor in MTA1. A, Lys-509 is identified as a major SUMO acceptor in vitro. ³⁵S-Labeled, in vitro translated MTA1-WT or MTA1-K509R mutant was incubated in SUMOylation reactions containing SUMO2 and 1.5 μg (left panel) or 0.25 μg (right panel) of GST-UBC9. The reaction products were resolved by SDS-PAGE and detected by autoradiography. The bands representing SUMO2 conjugation on Lys-509 and the lysine residue within the non-consensus site are indicated by an arrowhead and an asterisk, respectively. B, Lys-509 is identified as a major SUMO acceptor in vivo. COS-1 cells were co-transfected with the indicated expression vectors. HA-His-SUMO2 conjugates were purified by Ni-NTA beads and analyzed by Western blotting (WB) with anti-Myc mouse antibody. Expression levels of Myc-tagged MTA1 and vinculin are shown as input. The band representing SUMO2 conjugation on Lys-509 is indicated by an arrowhead. C, identification of potential SUMOylation sites other than Lys-509 in MTA1 in vitro. MTA1-WT or its indicated mutants were generated by ³⁵S-labeled in vitro translation and incubated in SUMOylation reactions containing SUMO2 and 0.25 μg of GST-UBC9. The reaction products were resolved by SDS-PAGE and detected by autoradiography.