Figure 1.

OTS1 Has SUMO Protease Activity.

(A) Alignment of the core protease domain of Arabidopsis OTS1, OTS2, and ESD4 and yeast (Saccharomyces cerevisiae) Ulp1. Asterisks indicate the conserved amino acids forming the catalytic triad. Numbers refer to the amino acid residues of each protein.

(B) and (C) In vitro SUMO protease activity assay. Equal amounts (20 μg) of soluble proteins from E. coli cells transformed with expression vector control (Vector), His:ots1(C526S), His:OTS1, and His:ESD4 were mixed with 200 ng of purified SUMO substrate SUMO1 (His:SUMO1:FLC). Individual components were incubated in the indicated combinations, and the mixture was resolved by 12% SDS-PAGE and probed with anti-His or anti-SUMO1/2 antibodies (C). Individual bands corresponding to different proteins are designated at right. Asterisks indicate additional bands likely derived from nonspecific degradation of His:SUMO1:FLC or His:OTS1/His:ots1(C526S). Arrowheads refer to cross reactions of nonspecific SUMO1 antibodies.