A new 30-kDa ubiquitin-related SUMO-1 hydrolase from bovine brain

SUMO-1 is a ubiquitin-like protein functioning as an important reversible p rotein modifier. To date there is no report on a SUMO-1 hydrolase/isopeptid ase catalyzing the release of SUMO-1 from its precursor or SUMO-1-ligated p roteins in mammalian tissues. Here we found multiple activities that cleave the SUMO-1 moiety from two model substrates, I-125-SUMO-1-alpha NH-HSTVG-S MHISPPEPESEEEEEHYC and/or GST-SUMO-1-S-35-RanGAP1 conjugate, in bovine brai n extracts. Of them, a major SUMO-1 C-terminal hydrolase had been partially purified by successive chromatographic operations. The enzyme had the abil ity to cleave SUMO-1 not only from its precursor but also from a SUMO-1-lig ated RanGAP1 but did not exhibit any significant cleavage of the ubiquitin- and NEDD8-precursor, The activity of SUMO-1 hydrolase was almost completel y inhibited by N-ethylmaleimide, but not by phenylmethanesulfonyl fluoride, EDTA, and ubiquitin-aldehyde known as a potent inhibitor of deubiquitinyla ting enzymes. Intriguingly, the apparent molecular mass of the isolated SUM O-1 hydrolase was approximately 30 kDa, which is significantly smaller than the recently identified yeast Smt3/SUMO-1 specific protease Ulp1. These re sults indicate that there are multiple SUMO-1 hydrolase/isopeptidases in ma mmalian cells and that the 30-kDa small SUMO-1 hydrolase plays a central ro le in processing of the SUMO-1-precursor.