Purification and characterization of UBP6, a new ubiquitin-specific protease in Saccharomyces cerevisiae

Ubiquitin-specific protease-6 (UBP6) in Saccharomyces cerevisiae was expressed in Escherichia coli and purified from the cells using I-125-labeled ubiquitin-alpha NH-MHISPPEPESEEEEEHYC as a model substrate. The purified UBP6 behaved as a 58-kDa under both nondenaturing and denaturing conditions, indicating that the enzyme comprises a single polypeptide. It was maximally active at pH levels between 8.5 and 9, but showed little or no activity at pH below 7 and above 9.5. As with other UBPs, its activity was strongly inhibited by sulfhydryl-blocking reagents, such as N-ethylmaleimide, and by ubiquitin-aldehyde. In addition to the model substrate, UBP6 hydrolyzed ubiquitin-alpha NH-protein extensions, such as the ubiquitin-alpha NH-carboxyl extension protein of 80 amino acids and ubiquitin-alpha NH-dihydrofolate reductase, but not poly-His-tagged diubiquitin. It was also capable of releasing free ubiquitin from branched polyubiquitin chains that are ligated to proteins through epsilon NH-isopeptide bonds, although to a limited extent. These results suggest that UBP6 may play an important role in the generation of free ubiquitins and certain ribosomal proteins from ubiquitin-ribosomal fusion proteins as well as in deubiquitination of certain polyubiquitinated proteins targeted for degradation by the 26S proteasomes. (C) 1997 Academic Press.