Purification and Characterization of UBP6, a New Ubiquitin-Specific Protease inSaccharomyces cerevisiae

Ubiquitin-specific protease-6 (UBP6) inSaccharomyces cerevisiaewas expressed inEscherichia coliand purified from the cells using125I-labeled ubiquitin-αNH-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 asN-ethylmaleimide, and by ubiquitin-aldehyde. In addition to the model substrate, UBP6 hydrolyzed ubiquitin-αNH-protein extensions, such as the ubiquitin-αNH-carboxyl extension protein of 80 amino acids and ubiquitin-αNH-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 ϵNH-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.