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 125I-labeled ubiquitin-alphaNH-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-alphaNH-protein extensions, such as the ubiquitin-alphaNH-carboxyl extension protein of 80 amino acids and ubiquitin-alphaNH-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 epsilonNH-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.
Park
K C
KC
College of Natural Sciences, Seoul National University, Seoul, 151-742, Korea.
Woo
S K
SK
Yoo
Y J
YJ
Wyndham
A M
AM
Baker
R T
RT
Chung
C H
CH
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism; D011480:Protease Inhibitors / Q000737:chemistry / Q000494:pharmacology; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity; D014452:Ubiquitins / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology
D010446:Peptide Fragments; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014452:Ubiquitins; C051927:ubiquitin-aldehyde; D010450:Endopeptidases; C109028:UBP6 protein, S cerevisiae