Ubiquitin C-terminal hydrolase activity associated with the 26 S protease complex.
In the ubiquitin (Ub) system for protein degradation, proteins ligated to Ub are degraded by an ATP-dependent 26 S protease complex. During or after proteolysis, free Ub is regenerated, but the mechanisms of Ub release remained unknown. It was previously observed that free Ub is released from a Ub-histone conjugate by an ATP-dependent activity present in partially purified preparations of 26 S complex, but the relationship of this activity to protein breakdown was not established. We now show that purified preparations of 26 S complex release free Ub from conjugates that are good substrates for proteolysis, such as conjugates of lysozyme with reductively methylated Ub. The activity that releases free Ub co-migrates with the 26 S protease complex in glycerol density gradient centrifugation, indicating that the responsible Ub C-terminal hydrolase is an integral part of the 26 S complex. Complex-associated hydrolase can also act on adducts in which a single Ub unit is attached to protein, such as a bacterially expressed construct in which the C terminus of Ub is fused to the alpha-NH2 group of a fragment of Ub that contains 60% of its N-terminal region. In all cases, Ub release is insensitive to Ub-aldehyde (an inhibitor of some Ub C-terminal hydrolases) and is stimulated by MgATP. ATP cannot be replaced by beta, gamma-nonhydrolyzable analogs, but it can be substituted by CTP and GTP. The nucleotide specificity of Ub release by the 26 S complex is similar to that observed previously for conjugate proteolysis and nucleotide hydrolysis. It thus seems that the activity of the Ub C-terminal hydrolase associated with the 26 S complex is tightly coupled to the proteolytic action of the complex, and it may have a role in the release of Ub from linkage to amino groups of the protein substrate at the final stages of the Ub proteolytic pathway.
Biochemistry Department, Technion-Israel Institute of Technology, Haifa.
E
E
Eytan
T
T
Armon
H
H
Heller
S
S
Beck
A
A
Hershko
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010447:Peptide Hydrolases / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D013869:Thiolester Hydrolases / Q000378:metabolism*; D043222:Ubiquitin Thiolesterase; D014452:Ubiquitins / Q000378:metabolism*
D009838:Oligodeoxyribonucleotides; D014452:Ubiquitins; D000255:Adenosine Triphosphate; D013869:Thiolester Hydrolases; D010447:Peptide Hydrolases; D043222:Ubiquitin Thiolesterase; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease