Ubiquitin C-terminal hydrolase activity associated with the 26 S protease complex.

Abstract

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.