Assaying degradation and deubiquitination of a ubiquitinated substrate by purified 26S proteasomes.

The 26S proteasome is a multisubunit complex that catalyzes ATP-dependent proteolysis of cellular proteins. It eliminates misfolded proteins, as well as labile regulatory proteins, thereby serving a central role in maintaining cellular homeostasis. The bulk of the known substrates of the 26S proteasome are earmarked for proteolysis by covalent modification with a multiubiquitin chain, which is recognized by specific receptors. Once targeted, the substrate is deubiquitinated and degraded by the 26S proteasome. This chapter describes assays that monitor ATP- and ubiquitin-dependent proteolysis of the S-Cdk inhibitor Sic1.