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.