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.
Verma
Rati
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Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.
Deshaies
R J
RJ
D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D004926:Escherichia coli / Q000378:metabolism; D009842:Oligopeptides; D046988:Proteasome Endopeptidase Complex / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013379:Substrate Specificity; D025801:Ubiquitin / Q000378:metabolism*
D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D009842:Oligopeptides; C086926:SIC1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D046988:Proteasome Endopeptidase Complex; C078846:epoxomicin