title: Proteasomes: Isolation and Activity Assays.
abstract: In eukaryotes, damaged or unneeded proteins are typically degraded by the ubiquitin-proteasome system. In this system, the protein substrate is often first covalently modified with a chain of ubiquitin polypeptides. This chain serves as a signal for delivery to the 26S proteasome, a 2.5-MDa, ATP-dependent multisubunit protease complex. The proteasome consists of a barrel-shaped 20S core particle (CP) that is capped on one or both of its ends by a 19S regulatory particle (RP). The RP is responsible for recognizing the substrate, unfolding it, and translocating it into the CP for destruction. Here we describe simple, one-step purifications scheme for isolating the 26S proteasome and its 19S RP and 20S CP subcomplexes from the yeast Saccharomyces cerevisiae, as well as assays for measuring ubiquitin-dependent and ubiquitin-independent proteolytic activity in vitro.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut.
authors.forename: Yanjie
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut.|Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee, Florida.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Tomko
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut.
authors.forename: Mark
authors.initials: M
authors.lastname: Hochstrasser
mesh_terms: D001681:Biological Assay / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D010447:Peptide Hydrolases / Q000378:metabolism; D025821:Polyubiquitin / Q000378:metabolism; D011208:Powders; D046988:Proteasome Endopeptidase Complex / Q000302:isolation & purification* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000254:growth & development / Q000378:metabolism; D057149:Ubiquitinated Proteins / Q000378:metabolism
chemical_list: D011208:Powders; D057149:Ubiquitinated Proteins; D025821:Polyubiquitin; D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex
keywords: ATPase; proteasome; proteolytic activity; purification; ubiquitin