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 purification schemes for isolating the 26S proteasome and its 19S RP and 20S CP subcomplexes from the yeast Saccharomyces cerevisiae. A gel filtration step can be added to further enhance purity. We also describe assays for measuring ubiquitin-dependent and ubiquitin-independent proteolytic activity in vitro. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Purification of active 26S proteasomes Support Protocol 1: Growth of yeast strains and production of yeast cell powder Support Protocol 2: Regeneration of anti-flag M2 affinity gel Basic Protocol 2: Purification of the 19S regulatory particle (RP) Basic Protocol 3: Purification of active 20S CP Basic Protocol 4: In-gel peptidase activity assay for 20S CP and 26S proteasomes Basic Protocol 5: In-solution peptidase activity assay for 20S and 26S proteasomes Basic Protocol 6: Measuring degradation of polyubiquitinated SIC1^PY Basic Protocol 7: Gel filtration of purified proteasomes and subcomplexes.

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Li Y, Tomko RJ, Hochstrasser M

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