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Curr Protoc Cell Biol. 2015 Jun 1;67:3.43.1-20. doi: 10.1002/0471143030.cb0343s67.

Proteasomes: Isolation and Activity Assays.

Author information

1
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut.
2
Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee, Florida.

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.

KEYWORDS:

ATPase; proteasome; proteolytic activity; purification; ubiquitin

PMID:
26061243
PMCID:
PMC4484579
DOI:
10.1002/0471143030.cb0343s67
[Indexed for MEDLINE]
Free PMC Article

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