title: Purification of 26S Proteasomes and Their Subcomplexes from Plants.
abstract: The 26S proteasome is a highly dynamic, multisubunit, ATP-dependent protease that plays a central role in cellular housekeeping and many aspects of plant growth and development by degrading aberrant polypeptides and key cellular regulators that are first modified by ubiquitin. Although the 26S proteasome was originally enriched from plants over 30 years ago, only recently have significant advances been made in our ability to isolate and study the plant particle. Here, we describe two robust methods for purifying the 26S proteasome and its subcomplexes from Arabidopsis thaliana; one that involves conventional chromatography techniques to isolate the complex from wild-type plants, and another that employs the genetic replacement of individual subunits with epitope-tagged variants combined with affinity purification. In addition to these purification protocols, we describe methods commonly used to analyze the activity and composition of the complex.
authors.affiliation: Department of Genetics, University of Wisconsin-Madison, 425-G Henry Mall, Madison, WI, USA.|Department of Biology, Washington University in St Louis, Campus Box 1137, One Brookings Drive, St. Louis, MO, 63130, USA.
authors.forename: Richard S
authors.initials: RS
authors.lastname: Marshall
authors.affiliation: Department of Genetics, University of Wisconsin-Madison, 425-G Henry Mall, Madison, WI, USA.
authors.forename: David C
authors.initials: DC
authors.lastname: Gemperline
authors.affiliation: Department of Genetics, University of Wisconsin-Madison, 425-G Henry Mall, Madison, WI, USA. rdvierstra@wustl.edu.|Department of Biology, Washington University in St Louis, Campus Box 1137, One Brookings Drive, St. Louis, MO, 63130, USA. rdvierstra@wustl.edu.
authors.forename: Richard D
authors.initials: RD
authors.lastname: Vierstra
mesh_terms: D017360:Arabidopsis / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D002458:Cell Fractionation / Q000295:instrumentation / Q000379:methods*; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D057075:Enzyme Assays; D015870:Gene Expression; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D059748:Proteolysis; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D036226:Seedlings / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D009842:Oligopeptides; D021122:Protein Subunits; D025801:Ubiquitin; C085706:FLAG peptide; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease
keywords: Affinity purification; Arabidopsis; Core protease; Proteasome; Proteolysis; Regulatory particle; Ubiquitin