Reconstitution of PA700, the 19S regulatory particle, from purified precursor complexes

Research output: Chapter in Book/Report/Conference proceedingChapter

3 Scopus citations

Abstract

Here, we describe methodology for the in vitro reconstitution of PA700, the 19S regulatory particle of the 26S proteasome, from three purified subcomplexes that closely represent cellular assembly intermediates. These PA700 subcomplexes (denoted PS-1, PS-2, and PS-3) account for all subunits present in purified PA700 but have no overlapping or non-PA700 components. The reconstituted PA700 displays functional features indistinguishable from independently purified PA700, including ATPase activity, deubiquitylating activity, and ATP-dependent binding and activation of the 20S proteasome. This reconstitution assay -provides a platform for exploration of critical biochemical and molecular features of PA700 assembly and for insights to 26S proteasome assembly in intact cells.

Original languageEnglish (US)
Title of host publicationUbiquitin Family Modifiers and the Proteasome
Subtitle of host publicationReviews and Protocols
EditorsJurgen Dohmen, Martin Scheffner
Pages443-452
Number of pages10
DOIs
StatePublished - 2012

Publication series

NameMethods in Molecular Biology
Volume832
ISSN (Print)1064-3745

Keywords

  • 19S regulator
  • 26S proteasome
  • AAA proteins
  • PA700
  • Rpt subunits

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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