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

Research output: Chapter in Book/Report/Conference proceedingChapter

3 Citations (Scopus)

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 publicationMethods in Molecular Biology
Pages443-452
Number of pages10
Volume832
DOIs
StatePublished - 2012

Publication series

NameMethods in Molecular Biology
Volume832
ISSN (Print)10643745

Fingerprint

Proteasome Endopeptidase Complex
Adenosine Triphosphatases
Adenosine Triphosphate
In Vitro Techniques
ATP dependent 26S protease
26S proteasome non-ATPase regulatory subunit 13

Keywords

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

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Demartino, G. N. (2012). Reconstitution of PA700, the 19S regulatory particle, from purified precursor complexes. In Methods in Molecular Biology (Vol. 832, pp. 443-452). (Methods in Molecular Biology; Vol. 832). https://doi.org/10.1007/978-1-61779-474-2_31

Reconstitution of PA700, the 19S regulatory particle, from purified precursor complexes. / Demartino, George N.

Methods in Molecular Biology. Vol. 832 2012. p. 443-452 (Methods in Molecular Biology; Vol. 832).

Research output: Chapter in Book/Report/Conference proceedingChapter

Demartino, GN 2012, Reconstitution of PA700, the 19S regulatory particle, from purified precursor complexes. in Methods in Molecular Biology. vol. 832, Methods in Molecular Biology, vol. 832, pp. 443-452. https://doi.org/10.1007/978-1-61779-474-2_31
Demartino GN. Reconstitution of PA700, the 19S regulatory particle, from purified precursor complexes. In Methods in Molecular Biology. Vol. 832. 2012. p. 443-452. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-61779-474-2_31
Demartino, George N. / Reconstitution of PA700, the 19S regulatory particle, from purified precursor complexes. Methods in Molecular Biology. Vol. 832 2012. pp. 443-452 (Methods in Molecular Biology).
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