Analysis of proteasome-associated ubiquitin ligase activity

Research output: Chapter in Book/Report/Conference proceedingChapter


The ubiquitin-proteasome system (UPS) is the predominant protein degradation machinery in eukaryotic cells. It is highly conserved among eukaryotes and essential for their survival. Through regulated proteolysis the UPS plays a key role in a myriad of cellular functions, including developmental and stress signaling, cell differentiation, and cell death. Attachment of a ubiquitin chain to a substrate can trigger its recruitment to the proteasome for proteolysis. To efficiently degrade substrates, however, the proteasome employs HECT-type ubiquitin ligases that can further remodel ubiquitin chains of proteasome-captured substrates. It is thought that this remodeling process is necessary to maintain substrate affinity for the proteasome and to completely translocate the substrate into the 20S proteolytic barrel. Here, we describe a protocol for purifying proteasomes and their associated accessory proteins and provide a practical way to detect proteasome-associated E3 ligase activity. This assay is reliable and efficient for assessing the ability of proteasomes to form ubiquitin conjugates and is applicable to a wide range of eukaryotic species.
Original languageEnglish
Title of host publicationPlant Proteostasis
Subtitle of host publication Methods in Molecular Biology
PublisherSpringer Nature
ISBN (Electronic)978-1-0716-2784-6
ISBN (Print)978-1-0716-2783-9
Publication statusPublished - 23 Nov 2022

Publication series

NamePlant Proteostasis
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029
NameMethods in Molecular Biology
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029


  • HECT-type ligases
  • posttranslational modifications
  • proteasome
  • proteasome-associated ubiquitin E3 ligases
  • ubiquitin


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