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Updated: Jul 20, 2025

In Vitro Analysis of E3 Ubiquitin Ligase Function
Published on: May 14, 2021
In vitro autoubiquitination activity of E3 ubiquitin ligases of the N-degron pathway
Alexander Sandmann1, Nico Dissmeyer1
1Department of Plant Physiology and Protein Metabolism Laboratory, University of Osnabruck, Osnabruck, Germany; CellNanOs-Center of Cellular Nanoanalytics, University of Osnabruck, Osnabruck, Germany; ScienceCampus Halle-Plant-Based Bioeconomy, Halle (Saale), Germany.
Abstract:
As a part of the ubiquitin-proteasome system, E3 ubiquitin ligases play an important role in the regulation of the proteome in eukaryotic cells. These enzymes are extensively studied because of their crucial function, however it can be challenging to observe E3 ubiquitin ligases in action. Here, we outline a method for determining whether a known or potential E3 ubiquitin ligase exhibits autoubiquitination activity in vitro using PROTEOLYSIS1 (PRT1, AT3G24800), the first identified N-degron pathway E3 ubiquitin ligase from plants as an example. The approach provided here makes it possible to analyze mutations that could reduce or eliminate activity, to test for interaction with E2 ubiquitin conjugating enzymes, as well as to check for in vitro substrate ubiquitination.
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