In vitro autoubiquitination activity of E3 ubiquitin ligases of the N-degron pathway

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.