Characterising Plant Deubiquitinases with in vitro Activity-based Labelling and Ubiquitin Chain Disassembly Assays

Post-translational modification of proteins by ubiquitin is an essential cellular signaling mechanism in all eukaryotes. Ubiquitin is removed from target proteins by a wide range of deubiquitinase (DUB) enzymes with different activities and substrate specificities. Understanding how DUBs function in vitro is a vital first step to uncovering their cellular roles. Here, we provide protocols for the rapid analysis of DUB activity in vitro by activity-based labelling with the suicide probe, HA-ubiquitin vinyl sulfone (HA-UbVS), and ubiquitin chain disassembly assays. We have previously used these methods to analyse the activity of the Arabidopsis thaliana DUB, UBP6, but in principle, these protocols are applicable to any DUB of interest.

Automated summary

Post-translational modification of proteins by ubiquitin is a crucial cellular signaling mechanism in eukaryotes. Deubiquitinase enzymes play a key role in removing ubiquitin from target proteins, and understanding their function in vitro is an important step in uncovering their cellular roles. The protocols provided in this study for analyzing DUB activity in vitro can be applied to any DUB of interest.