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

HECT ubiquitin ligases as accessory proteins of the plant proteasome

The proteasome plays vital roles in eukaryotic cells by orchestrating the regulated degradation of large repertoires of substrates involved in numerous biological processes. Proteasome dysfunction is associated with a wide variety of human pathologies and in plants severely affects growth, development and responses to stress. The activity of E3 ubiquitin ligases marks proteins fated for degradation with chains of the post-translational modifier, ubiquitin. Proteasomal processing of ubiquitinated substrates involves ubiquitin chain recognition, deubiquitination, ATP-mediated unfolding and translocation, and proteolytic digestion. This complex series of steps is made possible not only by the many specialised subunits of the 1.5 MDa proteasome complex but also by a range of accessory proteins that are recruited to the proteasome. A surprising class of accessory proteins are members of the HECT-type family of ubiquitin ligases that utilise a unique mechanism for post-translational attachment of ubiquitin to their substrates. So why do proteasomes that already contain all the necessary machinery to recognise ubiquitinated substrates, harbour HECT ligase activity? It is now clear that some ubiquitin ligases physically relay their substrates to proteasome-associated HECT ligases, which prevent substrate stalling at the proteasome. Moreover, HECT ligases ubiquitinate proteasome subunits, thereby modifying the proteasome’s ability to recognise substrates. They may therefore enable proteasomes to be both non-specific and extraordinarily selective in a complex substrate environment. Understanding the relationship between the proteasome and accessory HECT ligases will reveal how the proteasome controls so many diverse plant developmental and stress responses

Transcriptional regulation by complex interplay between posttranslational modifications

Transcriptional reprogramming in response to developmental changes or environmental inputs is regulated by a wide variety of transcription factors and cofactors. In plants, the stability of many transcriptional regulators is mediated by the ubiquitin-mediated proteasome. Recent reports suggest that additional post-translational modifications modulate the ubiquitination and thus stability of transcriptional regulators. In addition to well-recognized phosphorylative control, particularly conjugation to the ubiquitin-like protein SUMO as well as thiol modification by nitric oxide to yield S-nitrosothiols, are emerging as key regulatory steps for governing protein ubiquitination in the nucleus. Complex interplay between these different post-translational modifications may provide robust control mechanisms to fine tune developmental and stress-responsive transcriptional programs

Proteasome-associated HECT-type ubiquitin ligase activity is required for plant immunity

<div><p>Regulated degradation of proteins by the 26S proteasome plays important roles in maintenance and signalling in eukaryotic cells. Proteins are marked for degradation by the action of E3 ligases that site-specifically modify their substrates by adding chains of ubiquitin. Innate immune signalling in plants is deeply reliant on the ubiquitin-26S proteasome system. While progress has been made in understanding substrate ubiquitination during plant immunity, how these substrates are processed upon arrival at the proteasome remains unclear. Here we show that specific members of the HECT domain-containing family of ubiquitin protein ligases (UPL) play important roles in proteasomal substrate processing during plant immunity. Mutations in <i>UPL1</i>, <i>UPL3</i> and <i>UPL5</i> significantly diminished immune responses activated by the immune hormone salicylic acid (SA). In depth analyses of <i>upl3</i> mutants indicated that these plants were impaired in reprogramming of nearly the entire SA-induced transcriptome and failed to establish immunity against a hemi-biotrophic pathogen. UPL3 was found to physically interact with the regulatory particle of the proteasome and with other ubiquitin-26S proteasome pathway components. In agreement, we demonstrate that UPL3 enabled proteasomes to form polyubiquitin chains, thereby regulating total cellular polyubiquitination levels. Taken together, our findings suggest that proteasome-associated ubiquitin ligase activity of UPL3 promotes proteasomal processivity and is indispensable for development of plant immunity.</p></div