Date Published: November 20, 2018
Publisher: Public Library of Science
Author(s): James J. Furniss, Heather Grey, Zhishuo Wang, Mika Nomoto, Lorna Jackson, Yasuomi Tada, Steven H. Spoel, Libo Shan.
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 UPL1, UPL3 and UPL5 significantly diminished immune responses activated by the immune hormone salicylic acid (SA). In depth analyses of upl3 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.
The ubiquitin-26S proteasome system (UPS) plays an essential cellular role in selective degradation of proteins that are short-lived or damaged. Degradation of proteins is mediated by an enzymatic cascade in which a small and highly conserved ubiquitin molecule is covalently attached to the substrate. Typically an ubiquitin-activating E1 enzyme forms a high-energy thioester bond to an ubiquitin adduct, which is then transferred onto the active site of an ubiquitin conjugating E2 enzyme. In partnership with an E3 ligase that recruits a specific substrate, the E2 enzyme facilitates formation of an isopeptide bond between the ε-amino group of a lysine residue within the substrate and the carboxy-terminal group of ubiquitin. Reiterations of this reaction cycle result in subsequent ubiquitin molecules being similarly attached to internal lysines of the preceding ubiquitin moiety, thereby generating a polyubiquitin chain on the substrate [1, 2]. Lysine 48-linked chains of four or more ubiquitins show high affinity for ubiquitin receptors within the 19S regulatory cap of the proteasome . Substrate degradation involves its unfolding by chaperone activity of the 19S particle, cleavage and release of the polyubiquitin chain for recycling, and subsequent threading of the unfolded substrate into the 20S subunit of the proteasome, a barrel-shaped multi-catalytic proteinase .
The ubiquitin-26S proteasome system plays indispensable roles in transcriptional regulation of plant immune genes but how substrates are processed upon arrival at the proteasome remained unclear. Here we demonstrated that members of the HECT-domain family of UPL ubiquitin ligases play an important role in SA-dependent transcriptional responses and immunity. In particular we report that proteasomes harbour UPL3-dependent ubiquitin ligase activity that was necessary for total cellular substrate polyubiquitination as well as SA-responsive transcriptional reprogramming and immunity.