Spoel, Steven

Bayne, Elizabeth

Wang, Zhishuo

Biotechnology and Biological Sciences Research Council (BBSRC)

2022-02-23T11:50:36Z

2022-02-23T11:50:36Z

2022-02-23

Degradation of intracellular proteins by the ubiquitin-proteasome system (UPS) is a sophisticated mechanism that begins with anchoring ubiquitin molecules to a substrate and ends with proteasome-dependent proteolysis. Initiation of ubiquitination by E3 ligases is a key step in this pathway that selectively labels unstable or damaged proteins. The ubiquitinated substrate is then recognised by proteasome-associated ubiquitin receptors and subsequently degraded by the proteasome. Recent studies have identified several E3 ligases that surprisingly associate with the proteasome as accessory proteins. As substrates are already modified by ubiquitin when they arrive at the proteasome, it is unclear what the role of these proteasome-associated ligases are. In this study, the role of proteasome-associated ubiquitin ligases in proteasomal substrate degradation was characterised and the functional significance of ubiquitin chain remodelling at the proteasome were explored in planta. In Arabidopsis thaliana, HECT-type Ubiquitin Protein Ligases (UPLs) have been identified as proteasome-associated ubiquitin ligases that are required for salicylic acid (SA)-induced plant immunity. Accordingly, the mechanism behind regulation of plant immune response by UPLs is further studied in Chapter 3. Here, it is shown that UPLs control SA-dependent transcriptional reprogramming via regulating homeostasis of the SA-responsive coactivator NPR1. SA-induced accumulation of NPR1 was impaired in upl mutants, which resulted in diminished expression of immune genes. Additionally, proteasome-associated UPLs facilitated polyubiquitination of NPR1, and thereby promoted its proteasomal turnover. This process was indispensable for clearing inactive NPR1 from chromatin. Thus, UPL-mediated remodelling of NPR1-attached ubiquitin chains at the proteasome is required for maximum transcriptional activity of NPR1. In Chapter 4 I show that proteasome-associated UPLs also target other transcription activators, including the developmental and ethylene-responsive EIN3 activator. I demonstrate that by physically interacting with UPL3, the SCFEBF2 ubiquitin ligase complex directly escorted EIN3 to the proteasome. Subsequent ‘eleventh-hour’ ubiquitin chain remodelling by proteasome-associated UPL3/4 was required for processive degradation of EIN3 by the proteasome and was critical for removal of EIN3 from its target gene promoters. Besides targeting substrates destined for the proteasome, I show in Chapter 5 that UPL3 and UPL4 are also involved in polyubiquitination of other E3 ligases. UPL3/4 catalysed ubiquitination of the immune-responsive U-box E3 ligase, PUB22, and controlled its proteasomal turnover. Mutation of PUB22 and its homologues, PUB23 and PUB24, supressed the disease susceptibility phenotype of the upl3 upl4 mutant, indicating that UPL3/4 also regulate immunity via modulating homeostasis of PUB ligases. Overall, my findings indicate that unstable hormone-responsive transcriptional activators are sequentially polyubiquitinated by relays of ubiquitin ligases in which HECT-type ligases prevent the stalling of proteasome-bound substrates. On the other hand, HECT-type ligases also target other E3 ligases for degradation, thereby indirectly influencing substrate levels of these E3 ligases. Thus, my findings demonstrate that proteasomes unexpectedly influence the ubiquitination and stability of both E3 ligases and their substrates to regulate transcriptional programmes in plants.

https://hdl.handle.net/1842/38623

http://dx.doi.org/10.7488/era/1886

en

The University of Edinburgh

FURNISS, J. J., GREY, H., WANG, Z., NOMOTO, M., JACKSON, L., TADA, Y. & SPOEL, S. H. 2018. Proteasome-associated HECT-type ubiquitin ligase activity is required for plant immunity. PLoS Pathog, 14, e1007447

ubiquitin-proteasome system

UPS

cell signalling regulation

proteasome-associated ubiquitin ligases

substrate degradation

Functional analysis of proteasome-associated ubiquitin ligases in plants

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy