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