Project Summary
In the current paradigm of protein degradation by the ubiquitin-proteasome system, ubiquitination of protein
substrates has been considered as a major regulatory step. Hundreds of ubiquitinating enzymes regulate
polyubiquitination, to target proteins to the proteasome for degradation. This paradigm has led to remarkable
discoveries on how protein degradation is required for fundamental cellular processes, and also for destroying
misfolded or aberrant proteins, which can lead to cancers and neurodegenerative conditions. However, a
significant knowledge gap exists in the current paradigm, as to how the cell ensures the proper number of
functional proteasomes. Our research goal is to overcome this gap, by elucidating mechanisms of proteasome
assembly. We discovered multiple evolutionarily conserved chaperones that are dedicated to proteasome
assembly. In our model of proteasome assembly, chaperones hinder subunit addition until a given step of
assembly occurs correctly. This model suggests a new concept, that assembly is not simply a series of subunit
additions—it is also integrated with extensive quality control (QC). We will pursue this new direction of
research—unraveling mechanisms of chaperone-mediated QC during proteasome assembly. For QC, the
assembly process is a “moving target” as one step progresses to the next. How can these moving targets be
monitored? Chaperones can “mark” all of them, using a fundamental mechanism—specifically binding to
assembly intermediates. We will investigate how chaperones serve as components (e.g. adaptors) of extensive
QC of the proteasome, via three aims. First, we will examine chaperones’ new connections to proteasomal
nuclear localization signals (NLSs), which have been known for ~20 years to exist on proteasome subunits, but
their functions are poorly characterized. We hypothesize that proteasomal NLSs drive chaperones into the
nucleus, as a QC mechanism, to prevent defective proteasomes from forming in the cytoplasm. Second,
we will investigate the non-canonical role of ubiquitination via an E3 ligase, Not4, during proteasome assembly.
Bulky polyubiquitin sterically prevents the addition of the next subunits, blocking proteasome assembly, for QC.
We hypothesize that this non-canonical role of polyubiquitin depends on a specific chaperone, Nas6 (an
oncoprotein, Gankyrin in humans). Third, we will interrogate how chaperones help form proteasome storage
granules, which preserve functional proteasomes during nutritional stress. We hypothesize that chaperones
help distinguish functional proteasomes—via binding to sub-complexes—but in this case, resulting from
proteasome disassembly during nutritional stress. We will interrogate these hypotheses using our well-
established biochemical, cell biological, and proteomics strategies, together with our powerful yeast model to
assess proteasome functions in vivo. Proteasome assembly chaperones are dysregulated in many cancers,
altering proteasomal activities. Since the proteasome is a proven therapeutic target, we envision exploiting these
chaperones to modulate cellular protein degradation, to broaden the application of the proteasome as a target.