Abstract
The 26S proteasome conducts the majority of regulated protein catabolism in eukaryotes. At the heart of the proteasome is the barrel-shaped 20S core particle (CP), which contains two β-rings sandwiched between two α-rings. Whereas canonical CPs contain α-rings with seven subunits arranged α1-α7, a non-canonical CP in which a second copy of the α4 subunit replaces the α3 subunit occurs in both yeast and humans. The mechanisms that control canonical versus non-canonical CP biogenesis remain poorly understood. Here, we have repurposed a split-protein reporter to identify genes that can enhance canonical proteasome assembly in mutant yeast producing non-canonical α4-α4 CPs. We identified the proteasome subunit α1 as an enhancer of α3 incorporation, and find that elevating α1 protein levels preferentially drives canonical CP assembly under conditions that normally favor α4-α4 CP formation. Further, we demonstrate that α1 is stoichiometrically limiting for α-ring assembly, and that enhancing α1 levels is sufficient to increase proteasome abundance and enhance stress tolerance in yeast. Together, our data indicate that the abundance of α1 exerts multiple impacts on proteasome assembly and composition, and we propose that the limited α1 levels observed in yeast may prime cells for alternative proteasome assembly following environmental stimuli.
Highlights
The ubiquitin-proteasome system (UPS) is the primary mechanism for regulated protein catabolism in eukaryotes[1,2]
Yeast lacking α3 synthesize a non-canonical core particle (CP), referred to as the α4-α4 CP, in which a second copy of the canonical α4 subunit is incorporated into the position normally occupied by the absent α3 subunit[13]
We hypothesized that fusion of these dihydrofolate reductase (DHFR) fragments to pairs of α-subunits would report on their juxtaposition primarily within fully assembled CPs, which greatly outnumber proteasomal assembly intermediates in rapidly growing cells[13,15,17]
Summary
The ubiquitin-proteasome system (UPS) is the primary mechanism for regulated protein catabolism in eukaryotes[1,2]. The abundance of α4-α4 CPs is primarily controlled by a pair of evolutionarily conserved proteasome assembly chaperones, Pba[3] and Pba[4] (PAC3 and PAC4 in humans)[14,15,16,17,18,19] These chaperones form a heterodimer (Pba3-4) that promotes α3 incorporation over a second copy of α4 to yield canonical CPs. Under conditions commonly associated with the tumor phenotype, such as increased proteasome biogenesis[20,21,22] and oxidative stress[23], Pba[3,4] becomes limiting for proteasome assembly, resulting in enhanced formation of α4-α4 CPs14. It is unknown what governs whether α3 or α4 incorporates into the α3 position during such limiting chaperone activity
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