Two alternative mechanisms regulate the onset of chaperone-mediated assembly of the proteasomal ATPases

Abstract

The proteasome holoenzyme is a molecular machine that degrades most proteins in eukaryotes. In the holoenzyme, its heterohexameric ATPase injects protein substrates into the proteolytic core particle, where degradation occurs. The heterohexameric ATPase, referred to as 'Rpt ring', assembles through six ATPase subunits (Rpt1-Rpt6) individually binding to specific chaperones (Rpn14, Nas6, Nas2, and Hsm3). Here, our findings suggest that the onset of Rpt ring assembly can be regulated by two alternative mechanisms. Excess Rpt subunits relative to their chaperones are sequestered into multiple puncta specifically during early-stage Rpt ring assembly. Sequestration occurs during stressed conditions, for example heat, which transcriptionally induce Rpt subunits. When the free Rpt pool is limited experimentally, Rpt subunits are competent for proteasome assembly even without their cognate chaperones. These data suggest that sequestration may regulate amounts of individual Rpt subunits relative to their chaperones, allowing for proper onset of Rpt ring assembly. Indeed, Rpt subunits in the puncta can later resume their assembly into the proteasome. Intriguingly, when proteasome assembly resumes in stressed cells or is ongoing in unstressed cells, excess Rpt subunits are recognized by an alternative mechanism-degradation by the proteasome holoenzyme itself. Rpt subunits undergo proteasome assembly until the holoenzyme complex is generated at a sufficient level. The fully-formed holoenzyme can then degrade any remaining excess Rpt subunits, thereby regulating its own Rpt ring assembly. These two alternative mechanisms, degradation and sequestration of Rpt subunits, may help control the onset of chaperone-mediated Rpt ring assembly, thereby promoting proper proteasome holoenzyme formation. by guest on July 10, 2020 http://www.jbc.org/ Downloaded from Figure 1. Proteasome holoenzyme formation requires chaperone-mediated Rpt ring assembly during heat stress. A and B, upon heat stress, early-stage Rpt ring assembly requires the chaperones. Yeast strains were grown at 30°C (lanes 1 -4), and then exposed to heat stress at 37°C for 4 h (lanes 5-8). Proteasome activities were assessed by subjecting whole-cell lysates (60 g) to native PAGE and in-gel peptidase assays using the fluorogenic peptide substrate, LLVY-AMC (panel i). After imaging the native gels in panel i, 0.02% SDS was added to activate free CP in panel ii; SDS denatures the substrate entry gate of the CP (25). Native gels were then subjected to immunoblotting (IB) to detect levels of the Rpt5-Rpt4 modules (t5-t4 module) and proteasome holoenzymes (panel iii). Pgk1 is a loading control. B, Pgk1 blot in lanes 1-4 and 5-8 derives from two different gels, which were processed the same in parallel during immunoblotting and signal detection. C and D, ubiquitinated protein degradation requires chaperone-mediated proteasome assembly during heat stress. Whole-cell lysates (20 g) from samples as in A and B were subjected to 10% BisTris SDS-PAGE and immunoblotting for ubiquitin....

Dynamic Regulation of the 26S Proteasome: From Synthesis to Degradation

“…The main control point is through regulated expression of the corresponding suite of proteasome subunits and associated genes, which is tightly co-ordinated in an attempt to provide stoichiometric amounts of each polypeptide (Figure 2). How tight this regulation is within the collection of proteasome genes remains unclear, as excess subunits do not typically accumulate within cells as free forms (the exception being Rpn10), and appear to be rapidly degraded if they fail to integrate into their respective CP or RP sub-complexes (Peters et al, 2015; Nahar et al, 2019). Thus, while transcription and translation are modulated in an attempt to provide stoichiometric expression, an important arbiter dictating the final concentration of proteasomes might be the abundance of one or more factors in limiting supply.…”

“…A second pathway is the removal of non-functional proteasome subunits by the UPS itself prior to their integration into the holo-proteasome. Hsp42 was shown to be important in yeast by coalescing these polypeptides into cytoplasmic condensates from which they are cleared by active 26S proteasomes (Peters et al, 2015; Nahar et al, 2019).…”

Structure, Dynamics and Function of the 26S Proteasome

“…The second pathway is by the breakdown of dysfunctional proteasome subunits by the UPS itself to avoid their misassembly into the proteasome holoenzyme. It was shown that yeast Hsp42 coalesces the dysfunctional subunits into cytoplasmic condensates that can be removed by the proteasome itself (Peters et al 2015;Nahar et al 2019). The third pathway is by proteaphagy that relies on the autophagy system, which degrades large heterogeneous cytoplasmic constituents, such as organelles, protein aggregates and invasive pathogens Marshall et al 2016;.…”

Tagging the proteasome active site β5 causes tag specific phenotypes in yeast

“…Efforts by several labs in recent years have revealed a surprising complexity in the localization of proteasomes under different stress conditions in a number of organisms 9,19,20,24,25,37,38 . Monitoring the dynamic nature of proteasome assembly, proteasome autophagy, and PSG formation has relied on fluorescent tags fused to particular subunits.…”

“…This is because there is some functional redundancy within the proteasome, and regulatory mechanisms exists that compensate for reduced proteasome activity by upregulating proteasome levels. For instance, when the proteasome assembly chaperones Hsm3 or Nas2 are deleted individually, cells typically grow at rates similar to wild type 5 , 8 , 9 . However, yeast harboring deletions of both chaperones are very sensitive to heat and other protein folding stresses.…”