Hexameric assembly of the proteasomal ATPases is templated through their C termini

Abstract

Substrates of the proteasome are recognized and unfolded by the regulatory particle (RP), then translocated into the core particle (CP) to be degraded1. A hetero-hexameric ATPase ring, containing subunits Rpt1-Rpt6, is situated within the base subassembly of the RP1. The ATPase ring sits atop the CP, with the Rpt C-termini inserted into pockets in the CP2–6. We have identified a novel function of the Rpt proteins in proteasome biogenesis through deleting the C-terminal residue from each Rpt. Our results indicate that assembly of the hexameric ATPase ring is templated on the CP. We have also identified an apparent intermediate in base assembly, BP1, which contains Rpn1, three Rpts, and Hsm3, a chaperone for base assembly. The Rpt proteins with the strongest assembly phenotypes, Rpt4 and Rpt6, were absent from BP1. We propose that Rpt4 and Rpt6 form a nucleating complex to initiate base assembly, and that this complex is subsequently joined by BP1 to complete the Rpt ring. Our studies show that assembly of the proteasome base is a rapid yet highly orchestrated process.

Quantitative live-cell imaging reveals spatio-temporal dynamics and cytoplasmic assembly of the 26S proteasome

“…6c, right panel), suggesting that almost all proteasomes contained stoichiometric amounts of the fusion proteins. These observations support the CP template assembly model of the RP 39 . Furthermore, structural analysis by cryo-electron microscopy revealed that the Pre6-Rpt2-fused proteasome was almost indistinguishable from the wild-type proteasome (Fig.…”

“…Our FCS analysis did not detect proteasome assembly intermediates in wild-type cells, probably because such intermediates are not abundant [37][38][39][40] . Previous studies using a specific importin-a mutant, srp1-49, suggested that nucleocytoplasmic transport mediated by importin a/b is coupled to proteasome biogenesis [19][20][21]23 .…”

“…Rpn4 is a transcription factor that regulates proteasome concentrations; deletion of RPN4 significantly reduces the levels of proteasome subunits 39,41 . Consistent with this, the cytoplasmic concentration of Rpn7-GFP, measured by FCS, decreased to B160 nM in srp1-49 rpn4D double-mutant cells at both the permissive and non-permissive temperatures (Supplementary Fig.…”

Intracellular Dynamics of the Ubiquitin-Proteasome-System

“…Two competing models exist for RP assembly ( Funakoshi et al , 2009; Le Tallec et al , 2009; Park et al , 2009; Roelofs et al , 2009). The first posits that RP assembly occurs in modules independent of the CP with the help of four RP-dedicated chaperones, named Hsm3, Nas2, Nas6 and Rpn14 ( Funakoshi et al , 2009).…”

“…The first posits that RP assembly occurs in modules independent of the CP with the help of four RP-dedicated chaperones, named Hsm3, Nas2, Nas6 and Rpn14 ( Funakoshi et al , 2009). In contrast, the second model proposes that the CP serves as a scaffold for the heterohexameric ATPase ring of the RP base ( Park et al , 2009). The second model, however, appears less likely with regard to X-ray structure analysis showing that the RP-dedicated chaperones hinder the association between the RP base and CP α ring ( Barrault et al , 2012).…”