A Ubiquitin‐dependent Switch during Assembly of the Proteasomal ATPases Mediated by Not4 Ubiquitin Ligase

Abstract

In the proteasome holoenzyme, the hexameric ATPases (Rpt1‐Rpt6) enable degradation of ubiquitinated proteins by unfolding and translocating them into the proteolytic core particle. During early‐stage proteasome assembly, individual Rpt proteins assemble into the hexameric ‘Rpt ring’ through binding to their cognate chaperones: Nas2, Hsm3, Nas6 and Rpn14. Here, we show that Rpt ring assembly employs a specific ubiquitination‐mediated control. An E3 ligase, Not4, selectively ubiquitinates Rpt5 during Rpt ring assembly. To access Rpt5, Not4 competes with Nas2 until the penultimate step, and then with Hsm3 at the final step of Rpt ring completion. Using the known Rpt‐chaperone co‐crystal structures, we show that Not4‐mediated ubiquitination sites in Rpt5 are obstructed by Nas2 and Hsm3. Thus, Not4 can distinguish a Rpt ring that matures without these chaperones, based on its accessibility to Rpt5. Rpt5 ubiquitination does not destabilize the ring, but hinders incorporation of incoming subunits—Rpn1 ubiquitin receptor and Ubp6 deubiquitinase, thereby blocking progression of proteasome assembly and ubiquitin regeneration from proteasome substrates. Our findings reveal an assembly checkpoint where Not4 monitors chaperone actions during hexameric ATPase ring assembly, thereby ensuring the accuracy of proteasome holoenzyme maturation.Support or Funding InformationBoettcher Webb‐Waring Biomedical Research Award and 1R01GM127688‐01A1 from NIH (S.P.)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.