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 cocrystal 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.

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

“…Rpn10 is then recruited to complete assembly of the RP base. A checkpoint involving ubiquitylation of Rpt5 by the RING E3 Not4 helps ensure that the chaperone-bound modules are integrated in the correct order (Fu et al, 2018).…”

“…Extensive ubiquitylation of the yeast and Arabidopsis particles directs non-functional complexes for autophagic degradation via specific receptors that bind to both the ubiquitin moieties on the impacted proteasome subunits and ATG8 (Marshall et al, 2015, 2016; Cohen-Kaplan et al, 2016; see below). As mentioned above, specific ubiquitylation of the proteasomal ubiquitin receptors Rpn10 and Rpn13 suppresses their ability to recognize substrates (Isasa et al, 2010; Lipinszki et al, 2012; Jacobson et al, 2014; Zuin et al, 2015), while ubiquitylation of Rpt5 appears to be an important checkpoint during Rpt ring assembly (Fu et al, 2018).…”

“…However, analysis of yeast Δ ecm29 mutants suggested this it is not involved in proteaphagy (Marshall and Vierstra, 2018b). Several E3s have been detected in association with 26S proteasomes that could instead provide this quality control (Xie and Varshavsky, 2000; Crosas et al, 2006; Panasenko and Collart, 2011), some of which ubiquitylate specific subunits (Besche et al, 2014; Fu et al, 2018), but their function(s) in relation to proteaphagy, if any, remain to be determined. Further work is certainly required to fully unravel the mysteries surrounding this last chapter in the life of a proteasome.…”

The Ccr4-Not complex regulates TORC1 signaling and mitochondrial metabolism by promoting vacuole V-ATPase activity

“…TORC1 inhibition enhances Mpk1 signaling to induce expression of proteasomal subunits and chaperones, which enhances protein degradation and maintains cellular homeostasis [ 53 ]. Ccr4-Not also interacts with the proteasome [ 54 ], and Not4 is essential for correct proteasome assembly, catalytic activity, and cellular proteostasis [ 10 , 11 , 55 ]. Because Ccr4-Not loss represses TORC1 and deregulates Mpk1 expression, we analyzed the effect it has on proteasome activity by monitoring 20S core particle activity.…”

CCR4-NOT complex nuclease Caf1 is a novel shuttle factor involved in the degradation of ubiquitin-modified proteins by 26S proteasome

“…In line with that the RING type ubiquitin ligase Not4 has been reported in the proteolysis of translationally arrested peptides and misfolded proteins [22,33,34], however, the Not4 catalytic RING domain was shown to be dispensable for such function. In addition, Not4 has so far been known to monoubiquitylate Rpt5 subunit of the 26S proteasome in vitro [35], furthermore, the substrate at which Not4 polyubiquitylates remains to be explored. Besides, Not4 the function of other CCR4-NOT complex proteins Caf130, Caf40, Caf1, and Not2 in the UPS pathway is totally unexplored.…”