The proteasome is assembled via the nine-subunit lid, nine-subunit base, and 28-subunit core particle (CP). Previous work has shown that the chaperones Rpn14, Nas6, Hsm3, and Nas2 each bind a specific ATPase subunit of the base and antagonize base-CP interaction. Here, we show that the Nas6 chaperone also obstructs base-lid association. Nas6 alternates between these two inhibitory modes according to the nucleotide state of the base. When ATP cannot be hydrolyzed, Nas6 interferes with base-lid, but not base-CP, association. In contrast, under conditions of ATP hydrolysis, Nas6 obstructs base-CP, but not base-lid, association. Modeling of Nas6 into cryoelectron microscopy structures of the proteasome suggests that Nas6 controls both base-lid affinity and base-CP affinity through steric hindrance; Nas6 clashes with the lid in the ATP-hydrolysis-blocked proteasome, but clashes instead with the CP in the ATP-hydrolysis-competent proteasome. Thus, Nas6 provides a dual mechanism to control assembly at both major interfaces of the proteasome.proteasome | chaperone | AAA + ATPase | assembly | Nas6

The importance of histone variant H2A.Z in transcription regulation has been well established, yet its mechanism-of-action remains enigmatic. Conflicting evidence exists in support of both an activating and a repressive role of H2A.Z in transcription. Here we report cryo-electron microscopy (cryo-EM) structures of nucleosomes and chromatin fibers containing H2A.Z and those containing canonical H2A. The structures show that H2A.Z incorporation results in substantial structural changes in both nucleosome and chromatin fiber. While H2A.Z increases the mobility of DNA terminus in nucleosomes, it simultaneously enables nucleosome arrays to form a more regular and condensed chromatin fiber. We also demonstrated that H2A.Z’s ability to enhance nucleosomal DNA mobility is largely attributed to its characteristic shorter C-terminus. Our study provides the structural basis for H2A.Z-mediated chromatin regulation, showing that the increase flexibility of the DNA termini in H2A.Z nucleosomes is central to its dual-functions in chromatin regulation and in transcription.

In the proteasome, the proteolytic 20S core particle (CP) associates with the 19S regulatory particle (RP) to degrade polyubiquitinated proteins. Six ATPases (Rpt1-Rpt6) of the RP form a hexameric Rpt ring and interact with the heptameric α ring (α1–α7) of the CP via the Rpt C-terminal tails individually binding to the α subunits. Importantly, the Rpt6 tail has been suggested to be crucial for RP assembly. Here, we show that the interaction of the CP and Rpt6 tail promotes a CP-Rpt3 tail interaction, and that they jointly mediate proteasome activation via opening the CP gate for substrate entry. The Rpt6 tail forms a novel relationship with the Nas6 chaperone, which binds to Rpt3 and regulates the CP-Rpt3 tail interaction, critically influencing cell growth and turnover of polyubiquitinated proteins. CP-Rpt6 tail binding promotes the release of Nas6 from the proteasome. Based on disulfide crosslinking that detects cognate α3-Rpt6 tail and α2-Rpt3 tail interactions in the proteasome, decreased α3-Rpt6 tail interaction facilitates robust α2-Rpt3 tail interaction that is also strongly ATP-dependent. Together, our data support the reported role of Rpt6 during proteasome assembly, and suggest that its function switches from anchoring for RP assembly into promoting Rpt3-dependent activation of the mature proteasome.

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.