An Allosteric Interaction Network Promotes Conformation State-Dependent Eviction of the Nas6 Assembly Chaperone from Nascent 26S Proteasomes.
ABSTRACT: The 26S proteasome is the central ATP-dependent protease in eukaryotes and is essential for organismal health. Proteasome assembly is mediated by several dedicated, evolutionarily conserved chaperone proteins. These chaperones associate transiently with assembly intermediates but are absent from mature proteasomes. Chaperone eviction upon completion of proteasome assembly is necessary for normal proteasome function, but how they are released remains unresolved. Here, we demonstrate that the Nas6 assembly chaperone, homolog of the human oncogene gankyrin, is evicted from nascent proteasomes during completion of assembly via a conformation-specific allosteric interaction of the Rpn5 subunit with the proteasomal ATPase ring. Subsequent ATP binding by the ATPase subunit Rpt3 promotes conformational remodeling of the ATPase ring that evicts Nas6 from the nascent proteasome. Our study demonstrates how assembly-coupled allosteric signals promote chaperone eviction and provides a framework for understanding the eviction of other chaperones from this biomedically important molecular machine.
Project description:The proteasomal ATPase ring, comprising Rpt1-Rpt6, associates with the heptameric ?-ring of the proteasome core particle (CP) in the mature proteasome, with the Rpt carboxy-terminal tails inserting into pockets of the ?-ring. Rpt ring assembly is mediated by four chaperones, each binding a distinct Rpt subunit. Here we report that the base subassembly of the Saccharomyces cerevisiae proteasome, which includes the Rpt ring, forms a high-affinity complex with the CP. This complex is subject to active dissociation by the chaperones Hsm3, Nas6 and Rpn14. Chaperone-mediated dissociation was abrogated by a non-hydrolysable ATP analogue, indicating that chaperone action is coupled to nucleotide hydrolysis by the Rpt ring. Unexpectedly, synthetic Rpt tail peptides bound ?-pockets with poor specificity, except for Rpt6, which uniquely bound the ?2/?3-pocket. Although the Rpt6 tail is not visualized within an ?-pocket in mature proteasomes, it inserts into the ?2/?3-pocket in the base-CP complex and is important for complex formation. Thus, the Rpt-CP interface is reconfigured when the lid complex joins the nascent proteasome to form the mature holoenzyme.
Project description: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.
Project description:The proteasome is a protease that controls diverse processes in eukaryotic cells. Its regulatory particle (RP) initiates the degradation of ubiquitin-protein conjugates by unfolding the substrate and translocating it into the proteasome core particle (CP) to be degraded. The RP has 19 subunits, and their pathway of assembly is not understood. Here we show that in the yeast Saccharomyces cerevisiae three proteins are found associated with RP but not with the RP-CP holoenzyme: Nas6, Rpn14 and Hsm3. Mutations in the corresponding genes confer proteasome loss-of-function phenotypes, despite their virtual absence from the holoenzyme. These effects result from deficient RP assembly. Thus, Nas6, Rpn14 and Hsm3 are RP chaperones. The RP contains six ATPases-the Rpt proteins-and each RP chaperone binds to the carboxy-terminal domain of a specific Rpt. We show in an accompanying study that RP assembly is templated through the Rpt C termini, apparently by their insertion into binding pockets in the CP. Thus, RP chaperones may regulate proteasome assembly by directly restricting the accessibility of Rpt C termini to the CP. In addition, competition between the RP chaperones and the CP for Rpt engagement may explain the release of RP chaperones as proteasomes mature.
Project description:The 26S proteasome is an essential protease that selectively eliminates dysfunctional and short-lived regulatory proteins in eukaryotes. To define the composition of this proteolytic machine in plants, we tagged either the core protease (CP) or the regulatory particle (RP) sub-complexes in Arabidopsis to enable rapid affinity purification followed by mass spectrometric analysis. Studies on proteasomes enriched from whole seedlings, with or without ATP needed to maintain the holo-proteasome complex, identified all known proteasome subunits but failed to detect isoform preferences, suggesting that Arabidopsis does not construct distinct proteasome sub-types. We also detected a suite of proteasome-interacting proteins, including likely orthologs of the yeast and mammalian chaperones Pba1, Pba2, Pba3, and Pba4 that assist in CP assembly; Ump1 that helps connect CP half-barrels; Nas2, Nas6, and Hsm3 that assist in RP assembly; and Ecm29 that promotes CP-RP association. Proteasomes from seedlings exposed to the proteasome inhibitor MG132 accumulated assembly intermediates, reflecting partially built proteasome sub-complexes associated with assembly chaperones, and the CP capped with the PA200/Blm10 regulator. Genetic analyses of Arabidopsis UMP1 revealed that, unlike in yeast, this chaperone is essential, with mutants lacking the major UMP1a and UMP1b isoforms displaying a strong gametophytic defect. Single ump1 mutants were hypersensitive to conditions that induce proteotoxic, salt and osmotic stress, and also accumulated several proteasome assembly intermediates, consistent with its importance for CP construction. Insights into the chaperones reported here should enable study of the assembly events that generate the 26S holo-proteasome in Arabidopsis from the collection of 64 or more subunits.
Project description: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.
Project description: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.
Project description:The proteasome has a paramount role in eukaryotic cell regulation. It consists of a proteolytic core particle (CP) bound to one or two regulatory particles (RPs). Each RP is believed to include six different AAA+ ATPases in a heterohexameric ring that binds the CP while unfolding and translocating substrates into the core. No atomic-resolution RP structures are available. Guided by crystal structures of related homohexameric prokaryotic ATPases, we use disulfide engineering to show that the eukaryotic ATPases form a ring with the arrangement Rpt1-Rpt2-Rpt6-Rpt3-Rpt4-Rpt5 in fully assembled proteasomes. The arrangement is consistent with known assembly intermediates. This quaternary organization clarifies the functional overlap of specific RP assembly chaperones and led us to identify a potential RP assembly intermediate that includes four ATPases (Rpt6-Rpt3-Rpt4-Rpt5) and their cognate chaperones (Rpn14, Nas6, and Nas2). Finally, the ATPase ring structure casts light on alternative RP structural models and the mechanism of RP action.
Project description:The ubiquitin-proteasome pathway is a major proteolytic system in eukaryotic cells and regulates various cellular processes. The 26 S proteasome, the central enzyme of this pathway, consists of a proteolytic core particle and two 19 S regulatory particles (RPs) composed of ATPase (Rpt) and non-ATPase (Rpn) subunits. Growing evidence indicates that proteasome assembly is assisted by a variety of chaperones. In particular, it has been reported recently that Nas2, Nas6, Rpn14, and Hsm3 bind specific Rpt subunits, thereby contributing to the formation of 19 S RP. Rpn14 transiently binds to the C-terminal domain of the Rpt6 subunit (Rpt6-C) during maturation of the ATPase ring of 19 S RP. In this study, we determined the crystal structure of yeast Rpn14 at 2.0 A resolution, which revealed that this chaperone consists of a unique N-terminal domain with unknown function and a C-terminal domain assuming a canonical seven-bladed beta-propeller fold. The Rpt6-binding site on Rpn14 was predicted based on structural comparison with the complex formed between Nas6 and Rpt3-C. The top face of Rpn14 exhibits a highly acidic surface area, whereas the putative interacting surface of Rpt6-C is basic. By inspection of structural data along with genetic and biochemical data, we determined the specific residues of Rpn14 and Rpt6 for complementary charge interactions that are required for 19 S RP assembly.
Project description:The 26S proteasome is the most downstream element of the ubiquitin-proteasome pathway of protein degradation. It is composed of the 20S core particle (CP) and the 19S regulatory particle (RP). The RP consists of 6 AAA-ATPases and at least 13 non-ATPase subunits. Based on a cryo-EM map of the 26S proteasome, structures of homologs, and physical protein-protein interactions we derive an atomic model of the AAA-ATPase-CP sub-complex. The ATPase order in our model (Rpt1/Rpt2/Rpt6/Rpt3/Rpt4/Rpt5) is in excellent agreement with the recently identified base-precursor complexes formed during the assembly of the RP. Furthermore, the atomic CP-AAA-ATPase model suggests that the assembly chaperone Nas6 facilitates CP-RP association by enhancing the shape complementarity between Rpt3 and its binding CP alpha subunits partners.
Project description:The 26S proteasome, a central enzyme for ubiquitin-dependent proteolysis, is a highly complex structure comprising 33 distinct subunits. Recent studies have revealed multiple dedicated chaperones involved in proteasome assembly both in yeast and in mammals. However, none of these chaperones is essential for yeast viability. PAC1 is a mammalian proteasome assembly chaperone that plays a role in the initial assembly of the 20S proteasome, the catalytic core of the 26S proteasome, but does not cause a complete loss of the 20S proteasome when knocked down. Thus, both chaperone-dependent and -independent assembly pathways exist in cells, but the contribution of the chaperone-dependent pathway remains unclear. To elucidate its biological significance in mammals, we generated PAC1 conditional knockout mice. PAC1-null mice exhibited early embryonic lethality, demonstrating that PAC1 is essential for mammalian development, especially for explosive cell proliferation. In quiescent adult hepatocytes, PAC1 is responsible for producing the majority of the 20S proteasome. PAC1-deficient hepatocytes contained normal amounts of the 26S proteasome, but they completely lost the free latent 20S proteasome. They also accumulated ubiquitinated proteins and exhibited premature senescence. Our results demonstrate the importance of the PAC1-dependent assembly pathway and of the latent 20S proteasomes for maintaining cellular integrity.