Crystal structure of human proteasome assembly chaperone PAC4 involved in proteasome formation

The 26S proteasome is a large protein complex, responsible for degradation of ubiquinated proteins in eukaryotic cells. Eukaryotic proteasome formation is a highly ordered process that is assisted by several assembly chaperones. The assembly of its catalytic 20S core particle depends on at least five proteasome‐specific chaperones, i.e., proteasome‐assembling chaperons 1–4 (PAC1–4) and proteasome maturation protein (POMP). The orthologues of yeast assembly chaperones have been structurally characterized, whereas most mammalian assembly chaperones are not. In the present study, we determined a crystal structure of human PAC4 at 1.90‐Å resolution. Our crystallographic data identify a hydrophobic surface that is surrounded by charged residues. The hydrophobic surface is complementary to that of its binding partner, PAC3. The surface also exhibits charge complementarity with the proteasomal α4–5 subunits. This will provide insights into human proteasome‐assembling chaperones as potential anticancer drug targets.     

Characterization of the 20S proteasome of the lepidopteran,Spodoptera frugiperda

Multiple complexes of 20S proteasomes with accessory factors play an essential role in proteolysis in eukaryotic cells. In this report, several forms of 20S proteasomes from extracts of Spodoptera frugiperda (Sf9) cells were separated using electrophoresis in a native polyacrylamide gel and examined for proteolytic activity in the gel and by Western blotting. Distinct proteasome bands isolated from the gel were subjected to liquid chromatography-tandem mass spectrometry and identified as free core particles (CP) and complexes of CP with one or two dimers of assembly chaperones PAC1-PAC2 and activators PA28γ or PA200. In contrast to the activators PA28γ and PA200 that regulate the access of protein substrates to the internal proteolytic chamber of CP in an ATP-independent manner, the 19S regulatory particle (RP) in 26S proteasomes performs stepwise substrate unfolding and opens the chamber gate in an ATP-dependent manner. Electron microscopic analysis suggested that spontaneous dissociation of RP in isolated 26S proteasomes leaves CPs with different gate sizes related presumably to different stages in the gate opening. The primary structure of 20S proteasome subunits in Sf9 cells was determined by a search of databases and by sequencing. The protein sequences were confirmed by mass spectrometry and verified by 2D gel electrophoresis. The relative rates of sequence divergence in the evolution of 20S proteasome subunits, the assembly chaperones and activators were determined by using bioinformatics. The data confirmed the conservation of regular CP subunits and PA28γ, a more accelerated evolution of PAC2 and PA200, and especially high divergence rates of PAC1.     

PACemakers of proteasome core particle assembly

The 26S proteasome mediates ubiquitin-dependent proteolysis in eukaryotic cells. A number of studies including very recent ones have revealed that assembly of its 20S catalytic core particle is an ordered process that involves several conserved proteasome assembly chaperones (PACs). Two heterodimeric chaperones, PAC1-PAC2 and PAC3-PAC4, promote the assembly of rings composed of seven alpha subunits. Subsequently, beta subunits join to form half-proteasome precursor complexes containing all but one of the 14 subunits. These complexes lack the beta7 subunit but contain UMP1, another assembly chaperone, and in yeast, at least to some degree, the activator protein Blm10. Dimerization of two such complexes is triggered by incorporation of beta7, whose C-terminal extension reaches out into the other half to stabilize the newly formed 20S particle. The process is completed by the maturation of active sites and subsequent degradation of UMP1 and PAC1-PAC2.     

Stable incorporation of ATPase subunits into 19 S regulatory particle of human proteasome requires nucleotide binding and C-terminal tails

The 26 S proteasome is a large multi-subunit protein complex that degrades ubiquitinated proteins in eukaryotic cells. Proteasome assembly is a complex process that involves formation of six- and seven-membered ring structures from homologous subunits. Here we report that the assembly of hexameric Rpt ring of the 19 S regulatory particle (RP) requires nucleotide binding but not ATP hydrolysis. Disruption of nucleotide binding to an Rpt subunit by mutation in the Walker A motif inhibits the assembly of the Rpt ring without affecting heterodimer formation with its partner Rpt subunit. Coexpression of the base assembly chaperones S5b and PAAF1 with mutant Rpt1 and Rpt6, respectively, relieves assembly inhibition of mutant Rpts by facilitating their interaction with adjacent Rpt dimers. The mutation in the Walker B motif which impairs ATP hydrolysis does not affect Rpt ring formation. Incorporation of a Walker B mutant Rpt subunit abrogates the ATPase activity of the 19 S RP, suggesting that failure of the mutant Rpt to undergo the conformational transition from an ATP-bound to an ADP-bound state impairs conformational changes in the other five wild-type Rpts in the Rpt ring. In addition, we demonstrate that the C-terminal tails of Rpt subunits possessing core particle (CP)-binding affinities facilitate the cellular assembly of the 19 S RP, implying that the 20 S CP may function as a template for base assembly in human cells. Taken together, these results suggest that the ATP-bound conformational state of an Rpt subunit with the exposed C-terminal tail is competent for cellular proteasome assembly.     

Substrate access and processing by the 20S proteasome core particle

Intracellular proteolysis is an essential process. In eukaryotes, most proteins in the cytosol and nucleus are degraded by the ubiquitin (Ub)-proteasome pathway. A major component within this system is the 26S proteasome, a 2.5MDa molecular machine, built from more than 31 different subunits. This complex is formed by a cylinder-shaped multimeric complex referred to as the proteolytic 20S proteasome (core particle, CP) capped at each end by another multimeric component called the 19S complex (regulatory particle, RP) or PA700. Structure, assembly and enzymatic mechanism have been elucidated only for the CP, whereas the organization of the RP is less well understood. The CP is composed of 28 subunits, which are arranged as an alpha7beta7beta7alpha7-complex in four stacked rings. The interior of the free core particle, which harbors the active sites, is inaccessible for folded and unfolded substrates and represents a latent state. This inhibition is relieved upon binding of the RP to the CP by formation of the 26S proteasome holoenzyme. This review summarizes the current knowledge of the structural features of 20S proteasomes.     

Order of the Proteasomal ATPases and Eukaryotic Proteasome Assembly

The 26S proteasome is responsible for a large fraction of the regulated protein degradation in eukaryotic cells. The enzyme complex is composed of a 20S proteolytic core particle (CP) capped on one or both ends with a 19S regulatory particle (RP). The RP recognizes and unfolds substrates and translocates them into the CP. The RP can be further divided into lid and base subcomplexes. The base contains a ring of six AAA+ ATPases (Rpts) that directly abuts the CP and is responsible for unfolding substrates and driving them into the CP for proteolysis. Although 120 arrangements of the six different ATPases within the ring are possible in principle, they array themselves in one specific order. The high sequence and structural similarity between the Rpt subunits presents special challenges for their ordered association and incorporation into the assembling proteasome. In this review, we discuss recent advances in our understanding of proteasomal RP base biogenesis, with emphasis on potential specificity determinants in ring arrangement, and the implications of the ATPase ring arrangement for proteasome assembly.     

C termini of proteasomal ATPases play nonequivalent roles in cellular assembly of mammalian 26 S proteasome

The 26 S proteasome comprises two multisubunit subcomplexes as follows: 20 S proteasome and PA700/19 S regulatory particle. The cellular mechanisms by which these subcomplexes assemble into 26 S proteasome and the molecular determinants that govern the assembly process are poorly defined. Here, we demonstrate the nonequivalent roles of the C termini of six AAA subunits (Rpt1-Rpt6) of PA700 in 26 S proteasome assembly in mammalian cells. The C-terminal HbYX motif (where Hb is a hydrophobic residue, Y is tyrosine, and X is any amino acid) of each of two subunits, Rpt3 and Rpt5, but not that of a third subunit Rpt2, was essential for assembly of 26 S proteasome. The C termini of none of the three non-HbYX motif Rpt subunits were essential for cellular 26 S proteasome assembly, although deletion of the last three residues of Rpt6 destabilized the 20 S-PA700 interaction. Rpt subunits defective for assembly into 26 S proteasome due to C-terminal truncations were incorporated into intact PA700. Moreover, intact PA700 accumulated as an isolated subcomplex when cellular 20 S proteasome content was reduced by RNAi. These results indicate that 20 S proteasome is not an obligatory template for assembly of PA700. Collectively, these results identify specific structural elements of two Rpt subunits required for 26 S proteasome assembly, demonstrate that PA700 can be assembled independently of the 20 S proteasome, and suggest that intact PA700 is a direct intermediate in the cellular pathway of 26 S proteasome assembly.     

An atomic model AAA-ATPase/20S core particle sub-complex of the 26S proteasome

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.     

Base-CP proteasome can serve as a platform for stepwise lid formation

26S proteasome, a major regulatory protease in eukaryotes, consists of a 20S proteolytic core particle (CP) capped by a 19S regulatory particle (RP). The 19S RP is divisible into base and lid sub-complexes. Even within the lid, subunits have been demarcated into two modules: module 1 (Rpn5, Rpn6, Rpn8, Rpn9 and Rpn11), which interacts with both CP and base sub-complexes and module 2 (Rpn3, Rpn7, Rpn12 and Rpn15) that is attached mainly to module 1. We now show that suppression of RPN11 expression halted lid assembly yet enabled the base and 20S CP to pre-assemble and form a base-CP. A key role for Regulatory particle non-ATPase 11 (Rpn11) in bridging lid module 1 and module 2 subunits together is inferred from observing defective proteasomes in rpn11–m1, a mutant expressing a truncated form of Rpn11 and displaying mitochondrial phenotypes. An incomplete lid made up of five module 1 subunits attached to base-CP was identified in proteasomes isolated from this mutant. Re-introducing the C-terminal portion of Rpn11 enabled recruitment of missing module 2 subunits. In vitro, module 1 was reconstituted stepwise, initiated by Rpn11–Rpn8 heterodimerization. Upon recruitment of Rpn6, the module 1 intermediate was competent to lock into base-CP and reconstitute an incomplete 26S proteasome. Thus, base-CP can serve as a platform for gradual incorporation of lid, along a proteasome assembly pathway. Identification of proteasome intermediates and reconstitution of minimal functional units should clarify aspects of the inner workings of this machine and how multiple catalytic processes are synchronized within the 26S proteasome holoenzymes.     

Affinity Purification of the Arabidopsis 26 S Proteasome Reveals a Diverse Array of Plant Proteolytic Complexes

Selective proteolysis in plants is largely mediated by the ubiquitin (Ub)/proteasome system in which substrates, marked by the covalent attachment of Ub, are degraded by the 26 S proteasome. The 26 S proteasome is composed of two subparticles, the 20 S core protease (CP) that compartmentalizes the protease active sites and the 19 S regulatory particle that recognizes and translocates appropriate substrates into the CP lumen for breakdown. Here, we describe an affinity method to rapidly purify epitope-tagged 26 S proteasomes intact from Arabidopsis thaliana. In-depth mass spectrometric analyses of preparations generated from young seedlings confirmed that the 2.5-MDa CP-regulatory particle complex is actually a heterogeneous set of particles assembled with paralogous pairs for most subunits. A number of these subunits are modified post-translationally by proteolytic processing, acetylation, and/or ubiquitylation. Several proteasome-associated proteins were also identified that likely assist in complex assembly and regulation. In addition, we detected a particle consisting of the CP capped by the single subunit PA200 activator that may be involved in Ub-independent protein breakdown. Taken together, it appears that a diverse and highly dynamic population of proteasomes is assembled in plants, which may expand the target specificity and functions of intracellular proteolysis.     

The C terminus of Rpt3, an ATPase subunit of PA700 (19 S) regulatory complex, is essential for 26 S proteasome assembly but not for activation

PA700, the 19 S regulatory subcomplex of the 26 S proteasome, contains a heterohexameric ring of AAA subunits (Rpt1 to -6) that forms the binding interface with a heteroheptameric ring of α subunits (α1 to -7) of the 20 S proteasome. Binding of these subcomplexes is mediated by interactions of C termini of certain Rpt subunits with cognate binding sites on the 20 S proteasome. Binding of two Rpt subunits (Rpt2 and Rpt5) depends on their last three residues, which share an HbYX motif (where Hb is a hydrophobic amino acid) and open substrate access gates in the center of the α ring. The relative roles of other Rpt subunits for proteasome binding and activation remain poorly understood. Here we demonstrate that the C-terminal HbYX motif of Rpt3 binds to the 20 S proteasome but does not promote proteasome gating. Binding requires the last three residues and occurs at a dedicated site on the proteasome. A C-terminal peptide of Rpt3 blocked ATP-dependent in vitro assembly of 26 S proteasome from PA700 and 20 S proteasome. In HEK293 cells, wild-type Rpt3, but not Rpt3 lacking the HbYX motif was incorporated into 26 S proteasome. These results indicate that the C terminus of Rpt3 was required for cellular assembly of this subunit into 26 S proteasome. Mutant Rpt3 was assembled into intact PA700. This result indicates that intact PA700 can be assembled independently of association with 20 S proteasome and thus may be a direct precursor for 26 S proteasome assembly under normal conditions. These results provide new insights to the non-equivalent roles of Rpt subunits in 26 S proteasome function and identify specific roles for Rpt3.     

Fub1p, a novel protein isolated by boundary screening, binds the proteasome complex

Silenced chromatin domains are restricted to specific regions. Eukaryotic chromosomes are organized into discrete domains delimited by domain boundaries. From approximately 6,000 genes in Saccharomyces cerevisiae, we previously isolated 55 boundary genes. In this study, we focus on the molecular function of one of boundary genes, YCR076C/FUB1 (function of boundary), whose function has not been clearly defined in vivo. Biochemical analysis of Fub1p revealed that it interacted with multiple subunits of the 20S proteasome core particle (20S CP). To further clarify the functional link between Fub1p and proteasome, several proteasome mutants were analyzed. Although only 20S CP subunits were isolated as Fub1p interactors, a genetic interaction was also observed for component of 19S regulatory particle (19S RP) suggesting involvement of Fub1p with the whole proteasome. We also analyzed the mechanism of boundary establishment by using proteasome composition factor-deficient strains. Deletion of pre9 and ump1, whose products have effects on the 20S CP, resulted in a decrease in boundary function. Domain analyses of Fub1p identified a minimum functional domain in the C terminus that was essential for boundary establishment and showed a limited sequence homology to the human PSMF1, which is known to inhibit proteasome activity. Finally, boundary assay showed that human PSMF1 also exhibited boundary establishment activity in yeast. Our results defined the functional correlation between Fub1p and PSMF1.     

PAC1 Gene Knockout Reveals an Essential Role of Chaperone-Mediated 20S Proteasome Biogenesis and Latent 20S Proteasomes in Cellular Homeostasis

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.The 26S proteasome is a eukaryotic ATP-dependent protease responsible for the degradation of proteins tagged with polyubiquitin chains (21). The ubiquitin-dependent proteolysis by the proteasome plays a pivotal role in various cellular processes by catalyzing the selective degradation of short-lived regulatory proteins as well as damaged proteins. Thus, the proteasome is essential for the viability of all eukaryotic cells.The 26S proteasome is a large protein complex consisting of two portions; one is the catalytic 20S proteasome of approximately 700 kDa (also called the 20S core particle), and the other is the 19S regulatory particle (RP; also called PA700) of approximately 900 kDa, both of which are composed of a set of multiple distinct subunits (70). The 20S proteasome is a cylindrically shaped stack of four heptameric rings, where the outer and inner rings each are composed of seven homologous α subunits (α1 to α7) and seven homologous β subunits (β1 to β7), respectively (5). The proteolytic active sites reside within the central chamber enclosed by the two inner β-rings, while a small channel formed by the outer α-ring, which is primarily closed, restricts the access of native proteins to the catalytic chamber. Thus, the 20S proteasome is a latent enzyme. Appending 19S RP, which consists of 19 different subunits, to the α-ring enables the 20S proteasome to degrade native proteins; 19S RP accepts ubiquitin chains of substrate proteins, removes ubiquitin chains while unfolding the substrates, and feeds the substrates into the interior proteolytic chamber of the 20S proteasome through the α-ring that is opened when the C-terminal tails of the ATPase subunits of 19S RP are inserted into the intersubunit spaces of the α-ring (24, 62, 74). However, it also has been reported that some denatured or unstructured proteins can be degraded directly by the 20S proteasome even in the absence of 19S RP and ubiquitination (37, 39).Much attention has been focused on how such a highly elaborate structure is achieved. Recent studies have identified various proteasome-dedicated chaperones that assist in the assembly of the proteasome in eukaryotic cells (23, 40, 56, 57, 65, 66). In yeast, while most of the proteasome subunits are essential for viability, the deletion of any of these chaperones does not cause lethality. In fact, many, if not all, of the deletions exhibit subtle phenotypes. In mammalian cells, although the knockdown of the assembly chaperones reduced proteasome assembly and thus proteasome activity, leading to slow cell growth, the degree of reduction was much lower than that which occurred following the knockdown of the proteasome subunit itself (33, 35, 40). These results indicate that the assembly chaperones play an auxiliary role in proteasome biogenesis.Proteasome assembly chaperone 1 (PAC1) is one of the assembly chaperones originally identified in mammalian cells (34). PAC1 plays a role in α-ring formation that occurs during the initial assembly of the 20S proteasome; it also prevents the aberrant dimerization of the α-ring. As is the case for most assembly chaperones, the knockdown of PAC1 in mammalian cells decreases proteasome activity but to a lesser extent than that in, for example, β2 knockdown (34, 35). Therefore, both PAC1-dependent and -independent assembly pathways exist in cells, but the importance of the PAC1-dependent pathway remains elusive. To further elucidate the biological significance of PAC1 and PAC1-dependent proteasome biogenesis, we generated conditional mouse mutants carrying an inactivating mutation in Psmg1, the gene coding for PAC1 protein, in the whole body, the nervous system, and in the liver. Our results demonstrate that PAC1 is essential for the development of a mouse, and that it plays important roles in maintaining cellular integrity in quiescent tissue. Our study revealed for the first time the importance of chaperone-mediated proteasome biogenesis in a whole-body mammalian system and may provide valuable knowledge in medical drug development targeting proteasomes.     

The substrate translocation channel of the proteasome

The core particle (CP) of the yeast proteasome is composed of four heptameric rings of subunits arranged in a hollow, barrel-like structure. We have found that the CP is autoinhibited by the N-terminal tails of the outer (alpha) ring subunits. Crystallographic analysis showed that deletion of the tail of the alpha3 subunit opens a channel into the proteolytically active interior chamber of the CP, thus derepressing peptide hydrolysis. In the latent state of the particle, the tails prevent substrate entry by imposing topological closure on the CP. Inhibition by the alpha subunit tails is relieved upon binding of the regulatory particle to the CP to form the proteasome holoenzyme. Opening of the CP channel by assembly of the holoenzyme is regulated by the ATPase domain of Rpt2, one of 17 subunits in the RP. Thus, open-channel mutations in CP subunits suppress the closed-channel phenotype of an rpt2 mutant. These results identify a specific mechanism for allosteric regulation of the CP by the RP.     

Proteasome disassembly and downregulation is correlated with viability during stationary phase

During prolonged starvation, yeast cells enter a stationary phase (SP) during which the synthesis of many proteins is dramatically decreased. We show that a parallel decrease in proteasome-dependent proteolysis also occurs. The reduction in proteolysis is correlated with disassembly of 26S proteasome holoenzymes into their 20S core particle (CP) and 19S regulatory particle (RP) components. Proteasomes are reassembled, and proteolysis resumes prior to cell cycle reentry. Free 20S CPs are found in an autoinhibited state in which the N-terminal tails from neighboring alpha subunits are anchored by an intricate lattice of interactions blocking the channel that leads into the 20S CPs. By deleting channel gating residues of CP alpha subunits, we generated an "open channel" proteasome that exhibits faster rates of protein degradation both in vivo and in vitro, indicating that gating contributes to regulation of proteasome activity. This open channel mutant is delayed in outgrowth from SP and cannot survive following prolonged starvation. In summary, we have found that the ubiquitin-proteasome pathway can be subjected to global downregulation, that the proteasome is a target of this regulation, and that proteasome downregulation is linked to survival of SP cells. Maintaining high viability during SP is essential for evolutionary fitness, which may explain the extreme conservation of channel gating residues in eukaryotic proteasomes.     

Eukaryotic 20S proteasome catalytic subunit propeptides prevent active site inactivation by N-terminal acetylation and promote particle assembly.

Proteins targeted for degradation by the ubiquitin-proteasome system are degraded by the 26S proteasome. The core of this large protease is the 20S proteasome, a barrel-shaped structure made of a stack of four heptameric rings. Of the 14 different subunits that make up the yeast 20S proteasome, three have proteolytic active sites: Doa3/beta5, Pup1/beta2 and Pre3/beta1. Each of these subunits is synthesized with an N-terminal propeptide that is autocatalytically cleaved during particle assembly. We show here that the propeptides have both common and distinct functions in proteasome biogenesis. Unlike the Doa3 propeptide, which is crucial for proteasome assembly, the Pre3 and Pup1 propeptides are dispensable for cell viability and proteasome formation. However, mutants lacking these propeptide-encoding elements are defective for specific peptidase activities, are more sensitive to environmental stresses and have subtle defects in proteasome assembly. Unexpectedly, a critical function of the propeptide is the protection of the N-terminal catalytic threonine residue against Nalpha-acetylation. For all three propeptide-deleted subunits, activity of the affected catalytic center is fully restored when the Nat1-Ard1 Nalpha-acetyltransferase is mutated. In addition to delineating a novel function for proteasome propeptides, these data provide the first biochemical evidence for the postulated participation of the alpha-amino group in the proteasome catalytic mechanism.     

Differential roles of the COOH termini of AAA subunits of PA700 (19 S regulator) in asymmetric assembly and activation of the 26 S proteasome

The 26 S proteasome is an energy-dependent protease that degrades proteins modified with polyubiquitin chains. It is assembled from two multi-protein subcomplexes: a protease (20 S proteasome) and an ATPase regulatory complex (PA700 or 19 S regulatory particle) that contains six different AAA family subunits (Rpt1 to -6). Here we show that binding of PA700 to the 20 S proteasome is mediated by the COOH termini of two (Rpt2 and Rpt5) of the six Rpt subunits that constitute the interaction surface between the subcomplexes. COOH-terminal peptides of either Rpt2 or Rpt5 bind to the 20 S proteasome and activate hydrolysis of short peptide substrates. Simultaneous binding of both COOH-terminal peptides had additive effects on peptide substrate hydrolysis, suggesting that they bind to distinct sites on the proteasome. In contrast, only the Rpt5 peptide activated hydrolysis of protein substrates. Nevertheless, the COOH-terminal peptide of Rpt2 greatly enhanced this effect, suggesting that proteasome activation is a multistate process. Rpt2 and Rpt5 COOH-terminal peptides cross-linked to different but specific subunits of the 20 S proteasome. These results reveal critical roles of COOH termini of Rpt subunits of PA700 in the assembly and activation of eukaryotic 26 S proteasome. Moreover, they support a model in which Rpt subunits bind to dedicated sites on the proteasome and play specific, nonequivalent roles in the asymmetric assembly and activation of the 26 S proteasome.     

Putting it all together: intrinsic and extrinsic mechanisms governing proteasome biogenesis

Background

The 26S proteasome is at the heart of the ubiquitin-proteasome system, which is the key cellular pathway for the regulated degradation of proteins and enforcement of protein quality control. The 26S proteasome is an unusually large and complicated protease comprising a 28-subunit core particle (CP) capped by one or two 19-subunit regulatory particles (RP). Multiple activities within the RP process incoming ubiquitinated substrates for eventual degradation by the barrel-shaped CP. The large size and elaborate architecture of the proteasome have made it an exceptional model for understanding mechanistic themes in macromolecular assembly.

Objective

In the present work, we highlight the most recent mechanistic insights into proteasome assembly, with particular emphasis on intrinsic and extrinsic factors regulating proteasome biogenesis. We also describe new and exciting questions arising about how proteasome assembly is regulated and deregulated in normal and diseased cells.

Methods

A comprehensive literature search using the PubMed search engine was performed, and key findings yielding mechanistic insight into proteasome assembly were included in this review.

Results

Key recent studies have revealed that proteasome biogenesis is dependent upon intrinsic features of the subunits themselves as well as extrinsic factors, many of which function as dedicated chaperones.

Conclusion

Cells rely on a diverse set of mechanistic strategies to ensure the rapid, efficient, and faithful assembly of proteasomes from their cognate subunits. Importantly, physiological as well as pathological changes to proteasome assembly are emerging as exciting paradigms to alter protein degradation in vivo.

     

The catalytic activity of Ubp6 enhances maturation of the proteasomal regulatory particle

The 26S proteasome is a 2.5 MDa macromolecular machine responsible for targeted protein degradation. Recently, four chaperones were identified that promote the assembly of the 19S regulatory particle (RP). Here, we probe the dynamic architecture of the proteasome by applying quantitative proteomics and mass spectrometry (MS) of intact complexes to provide a detailed characterization of how Ubp6 assists this assembly process. Our MS data demonstrate stoichiometric binding of chaperones and Ubp6 to the basal part of the RP. Genetic interactions of Ubp6 with Hsm3, but not with the other chaperones, indicate a functional overlay with Hsm3. Our biochemical data identified Ubp6 as an additional member of the Hsm3 module. Deletions of ubp6 with hsm3 perturb 26S proteasome assembly, which we attribute to an accumulation of ubiquitylated substrates on these assembly precursors. We therefore propose that Ubp6 facilitates proteasomal assembly by clearing ubiquitylated substrates from assembly precursors by its deubiquitylating activity.     

Dissecting beta-ring assembly pathway of the mammalian 20S proteasome

The 20S proteasome is the catalytic core of the 26S proteasome. It comprises four stacked rings of seven subunits each, alpha(1-7)beta(1-7)beta(1-7)alpha(1-7). Recent studies indicated that proteasome-specific chaperones and beta-subunit appendages assist in the formation of alpha-rings and dimerization of half-proteasomes, but the process involved in the assembly of beta-rings is poorly understood. Here, we clarify the mechanism of beta-ring formation on alpha-rings by characterizing assembly intermediates accumulated in cells depleted of each beta-subunit. Starting from beta2, incorporation of beta-subunits occurs in an orderly manner dependent on the propeptides of beta2 and beta5, and the C-terminal tail of beta2. Unexpectedly, hUmp1, a chaperone functioning at the final assembly step, is incorporated as early as beta2 and is required for the structural integrity of early assembly intermediates. We propose a model in which beta-ring formation is assisted by both intramolecular and extrinsic chaperones, whose roles are partially different between yeast and mammals.