Geng Tian

Structural Insights into the Regulatory Particle of the Proteasome from Methanocaldococcus jannaschii

Eukaryotic proteasome consists of a core particle (CP), which degrades unfolded protein, and a regulatory particle (RP), which is responsible for recognition, ATP-dependent unfolding, and translocation of polyubiquitinated substrate protein. In the archaea Methanocaldococcus jannaschii, the RP is a homohexameric complex of proteasome-activating nucleotidase (PAN). Here, we report the crystal structures of essential elements of the archaeal proteasome: the CP, the ATPase domain of PAN, and a distal subcomplex that is likely the first to encounter substrate. The distal subcomplex contains a coiled-coil segment and an OB-fold domain, both of which appear to be conserved in the eukaryotic proteasome. The OB domains of PAN form a hexameric ring with a 13 A pore, which likely constitutes the outermost constriction of the substrate translocation channel. These studies reveal structural codes and architecture of the complete proteasome, identify potential substrate-binding sites, and uncover unexpected asymmetry in the RP of archaea and eukaryotes.

Chaperone-mediated pathway of proteasome regulatory particle assembly.

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.

Mechanism of substrate unfolding and translocation by the regulatory particle of the proteasome from Methanocaldococcus jannaschii.

In the archaebacterium Methanocaldococcus jannaschii (M. jannaschii), the proteasomal regulatory particle (RP), a homohexameric complex of proteasome-activating nucleotidase (PAN), is responsible for target protein recognition, followed by unfolding and translocation of the bound protein into the core particle (CP) for degradation. Guided by structure-based mutagenesis, we identify amino acids and structural motifs that are essential for PAN function. Key residues line the axial channel of PAN, defining the apparent pathway of substrate translocation. Subcomplex II of PAN, comprising the ATPase domain, associates with the CP and drives ATP-dependent unfolding of the substrate protein, whereas the distal subcomplex I forms the entry port of the substrate translocation channel. A linker segment between subcomplexes I and II is essential for PAN function, implying functional and perhaps mechanical coupling between these domains. Sequence conservation suggests that the principles of PAN function are likely to apply to the proteasomal RP of eukaryotes.

Rpn1 provides adjacent receptor sites for substrate binding and deubiquitination by the proteasome.

The yin and yang of proteasomal regulation The ubiquitin-proteasome pathway regulates myriad proteins through their selective proteolysis. The small protein ubiquitin is attached, typically in many copies, to the target protein, which is then recognized and broken down by the proteasome. Shi et al. found a repeat structure in the proteasome for recognizing ubiquitin as well as ubiquitin-like (UBL) proteins. Tandem binding sites allow the proteasome to dock multiple proteins. One of the bound UBL proteins is an enzyme that cleaves ubiquitin-protein conjugates, which antagonizes degradation. Thus, the repetition of related binding sites with distinct specificity achieves a balance of positive and negative regulation of the proteasome. Science, this issue p. 10.1126/science.aad9421 Tandem ligand-binding sites in the proteasome subunit Rpn1 modulate proteasome activity both positively and negatively. INTRODUCTION The ubiquitin-proteasome system comprises hundreds of distinct pathways of degradation, which converge at the step of ubiquitin recognition by the proteasome. Five proteasomal ubiquitin receptors have been identified, two that are intrinsic to the proteasome (Rpn10 and Rpn13) and three reversibly associated proteasomal ubiquitin receptors (Rad23, Dsk2, and Ddi1). RATIONALE We found that the five known proteasomal ubiquitin receptors of yeast are collectively nonessential for ubiquitin recognition by the proteasome. We therefore screened for additional ubiquitin receptors in the proteasome and identified subunit Rpn1 as a candidate. We used nuclear magnetic resonance (NMR) spectroscopy to characterize the structure of the binding site within Rpn1, which we term the T1 site. Mutational analysis of this site showed its functional importance within the context of intact proteasomes. T1 binds both ubiquitin and ubiquitin-like (UBL) proteins, in particular the substrate-delivering shuttle factor Rad23. A second site within the Rpn1 toroid, T2, recognizes the UBL domain of deubiquitinating enzyme Ubp6, as determined by hydrogen-deuterium exchange mass spectrometry analysis and validated by amino acid substitution and functional assays. The Rpn1 toroid thus serves a critical scaffolding role within the proteasome, helping to assemble multiple proteasome cofactors, as well as substrates. RESULTS Our results indicate that proteasome subunit Rpn1 can recognize both ubiquitin and UBL domains of substrate shuttling factors that themselves bind ubiquitin and function as reversibly associated proteasomal ubiquitin receptors. Recognition is mediated by the T1 site within the Rpn1 toroid, which supports proteasome function in vivo. We found that the capacity of T1 to recognize both ubiquitin and UBL shuttling proteins was shared with Rpn10 and Rpn13. The surprising multiplicity of ubiquitin-recognition domains within the proteasome may promote enhanced, multipoint binding of ubiquitin chains. The structures of the T1 site in its free state and in complex with monoubiquitin or lysine 48 (K48)–linked diubiquitin were solved, which revealed that three neighboring outer helices from the T1 toroid engage two ubiquitins. This ubiquitin-binding domain is structurally distinct from those of Rpn10 and Rpn13, despite their common ligands. Moreover, the Rpn1-binding mode leads to a preference for certain ubiquitin chain types, especially K6- and K48-linked chains, in a distinct configuration that can position substrates close to the entry port of the proteasome. The fate of proteasome-docked ubiquitin conjugates is determined by a competition between substrate degradation and deubiquitination; the latter leads to premature release of substrates. Proximal to the T1 site within the Rpn1 toroid is a second UBL-binding site, T2, that does not assist in ubiquitin chain recognition but, rather, in chain disassembly, by binding to the UBL domain of deubiquitinating enzyme Ubp6. Note that the UBL interactors at T1 and T2 are distinct and assign substrate localization to T1 and substrate deubiquitination to T2. CONCLUSION A ligand-binding hotspot was identified in the Rpn1 toroid, consisting of two adjacent receptor sites, referred to as T1 and T2. The Rpn1 toroid represents a distinct class of binding domains for ubiquitin and UBL proteins. The T1 site functions to recruit substrates directly by binding to ubiquitin itself and indirectly by binding to UBL shuttling factors, a feature shared by Rpn10 and Rpn13 despite a lack of structural similarity among these receptors. The T2 site also binds to a UBL domain protein, in this case deubiquitinating enzyme Ubp6. This study thus defines a two-site recognition domain intrinsic to the proteasome that uses distinct ubiquitin-fold ligands to assemble substrates, substrate shuttling factors, and a deubiquitinating enzyme in close proximity. A ligand-binding hotspot in the proteasome for assembling substrates and cofactors. Schematic (top) and model structure (bottom, left) mapping the UBL-binding Rpn1 T1 (indigo) and T2 (orange) sites. (Bottom, right) Enlarged region of the proteasome designed to illustrate Rpn1 T1 and T2 sites bound to a ubiquitinated (yellow) substrate (beige) and deubiquitinating enzyme Ubp6 (green), respectively. Aided by PDB 4CR2, 1WGG, 1VJV, and 2B9R. Hundreds of pathways for degradation converge at ubiquitin recognition by a proteasome. Here, we found that the five known proteasomal ubiquitin receptors in yeast are collectively nonessential for ubiquitin recognition and identified a sixth receptor, Rpn1. A site (T1) in the Rpn1 toroid recognized ubiquitin and ubiquitin-like (UBL) domains of substrate shuttling factors. T1 structures with monoubiquitin or lysine 48 diubiquitin show three neighboring outer helices engaging two ubiquitins. T1 contributes a distinct substrate-binding pathway with preference for lysine 48–linked chains. Proximal to T1 within the Rpn1 toroid is a second UBL-binding site (T2) that assists in ubiquitin chain disassembly, by binding the UBL of deubiquitinating enzyme Ubp6. Thus, a two-site recognition domain intrinsic to the proteasome uses distinct ubiquitin-fold ligands to assemble substrates, shuttling factors, and a deubiquitinating enzyme.

An asymmetric interface between the regulatory and core particles of the proteasome

The Saccharomyces cerevisiae proteasome comprises a 19-subunit regulatory particle and a 28-subunit core particle. To be degraded, substrates must cross the core particle–regulatory particle interface, a site for complex conformational changes and regulatory events. This interface includes two aligned heteromeric rings, one formed by the six ATPase (Rpt) subunits of the regulatory particle and the other by the seven α subunits of the core particle. The Rpt C termini bind to intersubunit cavities in the α-ring, thus directing core particle gating and proteasome assembly. We mapped the Rpt C termini to the α subunit pockets, using a cross-linking approach that revealed an unexpected asymmetry: one side of the ring shows 1:1 contacts of Rpt2-α4, Rpt6-α3 and Rpt3-α2, whereas on the opposite side, the Rpt1, Rpt4 and Rpt5 tails each cross-link to multiple α pockets. Rpt–core particle cross-links are all sensitive to nucleotides, implying that ATP hydrolysis drives dynamic alterations at the core particle–regulatory particle interface.

USP14 deubiquitinates proteasome-bound substrates that are ubiquitinated at multiple sites.

USP14 is a major regulator of the proteasome and one of three proteasome-associated deubiquitinating enzymes. Its effects on protein turnover are substrate-specific, for unknown reasons. We report that USP14 shows a marked preference for ubiquitin–cyclin B conjugates that carry more than one ubiquitin modification or chain. This specificity is conserved from yeast to humans and is independent of chain linkage type. USP14 has been thought to cleave single ubiquitin groups from the distal tip of a chain, but we find that it removes chains from cyclin B en bloc, proceeding until a single chain remains. The suppression of degradation by USP14’s catalytic activity reflects its capacity to act on a millisecond time scale, before the proteasome can initiate degradation of the substrate. In addition, single-molecule studies showed that the dwell time of ubiquitin conjugates at the proteasome was reduced by USP14-dependent deubiquitination. In summary, the specificity of the proteasome can be regulated by rapid ubiquitin chain removal, which resolves substrates based on a novel aspect of ubiquitin conjugate architecture.

Reconfiguration of the proteasome during chaperone-mediated assembly

The proteasomal ATPase ring, comprising Rpt1–Rpt6, associates with the heptameric α-ring of the proteasome core particle (CP) in the mature proteasome, with the Rpt carboxy-terminal tails inserting into pockets of the α-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 α-pockets with poor specificity, except for Rpt6, which uniquely bound the α2/α3-pocket. Although the Rpt6 tail is not visualized within an α-pocket in mature proteasomes, it inserts into the α2/α3-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.

Structural defects in the regulatory particle-core particle interface of the proteasome induce a novel proteasome stress response.

Background: Ecm29, a proteasome component, has the unusual property of binding both the regulatory particle (RP) and core particle (CP). Results: Ecm29 loads onto proteasomes in response to various mutational perturbations at the RP-CP interface. Conclusion: Ecm29 is a regulator of RP assembly and CP gating. Significance: These studies reveal significant new aspects of the cellular response to proteasome stress. Proteasomes consist of a 19-subunit regulatory particle (RP) and 28-subunit core particle (CP), an α7β7β7α7 structure. The RP recognizes substrates and translocates them into the CP for degradation. At the RP-CP interface, a heterohexameric Rpt ring joins to a heteroheptameric CP α ring. Rpt C termini insert individually into the α ring pockets to form a salt bridge with a pocket lysine residue. We report that substitutions of α pocket lysine residues produce an unexpected block to CP assembly, arising from a late stage defect in β ring assembly. Substitutions α5K66A and α6K62A resulted in abundant incorporation of immature CP β subunits, associated with a complete β ring, into proteasome holoenzymes. Incorporation of immature CP into the proteasome depended on a proteasome-associated protein, Ecm29. Using ump1 mutants, we identified Ecm29 as a potent negative regulator of RP assembly and confirmed our previous findings that proper RP assembly requires the CP. Ecm29 was enriched on proteasomes of pocket lysine mutants, as well as those of rpt4-Δ1 and rpt6-Δ1 mutants, in which the C-terminal residue, thought to contact the pocket lysine, is deleted. In both rpt6-Δ1 and α6K62A proteasomes, Ecm29 suppressed opening of the CP substrate translocation channel, which is gated through interactions between Rpt C termini and the α pockets. The ubiquitin ligase Hul5 was recruited to these proteasomes together with Ecm29. Proteasome remodeling through the addition of Ecm29 and Hul5 suggests a new layer of the proteasome stress response and may be a common response to structurally aberrant proteasomes or deficient proteasome function.

Assembly manual for the proteasome regulatory particle: the first draft.

The proteasome is the most complex protease known, with a molecular mass of approx. 3 MDa and 33 distinct subunits. Recent studies reported the discovery of four chaperones that promote the assembly of a 19-subunit subcomplex of the proteasome known as the regulatory particle, or RP. These and other findings define a new and highly unusual macromolecular assembly pathway. The RP mediates substrate selection by the proteasome and injects substrates into the CP (core particle) to be degraded. A heterohexameric ring of ATPases, the Rpt proteins, is critical for RP function. These ATPases abut the CP and their C-terminal tails help to stabilize the RP-CP interface. ATPase heterodimers bound to the chaperone proteins are early intermediates in assembly of the ATPase ring. The four chaperones have the common feature of binding the C-domains of Rpt proteins, apparently a remarkable example of convergent evolution; each chaperone binds a specific Rpt subunit. The C-domains are distinct from the C-terminal tails, but are proximal to them. Some, but probably not all, of the RP chaperones appear to compete with CP for binding of the Rpt proteins, as a result of the proximity of the tails to the C-domain. This competition may underlie the release mechanism for these chaperones. Genetic studies in yeast point to the importance of the interaction between the CP and the Rpt tails in assembly, and a recent biochemical study in mammals suggests that RP assembly takes place on pre-assembled CP. These results do not exclude a parallel CP-independent pathway of assembly. Ongoing work should soon clarify the roles of both the CP and the four chaperones in RP assembly.

UBE2O remodels the proteome during terminal erythroid differentiation

Removing orphan proteins from the system The degradation of excess subunits of protein complexes is a major quality-control problem for the cell. How such “orphans” are recognized and tagged for degradation is poorly understood. Two papers identify a protein quality-control pathway that acts on some of the most abundant protein complexes in the human body: hemoglobin and ribosomes (see the Perspective by Hampton and Dargemont). Yanagitani et al. show that the central player in this process is an unusual enzyme (UBE2O) that recognizes substrates and tags them for destruction. Other quality-contr ol pathways tend to use separate factors for target selection (often a chaperone), ubiquitin donation (an E2), and ubiquitin conjugati on (an E3). Encoding all three activities in a single factor whose function can be reconstituted in a purified system provides a tractable route to detailed mechanistic and structural dissection. Nguyen et al. show the importance of the UBE2O pathway in the differentiation of red blood cells. Science, this issue p. 472, p. eaan0218; see also p. 450 During terminal differentiation, a specialized state is achieved through the targeted elimination of preexisting proteins. INTRODUCTION The reticulocyte–red blood cell transition is a canonical example of terminal differentiation. The mature red blood cell has one of the simplest cellular proteomes known, with hemoglobin remarkably concentrated to ~98% of soluble protein. During reticulocyte maturation, the proteome is remodeled through the programmed elimination of most generic constituents of the cell, in parallel with abundant synthesis of cell type–specific proteins such as hemoglobin. The mechanisms that drive rapid turnover of soluble and normally stable proteins in terminally differentiating cells remain largely unclear. RATIONALE The ubiquitin-proteasome system (UPS) was discovered in reticulocytes, where it is highly active. However, its function in this developmental context has not been established. UBE2O is an E2 (ubiquitin-conjugating) enzyme that is co-induced with globin and expressed at elevated levels late in the erythroid lineage. We identified an anemic mouse line with a null mutation in Ube2o, and used multiplexed quantitative proteomics to identify candidate substrates of UBE2O in an unbiased and global manner. We found that the protein compositions of mutant and wild-type reticulocytes differed markedly, suggesting that UBE2O-dependent ubiquitination might target its substrates for degradation to effect remodeling of the proteome. To test whether UBE2O was sufficient for proteome remodeling, we engineered a non-erythroid cell line to inducibly express UBE2O above its basal level. Upon induction, we observed the decline of hundreds of proteins from these cells, in many cases the same proteins as those eliminated from reticulocytes. Overexpression of an active-site mutant of UBE2O did not show these effects. Therefore, a major component of the specificity underlying differentiation-linked proteome remodeling appears to be carried by UBE2O itself. These results also indicate that UBE2O may function as a hybrid enzyme with both E2 and E3 (ubiquitin-ligating) activities. In support of this model, candidate substrates identified by proteomics were ubiquitinated by purified UBE2O without the assistance of additional specificity factors. RESULTS The most prominent phenotypes of the Ube2o mutant are an anemia characterized by small cells with low hemoglobin content (microcytic hypochromic anemia), and a defect in the elimination of ribosomes, the latter being a key aspect of reticulocyte maturation. When we added recombinant UBE2O protein to reticulocyte lysates from the null mutant, ubiquitin was conjugated primarily to ribosomal proteins. Moreover, immunoblot analysis and quantitative proteomics revealed elevated levels of multiple ribosomal proteins in mutant reticulocytes. Sucrose gradient analysis indicated the persistence not only of ribosomal proteins but of ribosomes themselves during ex vivo differentiation of mutant reticulocytes. Accordingly, ribosomes were eliminated upon induction of UBE2O in non-erythroid cells. The elimination of organelles from reticulocytes, as exemplified by that of mitochondria, was not affected in the Ube2o mutant, indicating the specificity of its effects on programmed protein turnover. Free ribosomal proteins were ubiquitinated by purified UBE2O, which suggests that these proteins are true substrates of the enzyme. However, UBE2O substrates are diverse in nature and not limited to ribosomal proteins. Individual domains of UBE2O bound substrates with distinct specificities. Thus, the broad specificity of UBE2O reflects the presence of multiple substrate recognition domains within the enzyme. Proteasome inhibitors blocked the degradation of UBE2O-dependent substrates in reticulocytes, although UBE2O does not form polyubiquitin chains. Rather, UBE2O adds single ubiquitin groups to substrates at multiple sites. Proteasome inhibitor treatment ex vivo led to depletion of the pools of many amino acids; this result implies that the flux of ubiquitinated substrates through the reticulocyte proteasome is sufficient to supply amino acids needed for late-stage translation of mRNA. In late erythropoiesis, several ubiquitin-conjugating enzymes and ligases are induced together with Ube2o while most components of the UPS disappear. We propose that the UPS is not simply amplified during erythroid maturation, but is instead broadly reconfigured to promote remodeling of the proteome. CONCLUSION A highly specialized UPS is expressed in the reticulocyte and is used to remodel the proteome of these cells on a global scale. UBE2O, a hybrid E2-E3 enzyme, functions as a major specificity factor in this process. In reticulocytes, and perhaps in other differentiated cells such as in the lens, the induction of ubiquitinating factors may drive the transition from a complex to a simple proteome. UBE2O drives remodeling of the proteome during erythroid differentiation. The transformation of reticulocytes into erythrocytes involves the elimination of myriad proteins. UBE2O is an E2-E3 hybrid enzyme that directly recognizes and ubiquitinates proteins that are fated for elimination. The target protein is degraded by the proteasome; ubiquitin (Ub) is recycled. UBE2O substrates include ribosomal proteins, recognized in a free form or possibly within the ribosome complex. During terminal differentiation, the global protein complement is remodeled, as epitomized by erythrocytes, whose cytosol is ~98% globin. The erythroid proteome undergoes a rapid transition at the reticulocyte stage; however, the mechanisms driving programmed elimination of preexisting cytosolic proteins are unclear. We found that a mutation in the murine Ube2o gene, which encodes a ubiquitin-conjugating enzyme induced during erythropoiesis, results in anemia. Proteomic analysis suggested that UBE2O is a broad-spectrum ubiquitinating enzyme that remodels the erythroid proteome. In particular, ribosome elimination, a hallmark of reticulocyte differentiation, was defective in Ube2o−/− mutants. UBE2O recognized ribosomal proteins and other substrates directly, targeting them to proteasomes for degradation. Thus, in reticulocytes, the induction of ubiquitinating factors may drive the transition from a complex to a simple proteome.