Research Article

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

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Science  19 Feb 2016:
Vol. 351, Issue 6275, aad9421
DOI: 10.1126/science.aad9421

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

Structured Abstract


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).


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.


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.


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.

Protein fates are regulated on a global scale by the covalent conjugation of ubiquitin, which can direct target proteins to the proteasome for degradation. Misregulation of the ubiquitin-proteasome system is associated with a broad range of human diseases, such as cancer, developmental disorders, neurodegenerative diseases, immune disorders, and microbial infections. In the vast network of the ubiquitin-proteasome system, the recognition of ubiquitin by the proteasome constitutes a central node. Defects in proteasomal ubiquitin receptors have been implicated in both amyotrophic lateral sclerosis and Alzheimer’s disease (1, 2). Although the mechanisms by which the proteasome achieves its substrate specificity are largely unresolved, it is clear that myriad substrates are first committed to degradation through an initial receptor-mediated recognition step.

There are two distinct modes of ubiquitin recognition by a proteasome. The intrinsic recognition pathway uses specific proteasome subunits (Rpn10 and Rpn13) for ubiquitin binding (37), whereas the extrinsic recognition pathway uses shuttling factors (Rad23, Dsk2, and Ddi1 in yeast) to escort ubiquitinated substrates to the proteasome (8, 9). The shuttling factors are known as UBL-UBA proteins, because each binds the proteasome by means of a ubiquitin-like (UBL) domain while binding ubiquitin chains with a ubiquitin-associated domain (UBA). The intrinsic and extrinsic pathways are functionally redundant, at least in part, and cooperate in mediating substrate degradation by the proteasome (6, 8, 9). These receptors exhibit some substrate specificity but with a mechanistic basis that is poorly understood (913).

Existence of an additional ubiquitin receptor in the proteasome

To investigate ubiquitin binding by the proteasome, we first obtained an RPN13 allele that abrogates ubiquitin binding while having no discernible effect on proteasome assembly. [Existing alleles of Rpn13 decrease affinity for ubiquitin to one-eighth that of wild-type (6)]. Guided by nuclear magnetic resonance (NMR) data, we introduced two substitutions into the loop segments of the PRU domain, which, when combined with three previously characterized substitutions, completely abolished Rpn13 binding to ubiquitin, as judged by the absence of spectral changes in NMR titration experiments performed at high concentrations of ubiquitin and Rpn13 (fig. S1). The resulting rpn13 E41K E42K L43A F45A S93D mutant is referred to as rpn13-pru below.

To test whether the five known proteasomal ubiquitin receptors are together required for ubiquitin recognition by the proteasome, we introduced the rpn13-pru allele into a strain carrying mutations in the other four receptors, all of which appear to be nulls for ubiquitin recognition. Unexpectedly, this strain, hereafter known as the quintuple mutant, proved to be viable and only moderately defective in growth under standard conditions (Fig. 1A). This strain did display a range of physiological defects, including heat sensitivity, canavanine sensitivity, and high salt sensitivity (Fig. 1A); evidently, its ubiquitin-proteasome system is strongly attenuated. In summary, the viability of the quintuple mutant led us to hypothesize the existence of still unidentified ubiquitin receptors on the proteasome.

Fig. 1 Evidence for an additional ubiquitin receptor in yeast.

(A) Yeast strains with the indicated genotypes were serially diluted, transferred to agar plates, and incubated at 30°C. Media were yeast extract peptone dextrose (YPD), YPD with 1 M NaCl (top row), synthetic medium lacking arginine (Arg), or Arg with 6 μm/ml canavanine (bottom row). ΔUU designates the rad23Δ dsk2Δ ddi1Δ background. WT, wild type. (B) Proteasome association with ubiquitin conjugates evaluated by a mobility-shift assay. RP complexes were purified from an rpn10-uim rpn13-pru strain and incubated with core particle. Reconstituted proteasomes were incubated with autoubiquitinated Cdc34, resolved via native PAGE, and visualized with a fluorogenic activity stain. (C) Known and candidate ubiquitin receptor proteins were expressed in bacteria as GST fusion proteins and immobilized on glutathione resin. Ubiquitinated T7-PY-Sic1 conjugates were used as ligand. Resin-bound proteins were resolved by SDS-PAGE, blotted, and probed with antibodies as indicated. Direct loading of ubiquitinated T7-PY-Sic1 is also included (input). (D) 1H, 15N heteronuclear single-quantum coherence (HSQC) spectra of 15N Rpn1412-625 (black) and with equimolar ubiquitin (orange). A titration series is displayed to the right for D541 with increasing ubiquitin, as indicated. (E) Model ribbon diagram of the Rpn1 toroidal domain [PDB 4CR2 (47)] showing residues that shift after ubiquitin addition (D) in orange. (F) Ubiquitin amino acids with amide signals that shift by 1 SD above average after Rpn1 T1 addition, as shown in fig S8, B and D, are labeled and highlighted in orange on a ribbon diagram [PDB 2K39 (48)].

Proteasome subunit Dss1/Sem1 was recently proposed to be a novel ubiquitin receptor for this complex (14). In this study, recombinant Sem1 was shown to bind ubiquitin, but whether proteasomes lacking Sem1 are defective in ubiquitin recognition was not examined. To evaluate Sem1, we tested the ubiquitin-binding activity of sem1Δ proteasomes. We could not resolve any defect, even in genetic backgrounds in which other ubiquitin receptors had been eliminated (fig. S2), and thus, further studies may be required to validate Sem1 as a proteasomal ubiquitin receptor. Because the ubiquitin-binding surface of Sem1 overlaps with its proteasome-binding surface (14), simultaneous binding of the proteasome and ubiquitin by Sem1 might not be possible. Although mutations in the ubiquitin-binding region of Sem1 lead to hypomorphic proteasome phenotypes, these mutations are also associated with proteasome assembly defects.

To test further for additional proteasomal ubiquitin receptors, we characterized proteasomes in which all five known ubiquitin receptors were either functionally inactivated by making amino acid substitutions in their ubiquitin-binding sites or removed during purification by salt wash. When such proteasomes were electrophoresed on a native gel in the presence of ubiquitin conjugates, their migration—visualized by an in-gel activity assay—was retarded, which indicated productive binding (Fig. 1B). This binding activity was further localized to the nine-subunit base subcomplex by using native gel-shift assays (fig. S3). With the number of candidate receptors narrowed down in this way, we tested proteins individually for ubiquitin binding. Rpn1, in particular, proved to form a stable complex (Fig. 1C) with a model proteasome substrate, ubiquitinated PY-Sic1 (15). Rpn1 binding to this substrate was specific to the ubiquitin moiety, because nonubiquitinated Sic1, an internal control, failed to bind (Fig. 1C). Rpn1, the largest subunit of the base, has been previously suggested to serve in the recognition of UBL proteins, such as Rad23, by the proteasome (1618).

Rpn1 is paralogous to Rpn2, the structure of which has been solved by x-ray crystallography (19). Both proteins have a centrally located domain composed of 11 repeats of 30 to 40 residues each that form helix-turn-helix hairpins, known as PC repeats (20). Collectively, these repeats form a closed toroid (19). On the basis of these data, we generated a series of deletions in Rpn1. Using ubiquitinated PY-Sic1 as a ligand, the binding activity of Rpn1 was mapped to residues serine 376 (S376) to alanine 635 (A635) (fig. S4). This fragment was trimmed further to obtain a recombinant fragment suitable for NMR, residues glycine 412 (G412) to threonine 625 (T625) (fig. S5A). NMR titration experiments in which unlabeled ubiquitin was added to 15N-labeled Rpn1 (412–625) showed clear binding (Fig. 1D). The signals in the NMR spectrum were assigned to Rpn1 amino acids (fig. S5, A and B), as described below and in the supplementary materials (SM). In an Rpn1 model structure, those residues that contact ubiquitin according to the titration experiment (fig. S6) map to three outer helices of the toroid (Fig. 1E). We thus name this ubiquitin-binding region T1 (toroid 1). Plots of the spectral changes observed for each amino acid with ubiquitin addition revealed two distinct binding affinities, with amino acids from helix 26 (H26) presenting dissociation constant (Kd) values of 350 ± 70 μΜ and those from H28 and H30 with values of 120 ± 10 μM (fig. S7). Thus, two weak binding sites for monoubiquitin are suggested on T1.

When unlabeled Rpn1 (412–625) was added to 15N- or 13C-labeled ubiquitin, significant shifting was observed for amino acids located in the ubiquitin β-strand surface (Fig. 1F and fig. S8), which is recognized as well by Rpn10 and Rpn13 (7, 21). Included in this surface are specific residues of leucine, arginine, isoleucine, histidine, and valine L8, R42, I44, H68, V70, and R74, which shift significantly when Rpn1 is added.

The Rpn1 T1 site binds two ubiquitins with three outer toroid helices

Because only a model structure was available for Rpn1, we used NMR to solve the structure of the T1 site. The N- and C-terminal portions of Rpn1 (412–625) were poorly behaved, with Y608 to T625 absent from the recorded spectra and G412 to K484 disordered (fig. S5, B and C). In addition, we observed a concentration-dependent dimerization, as determined by dynamic light scattering (fig. S9A), which NMR data suggested to be mediated by the inner helices (H27, H29, and H31) of the toroid (Fig. 1E and fig. S9, B and C). These effects are no doubt the result of truncation artifacts, although larger deletions only destabilized the protein fold further (fig. S10). Nonetheless, by using standard NMR techniques, as well as stereoselective methyl labeling and four-dimensional (4D) nuclear Overhauser effect spectroscopy (NOESY) experiments to record interactions between methyl groups (fig. S11), we were able to solve the structure of the Rpn1 T1 site. This region folds into 3.5 PC repeats (Materials and Methods and table S1), with H25 forming an incomplete hairpin structure (Fig. 2A). H26 to H31, which forms three complete hairpins, converge to a backbone root mean square deviation (RMSD) of 0.8 Å. The 4D NOESY spectrum (fig. S11) and an evaluation based on chemical shift values (fig. S5C) revealed H25 to be helical, but loss of its hairpin partner (presumed to range from A470 to T479) apparently destabilized it and resulted in this helix adopting a dynamic orientation relative to H26 to H31.

Fig. 2 The Rpn1 T1 site engages two ubiquitin molecules in a mode suited for K48-linked chains.

(A) Backbone heavy atoms for the 10 lowest energy structures of the Rpn1 T1 site with H26 to H31 superimposed. The N- and C-terminal residues and the individual helices are labeled. (B) Ribbon diagram of the lowest energy structure of the Rpn1 T1 site (blue) bound to two monoubiquitin molecules (orange and yellow). Heavy atoms for G76, the M1 backbone, and lysine side chains are displayed with their oxygen and nitrogen atoms in red and blue respectively. Displayed side chains for the ubiquitin at H28/H30 are labeled in orange, whereas those from the ubiquitin centered at H26 are labeled in black. Dashed red lines are drawn between G76 from the ubiquitin at H28/H30 and the closest lysine side chains (K6 and K48) from the ubiquitin at H26; the distance between these two pairs of amino acids is <10 Å and the flexibility of the lysine side chain and C-terminal tail of monoubiquitin allows this distance to be readily shortened. (C) Zoomed-in view of (B) highlighting the Rpn1:ubiquitin contact surface with key amino acids displayed and labeled. Electrostatic interactions are indicated with red dashed lines. (D) Pull-down assay with His-scRpn1 full length protein and M1-, K6-, K11-, K27-, K29-, K33-, K48-, and K63-diubiquitin, as indicated (top and middle panels, left). Immunoblotting was done with antibody against ubiquitin (anti-ubiquitin) (top, left) or antibody against polyhistidine (anti-His) (middle, left). Intrinsically disordered protein SocB-His (49) was used as a negative control with K48 diubiquitin, as indicated. Direct loading for 15% of the diubiquitin input for each chain type with immunoblotting by ubiquitin-specific antibody is included (bottom, left). The pull-down assay was repeated four times, and the diubiquitin signal intensities were separately normalized to the strongest signal by using ImageJ. The average value and standard deviation are plotted (right). (E) Shifting for indicated Rpn1 T1 site residues plotted with increasing K48 diubiquitin and fitting to the listed Kd values. CSP, chemical shift perturbation. (F) ITC analysis of Rpn1412-625 binding to K48 diubiquitin. K48 diubiquitin (1.91 mM) was injected into a calorimeter cell containing 0.18 mM Rpn1412-625, and the data were fit to a two-site sequential binding mode to yield the indicated thermodynamic values. (G) As in (D) but with GST-hRpn1404-617 or GST (as a control). In this case, quantification on the right was done with two independent pull-down assays.

We next solved the structure of the Rpn1 T1 site in complex with ubiquitin, as described in Materials and Methods and (22). Spectra collected on this protein complex showed higher signal-to-noise ratio (fig. S12) compared with those recorded on free Rpn1 and indicated that the structure of the Rpn1 T1 site does not change upon binding to ubiquitin (fig. S13, A, B, and D). Moreover, spectra from NOESY recorded on the complex were used to generate new intramolecular constraints for Rpn1 to be used in the calculation of the Rpn1 T1:ubiquitin complex (table S2). Half-filtered NOESY experiments, specialized to record interactions between Rpn1 and ubiquitin, revealed many contacts involving ubiquitin L8, T9, I44, V70, L71, and L73 (fig. S14, A and B), as expected from the titration experiment (Fig. 1F). Intermolecular contacts involving these amino acids validated the presence of two binding sites. For example, L8 contacted both L508 and T544 (fig. S14A), which are separated by a 16-Å distance across the toroid (fig. S14C). Ultimately, 106 unambiguous intermolecular distance constraints were identified between Rpn1 and two ubiquitin molecules (table S2). Twenty-four intermolecular paramagnetic relaxation enhancement (PRE)–derived constraints were also collected with a spin label substituted for ubiquitin G75 (fig. S15A). The resulting structures converged to a backbone RMSD of 0.93 Å (fig. S16) with one ubiquitin at a surface formed by H28 and H30 (H28/H30) (Fig. 2B, orange) and the other at H26 (Fig. 2B, yellow). Individually, the H28/H30:ubiquitin region converged better than the H26:ubiquitin region, with backbone RMSD values of 0.58 and 0.74, respectively (table S2), owing to fewer detected interactions for the lower-affinity H26 site (figs. S14, A and B, and S7). Both sites use hydrophobic amino acids to engage ubiquitin L8-I44-V70 (Fig. 2C).

One of seven lysines or the N-terminal methionine in ubiquitin is used to generate ubiquitin chains, which differ in their signaling properties (23). To evaluate Rpn1 ubiquitin chain preference experimentally, resin-bound His-scRpn1 was incubated separately with diubiquitin of each linkage type, washed, and complex formation assayed by immunoblotting with ubiquitin-specific antibody. In this experiment, Rpn1 T1 demonstrated the greatest affinity for K6 and K48 diubiquitin (Fig. 2D), the latter of which is an established linkage type for signaling substrate proteolysis by the proteasome (23). Consistent with this result, G76 from ubiquitin at H28/H30 is spatially close to K48 and K6 of the ubiquitin at H26 (Fig. 2B and fig. S14D). An NMR titration experiment revealed Rpn1 to prefer K48-linked ubiquitin chains to monoubiquitin with 11- and 2.7-fold increased affinity at H28/H30 and H26, respectively (Fig. 2E), as supported by isothermal titration calorimetry (ITC, Fig. 2F). The 11 μM affinity of Rpn1 for K48 diubiquitin is similar to the 13 μM affinity reported for scRpn10 binding to K48 diubiquitin (24).

Human Rpn1 (404–617) is 43.8% identical to scRpn1 (412–625) (fig. S17). Glutathione S-transferase (GST) fused with hRpn1(GST-hRpn1 (404–617) or GST (as a negative control) was bound to glutathione S-Sepharose resin and incubated with diubiquitin of each linkage type. After removal of unbound proteins by washing, binding between hRpn1 T1 and ubiquitin was assessed by immunoblotting. This experiment confirmed that human Rpn1 also harbors the ubiquitin-binding T1 site, which prefers K6 and K48 linkages (Fig. 2G).

The T1 plays a dual role in binding ubiquitin chains and shuttle factor Rad23

Rpn1 H28 contacts both ubiquitins forming electrostatic interactions with D541, D548, and E552 (Fig. 2C). We replaced these residues in the Rpn1 T1 site to generate a D541A D548R E552R triple mutant. The structural integrity of this ARR mutant was maintained, as indicated by two-dimensional (2D) NMR analysis (fig. S18). These substitutions eliminated ubiquitin interaction at fivefold molar excess (Fig. 3A), as well as Rpn1 T1 binding to K6 and K48 diubiquitin in GST pull down (Fig. 3B) and ITC (fig. S19) experiments. Using the D541A D548R E552R mutant (hereafter rpn1-ARR), we tested in vitro ubiquitin binding activity in the context of full-length Rpn1. The Rpn1-ARR protein showed substantially decreased binding activity to ubiquitinated PY-Sic1 (Fig. 3C).

Fig. 3 H28 of the Rpn1 T1 site plays a dual role in binding ubiquitin chains and shuttle factor Rad23.

(A) Selected regions from 1H, 15N HSQC spectra of 15N wild-type Rpn1412-625 (left) and 15N Rpn1412-625–ARR (D541A/D548R/E552R, right) alone (black) and with ubiquitin (orange) at four- and fivefold molar excess, respectively. (B) GST pull-down assay with GST, GST–Rpn1412-625, GST–Rpn1412-625–ARR, or GST–Rpn1412-625 A514G-D517A-D541A-D548R-E552R (GAARR) and the indicated ubiquitin species. Immunoblotting was done with anti-ubiquitin (top) or anti-GST (bottom) antibodies. (C) Defective ubiquitin binding by the Rpn1-ARR mutant protein. Full-length GST-Rpn1 fusion protein was expressed, purified, and tested for binding to ubiquitinated T7–PY-Sic1. GST-Rpn10 and GST-Rpn10-uim were included as positive and negative controls, respectively. (D) Rad23 variants were tested for binding to Rpn1-WT and Rpn1-ARR. A mixture of recombinant Rad23-Flag and Rad23ΔUBL-Flag (serving as an internal negative control) were used as ligands. UBA domains were absent from both constructs. (E) Rpn1 amino acids at the Rad23 UBL domain contact surface, as determined by the data in fig. S20 are highlighted in orange on the ribbon diagram of the Rpn1 T1 site. (F) ITC analysis of the Rpn1 T1 site binding to Rad23 UBL. Rad23 UBL (0.407 mM) was injected into a calorimeter cell containing 0.036 mM Rpn1412-625, and the data were fit to a one-site binding mode with the indicated thermodynamic parameters.

We also tested the binding of Rad23 to the Rpn1-ARR protein. Rpn1-ARR proved to be almost completely defective in Rad23 binding as well (Fig. 3D). This binding was mediated by the UBL domain of Rad23, in agreement with previous studies of the Rad23-Rpn1 interaction (16, 18). Thus, the Rpn1-ARR mutation appears to define a binding site for both ubiquitin and UBL-UBA proteins. To test this model further, we added unlabeled Rad23 UBL domain to 15N-labeled Rpn1 T1 and used 2D NMR to visualize the affected amino acids. This experiment validated the positioning of H28 at the UBL contact surface (Fig. 3E and fig. S20). ITC demonstrated that Rpn1 (412–625) binds Rad23 UBL with an affinity of 64 ± 25 nM (Fig. 3F), and binding was not detectable with the triple mutant (fig. S21). Thus, H28 is a hotspot within the Rpn1 T1 site for directly and indirectly receiving ubiquitinated substrates.

Phenotypic effects of the rpn1-ARR mutation

To assess the ubiquitin-binding activity of Rpn1 in vivo, we integrated the ARR mutation into the genomic RPN1 locus. Rpn1-ARR proteasomes were found to resemble those of wild type in their assembly and stability, based on several assays: Proteasomes were purified from rpn1-ARR cells without apparent difference in composition or yield (fig. S22A); the amount of Rpn1-ARR protein was comparable to that of wild type (fig. S22); the relative amounts among different subcomplexes of the proteasome were unaffected by the rpn1-ARR mutation (fig. S22C); the SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and native gel profiles of wild-type and Rpn1-ARR proteasomes were indistinguishable (fig. S22, A to C); no mutant-specific fragmentation of the protein was observed for total yeast cell extracts probed with an Rpn1-specific antibody (fig. S22D).

To assess the affinity of the proteasome for ubiquitin conjugates and UBL proteins in a defined biochemical system, we used proteasomes reconstituted from their two major subassemblies, the 19-subunit regulatory particle (RP), which contains Rpn1, and the 28-subunit proteolytic core particle (CP). The RP was purified from strains bearing RPN1-WT or rpn1-ARR alleles in the rpn13-pru rpn10-uim background (fig. S23A) and was reconstituted into holoenzyme by adding back purified wild-type CP. Reconstituted proteasomes have a strong ubiquitin-binding activity that is lost in the rpn1-ARR mutant (Fig. 4A). This activity cannot be attributed to contaminating UBL-UBA proteins, because the molar ratio of Rad23 to RP is very low in these preparations (between 1:340 to 1:680) (fig. S23, B and C). Moreover, the dependence of ubiquitin binding on Rpn1 was also seen using RPs purified from the ubl-ubaΔ background (fig. S23D). These results show that the Rpn1 T1 site can function as a ubiquitin receptor on the proteasome and that this ubiquitin-binding surface in whole proteasomes is the same as that characterized by NMR.

Fig. 4 Rpn1, Rpn10, and Rpn13 play dual roles in recruiting ubiquitin conjugates and shuttling receptors to the proteasome.

(A) Proteasome association with ubiquitin conjugates evaluated by mobility-shift assay. RP complexes were purified from the indicated yeast strains, incubated with purified CP complex to form holoenzyme, and tested for association with ubiquitin conjugates as described for Fig. 1B. (B) Proteasomes were prepared, as described for (A), and assayed for degradation of a ubiquitinated fragment of cyclin B1 (Ubn-HA-NCyclinB). Rpn5 was probed as a loading control. (C) Synthetic canavanine sensitivity of rpn1-ARR with other intrinsic ubiquitin receptor mutants. All strains were prepared in the rad23Δ dsk2Δ ddi1Δ background and carry additional mutations as indicated. Yeast cultures were serially diluted and transferred to synthetic media agar plates lacking arginine (Arg) or Arg with 2 μg/ml canavanine, followed by incubation at 30°C. (D) Proteasome association with Rad23 evaluated by mobility-shift assay. Proteasomes were purified from rad23Δ dsk2Δ ddi1Δ yeast strains bearing the indicated intrinsic ubiquitin receptor mutations and incubated with ligand at 100-fold molar excess. Binding mixtures were resolved by native PAGE and proteasome complexes assayed as described. (E) Proteasomes were purified, in the absence of salt, from yeast strains bearing mutations in intrinsic ubiquitin receptors, as indicated. Proteasomes and their associated proteins were resolved by SDS-PAGE, blotted, and probed with antibodies to proteasome-associated UBL protein Rad23 and to proteasome subunit Rpn5, as a loading control. (F) In vivo degradation of proteasome substrate Gic2. Yeast strains rpn10-uim rpn13-pru bearing TAP-tagged Gic2 integrated at the native genomic locus, and other alleles as indicated, were treated with cycloheximide for the indicated times. Lysates were prepared and resolved by SDS-PAGE. Proteins were blotted and probed for Gic2-TAP, with Pgk1 as a loading control. (G) The rpn1-ARR allele confers sensitivity to 4-NQO. Cultures of strains carrying the indicated mutations were serially diluted, transferred to YPD plates either lacking or containing 0.1 μg/ml 4-NQO, and incubated at 30°C for 2 days.

To test whether conjugate binding to the T1 site could support protein degradation, we used an established in vitro degradation assay based on ubiquitinated cyclin B1 (2527); all proteasomes tested were from the rpn13-pru rpn10-uim ubl-ubaΔ background. The rpn1-ARR mutation led to stabilization of cyclin B1 in this assay (Fig. 4B). Stabilization was incomplete, however, which pointed to the possible existence of a fourth intrinsic ubiquitin receptor in the proteasome. Similar results were obtained using ubiquitinated PY-Sic1 (fig. S24).

We next examined the rpn1-ARR phenotype for signs of proteasome dysfunction in vivo. Proteasome function in yeast is typically assayed in the presence of the arginine analog canavanine, whose presence in proteins induces misfolding and consequent proteasome-mediated degradation. rpn1-ARR exhibited a canavanine-sensitive phenotype when in the presence of the rpn10-uim allele (Fig. 4C). Its synthetic interaction with rpn10-uim was similar to that of rpn13-pru. In these strains, all genes encoding UBL-UBA proteins had been deleted, so that proteasome output should be driven only by direct ubiquitin recognition by intrinsic ubiquitin receptors. Thus, the canavanine-sensitive phenotype of the rpn1-ARR allele suggests that Rpn1 can support ubiquitin-dependent substrate recognition in cells. Its ability to do so is at some level redundant with other proteasomal ubiquitin receptors, a common characteristic of these proteins.

As discussed above, the isolated toroid domain of Rpn1 can bind both ubiquitin and the UBL protein Rad23. To assess which of the intrinsic proteasomal ubiquitin receptors can also bind UBL proteins within the context of intact proteasomes, we purified proteasomes from yeast strains that express mutant forms of two of the three intrinsic receptors, which left only a single receptor functional. As a control, one strain carried mutant forms of all three receptors. Using a gel-shift assay, we found that all three receptors could bind Rad23 (Fig. 4D). Therefore, the dual ubiquitin-UBL–binding mode seen for Rpn1 is a general feature of proteasomal ubiquitin receptors. These data were confirmed by using endogenous material; copurification of Rad23 with rpn10-uim rpn13-pru proteasomes was reduced by the rpn1-ARR mutation (Fig. 4E). This effect is not due to a reduced level of Rad23 in the mutant strain (fig. S25). We also found, using recombinant material, that the T1 site was a Dsk2-binding site (fig. S26).

Notably, control proteasomes lacking ubiquitin- and/or UBL-binding sites in all three receptors showed no detectable interaction with Rad23 (Fig. 4D), which suggested that all major proteasomal receptors for this UBL-UBA protein have now been identified. The contribution of Rpn10 to Rad23 binding was unexpected, because several previous studies had found that Rpn10 did not appear to contribute to UBL protein recognition by intact proteasomes (16, 28, 29). The difficulty in resolving this binding interaction in the past was likely the result of high background binding due to Rpn1 and Rpn13. One possible caveat to these data is that some fraction of copurified Rad23 may be indirectly pulled down by ubiquitin conjugates by the ubiquitin-binding UBA domains that are present in Rad23. This is addressed, however, by the reconstitution experiments of Fig. 4D. In summary, our results reveal a deep interconnectivity between the intrinsic and extrinsic ubiquitin receptors of the proteasome, where each intrinsic receptor can dock ubiquitin conjugates either by direct ubiquitin recognition or indirectly by an extrinsic receptor.

In phenotypic studies, we found that the T1 site of Rpn1 may promote protein degradation, as well as DNA repair, through UBL binding. In particular, the proteasome substrate Gic2, in its tandem affinity purification (TAP)–tagged form (30), is strongly stabilized by the T1 site mutation, and this effect depends on the expression of UBL-UBA proteins (Fig. 4F). Similar effects were observed for Gcn4 (fig. S27). Mutation of the T1 site also leads to a phenotype of sensitivity to 4-nitroquinoline 1-oxide (4-NQO), which suggests a defect in DNA repair (Fig. 4G). No difference was seen between mutant and wild-type forms of T1 in the UBL-UBA null genetic background, consistent with mediation of the 4-NQO–sensitive phenotype by an interaction between the proteasome and Rad23, a known DNA repair factor (31). In summary, phenotypic data suggest that the T1 site functions by the docking of ubiquitin conjugates at the proteasome but can do so both directly and through docking of ubiquitin receptors.

Structure of Rpn1 T1:K48 diubiquitin reveals a pathway at the proteasome for ubiquitin chains

Because of the importance of K48 ubiquitin chains in targeting substrates for proteolysis, we characterized Rpn1 binding to this chain type more extensively. As observed with monoubiquitin (fig. S13, A and B), binding to K48 diubiquitin did not change the Rpn1 T1 fold, as demonstrated by contacts between methyl groups (fig. S13, A and C). NMR experiments designed to record intermolecular contacts between Rpn1 and each ubiquitin of the K48 dimer revealed the presence of two binding modes. In particular, each ubiquitin moiety can bind to either the H28/H30 or the H26 binding surface (fig. S28). We thus calculated two sets of Rpn1 T1:K48 diubiquitin structures (Materials and Methods and table S2). In addition to nuclear Overhauser effect (NOE)–derived distance constraints, we obtained intermolecular distances between the Rpn1 T1 site and each ubiquitin of the K48 dimer by using PRE data obtained by placing a spin label in the loop connecting H26 and H27 at L518 (fig. S15B). In one set of calculations, 104 NOE- and 19 PRE-derived intermolecular distance constraints placed distal ubiquitin at Rpn1 H28/H30 and proximal ubiquitin was centered at Rpn1 H26 (Fig. 5A and fig. S29A); proximal ubiquitin is so-designated for its free G76, which can, in principle, be conjugated to a substrate. We refer to this binding mode as “extended,” because addition of ubiquitin moieties at either end of the chain yields an opened configuration across the Rpn1 T1 site, as illustrated in a model structure of Rpn1482-612 bound to K48 tetraubiquitin (Fig. 5B, left).

Fig. 5 Structures of the Rpn1 T1:K48 diubiquitin complex suggest a pathway for ubiquitin chain recognition at the proteasome.

(A and C) Lowest-energy structures of Rpn1482-612:K48 diubiquitin in the extended (A) or contracted (C) binding mode. These structures were solved experimentally by using a suite of NMR experiments, as described in Materials and Methods by using the data listed in table S2. An enlarged view is included in (C) to illustrate restricted accessibility of proximal ubiquitin G76. Displayed amino acid side chains from distal ubiquitin (orange) are labeled in orange, whereas those from proximal ubiquitin (yellow) are labeled in black. Blue coloring is used for Rpn1, with helices labeled in gray. (B) Model of Rpn1 T1:K48 tetraubiquitin by adding a ubiquitin (yellow) to each end of the K48 diubiquitin chain for the extended (left) and contracted (right) experimentally determined Rpn1 T1:K48 diubiquitin structure. The Rpn1 T1:K48 tetraubiquitin structures were energy-minimized by using Schrödinger ( (D) Expanded view of the extended (top) or contracted (bottom) binding mode for Rpn1 T1:K48 diubiquitin to illustrate hydrophobic and electrostatic interactions at the contact surface. (E) Model of proteasome engaging a ubiquitinated substrate, generated with Rpn1 T1:K48 diubiquitin in the extended binding mode, Rpn13 Pru:ubiquitin (PDB 2Z59), hRpn10:K48 diubiquitin (PDB 2KDF), and human cyclin B1 (PDB 2B9R) placed into a proteasome cryo-EM–based model (PDB 4CR2). Rpn1, blue and indigo; ubiquitin, yellow; Rpn13 Pru, navy; Rpn10, light blue; substrate, beige; ATPase ring, burgundy; CP, gray; remaining RP, white.

The second set of structural calculations uses 100 NOE- and eight PRE-derived intermolecular distance constraints that place proximal ubiquitin at H28/30 and distal ubiquitin at H26 (Fig. 5C and fig. S29B). In this binding mode, G76 from ubiquitin at H28/H30 is spatially close to ubiquitin at H26 (Fig. 5C, expanded region), a configuration caused by a H-bond between proximal ubiquitin R74 and Rpn1 D541 and by van der Waals interactions between proximal ubiquitin L73 and Rpn1 L510 and A514 (Fig. 5C, expanded region). This binding orientation results in a “contracted” ubiquitin chain, best illustrated in a model structure with K48-linked tetraubiquitin (Fig. 5B, right).

Many electrostatic and hydrophobic interactions used to bind monoubiquitin are preserved when the Rpn1 T1 site binds K48 diubiquitin (Figs. 2C and 5D). The backbone RMSD between the contracted and extended binding mode is 0.49 and 0.36 Å for ubiquitin binding at Rpn1 H26 and H28/H30, respectively. This similarity reflects that contacts between ubiquitin L8, T9, I44, V70, L71, and L73 and Rpn1 H26, H28, and H30 are retained in all cases, as supported by the experimental data (figs. S14 and S28).

We computationally generated a model substrate (cyclin B1) with an attached K48 triubiquitin chain and docked it in the extended binding mode into the Rpn1 T1 site of a proteasome cryo–electron microscopy (cryo-EM)–based model structure (PDB 4CR2). The resulting model illustrated the substrate to be directed toward the adenosine triphosphatase (ATPase) ring and its substrate entry channel (fig. S30). We next adapted this model to include our Rpn13 Pru:ubiquitin (7) and hRpn10:K48 diubiquitin (32) structures (Fig. 5E). The full model represents an additional pathway for ubiquitin chain binding contributed by the Rpn1 T1 site that exists on the same proteasome face as the ubiquitin-binding pathway generated by hRpn13 and hRpn10. Additional experiments will be needed to test this model further.

A second UBL-recognizing site on the Rpn1 toroid binds a deubiquitinating enzyme

In transit from Rpn1 and the other proteasome ubiquitin receptors to the catalytic center, substrates are deubiquitinated before passing through a narrow gating mechanism, as reviewed in (33). We have previously suggested that the principal binding site for Ubp6 on the proteasome may be in Rpn1 (18, 34), and more recently Rpt1 has also been implicated as an interaction site (35, 36). Using purified Rpn1 and purified Ubp6, we were unable to map the putative Ubp6 binding site of Rpn1 by deletion analysis. We turned to hydrogen exchange mass spectrometry (HX MS), which has proven useful for structural characterization of protein systems intractable by other techniques. HX MS reports on backbone amide hydrogens of proteins, which undergo isotopic exchange in deuterated solvent (3739). Exchange rates can vary by many orders of magnitude (40) and are influenced by both hydrogen bonding of the backbone and solvent accessibility.

During HX MS analysis of free Rpn1, it became apparent that extensive regions of Rpn1 underwent localized cooperative unfolding, visualized as multiple populations that incorporate deuterium at different, nonsynchronized rates. Such exchange kinetics, termed EX1 (4143), are a hallmark of a heterogeneous protein population, often reporting on long-lived conformational fluctuations (44) that result from changes in the conformational ensemble during the deuterium labeling. We hypothesized that Rpn1 incorporated into the base complex might be stabilized by intermolecular interactions within this complex, and therefore attempted to map the Rpn1-Ubp6 interaction in the context of the base.

A base-Ubp6 complex was subjected to deuterium labeling, followed by proteolytic digestion, and the resulting peptide mixture was analyzed by UPLC, ion mobility spectrometry, and mass spectrometry (fig. S31). We identified and measured the temporal deuterium uptake for 40 Rpn1 peptides covering ~60% of the sequence (Fig. 6A and figs. S32 and S33). As hypothesized, when incorporated into the base complex, Rpn1 was dramatically stabilized in conformation relative to when it was free in solution (Fig. 6A). In particular, the EX1 uptake kinetics indicative of conformational heterogeneity was no longer observable. The addition of Ubp6 to the base provided further protection from exchange exclusively for a highly localized region, suggestive of a well-defined binding site rather than further stabilization of the Rpn1 conformation (Fig. 6B). HX was suppressed most strongly for peptic peptide 419–436, corresponding to inner helix 21 and outer helix 22 of the toroid (Fig. 6B and figs. S4 and S33). These experiments represent to our knowledge the largest unique-sequence multiprotein complex studied to date by HX MS.

Fig. 6 Evidence for a second UBL-specific receptor site on the Rpn1 toroid, which recognizes Ubp6.

(A) Deuteration of recombinant Rpn1 (Rpn1free) compared with deuteration of Rpn1 in the context of the base (Rpn1base). Peptide residue numbers are shown at left, as well as the Rpn1 domain organization as described in the SM. Differences at each time point were calculated using Eq. 6 (see SM) and color-coded according to the scale at the bottom. (B) Deuteration differences between Rpn1base when bound to Ubp6 minus Rpn1base without Ubp6. Peptide residue numbers are identical to those in (A), and deuteration differences are indicated by the scale at the bottom. (C) Proteasomes were purified from strains bearing wild-type or mutant Rpn1 alleles, as indicated, by using mild washes. The YY mutant is D431Y Q434Y; AAAA is L430A D431A Q434A Q435A; and AKAA is L430A D431K Q434A Q435A. Aliquots of extracts and purified proteasomes were resolved by 10% SDS-PAGE, blotted, and probed with antibodies against Rpn1, Ubp6, and Rpn12. (D) Activation of Ubp6 by wild-type and mutant RP. Ubp6 was incubated with RP purified from wild-type and rpn1-AKAA strains and assayed for Ub-AMC hydrolysis activity. Concentration of RP was constant at 1 nM, and the concentration of Ubp6 was graded. Curve-fitting, as shown, yields a Kd value of 4.7 nM for wild type. For the AKAA mutant, a concentration of Ubp6 29 times that of the wild type would be required to achieve a hydrolytic rate corresponding to half-maximal for wild type.

Having apparently localized the Ubp6 binding site to residues 419 to 436 of Rpn1, we introduced three sets of mutations into this region of the toroid and in all cases Ubp6 failed to copurify with proteasomes upon affinity purification (Fig. 6C). We refer to this site, a second and distinct UBL binding site within the Rpn1 toroid, as T2. We purified RP complexes from the T2 mutant rpn1-L430A D431K Q434A Q435A (hereafter rpn1-AKAA), and found, using the fluorogenic substrate ubiquitin-7-amino-4-methylcoumarin (ubiquitin-AMC) (34), that these mutations result in a dramatic loss of affinity for recombinant Ubp6 (Fig. 6D).

We next tested whether the T1 and T2 sites can be loaded independently and whether their specificities are truly distinct. Native gel binding assays carried out with T1 site and T2 site mutants showed that the two sites have nonoverlapping specificities (Fig. 7A). In particular Rpn1-AKAA demonstrated a specific defect in Ubp6 and not Rad23 binding, whereas Rpn1-ARR bound to Ubp6 but was defective for Rad23 binding (Fig. 7A). This experiment also mapped the T2 site interacting element of Ubp6 to its UBL domain (Fig. 7A), consistent with cryo-EM analysis of the proteasome (35, 36). The lack of effect of T1 site mutations on Ubp6 binding was confirmed with endogenous material (fig. S34). We tested whether the T1 site influences the activity of Ubp6 by using the ubiquitin-AMC assay. Neither the T1 site, nor the ubiquitin-binding sites of Rpn13 and Rpn10, influenced Ubp6-mediated deubiquitinating activity (Fig. 7B), thus demonstrating high specificity among these sites despite their common usage of UBL class domains as ligands. In summary, the Rpn1 toroid contains two proximal binding sites for ubiquitin or UBL domains, as shown in Fig. 7C, and thus Rpn1 serves as a key receptor and spatial organizer of both substrates and cofactors of the proteasome.

Fig. 7 The Rpn1 toroid spatially registers ubiquitin chains and Ubp6.

(A) Reconstituted proteasomes, prepared with RPs isolated from wild-type, rpn1-AKAA, or rpn1-ARR strains, were incubated with GST-Ubp6UBL or GST-Rad23UBL in molar excess, resolved by native PAGE, and assayed as described. (B) Ubp6 in RP complexes as indicated was evaluated for activation by the proteasome. Proteins were incubated with 1 μM Ub-AMC, and hydrolytic activity was monitored by the fluorescence of released AMC. (C) Toroidal domain of Rpn1 highlighting the T1 and T2 sites. Residues required for interaction with ubiquitin chains and Rad23 are shown in purple, and exposed residues implicated in Ubp6 binding are highlighted in orange. This image is generated by using PDB 4CR2. (D) Rpn1 T1 and T2 sites in the context of the proteasome, with sites colored as indicated in (C). This image was generated by docking the Ubp6 structure (PDB 1VJV and 1WGG) onto PDB 4CR2, on the basis of the experimental data of Fig. 6, B and C. The coloring scheme follows that of Fig. 5E and with Ubp6 in green. T2 amino acids L430, D431, Q434, and Q435 are highlighted in orange.

We used a molecular modeling program (HADDOCK 2.1) to dock the Ubp6 UBL domain against Rpn1 amino acids L430, D431, Q434, and Q435. Although the orientation of the UBL is not defined experimentally, the resulting model demonstrates the relative positioning of the T1 and T2 sites and was integrated within the overall architecture of the proteasome as determined by cryo-EM (Fig. 7D).


We report here a hotspot in the proteasome for assembling and localizing substrates and cofactors. This region is contributed by the toroid domain of Rpn1 and spans at least four of its eleven hairpin repeats. Interestingly, we were able to identify three distinct binding positions for ubiquitin-fold ligands, two of which combine to form a bifurcated binding site for a diubiquitin element, especially when linked by K48 or K6. This additional ubiquitin-binding motif in the Rpn1 toroid also binds to the UBL domain of substrate shuttling factor Rad23 and we name this substrate receptor site T1. The third UBL-binding site, which is adjacent to the T1 site, provides a tether point for deubiquitinating enzyme Ubp6. The location of the T1 and T2 sites within the proteasome places substrate ubiquitin chains and Ubp6 in the neighborhood of the ATPase hexamer, where the Ubp6 catalytic domain is known to form contacts with Rpt1 (35, 36). It is furthermore anticipated that the positioning of these sites on the same proteasome face as the ubiquitin-binding pathway generated by hRpn10 and hRpn13 allows for avidity effects that could increase proteasome affinity for ubiquitinated substrates, especially those that carry multiple ubiquitin chains. A recent single molecule study indeed demonstrates greater degradation efficiency for model substrates in which ubiquitin groups are dispersed into multiple chains (45). Like Rpn1, Rpn13 functions not only as a ubiquitin receptor but also as a receptor for Uch37, a second proteasomal deubiquitinating enzyme. Thus, both Ubp6 and Uch37 are positioned in apposition to ubiquitin receptors, and it will be interesting to explore the functional implications of this localization in future work.

The ability of the T1 site of Rpn1 to bind both ubiquitin and UBL proteins that shuttle ubiquitinated substrates to the proteasome applies to all intrinsic substrate receptor sites of the proteasome. The multiplicity of intrinsic substrate receptors on the proteasome, taken together with the combinatorial array of states associated with loading of multiple extrinsic receptors on any intrinsic receptor, should allow for tens and possibly hundreds of distinct receptor states for binding of ubiquitin conjugates. The use of multiple sites for substrate recognition, not all showing high affinity, is consistent with a multipoint, avid mode of ubiquitin chain recognition, which may be associated with highly dynamic interactions between ubiquitin receptors and the proteasome-bound substrate (45). Substrate binding is thought to be productive for proteolysis only if it results in the presentation of an unstructured initiation site into the substrate translocation channel of the ATPase ring (46). A dynamic mode of ubiquitin chain binding may allow for the body of the substrate to present alternative orientations to the proteasome so as to achieve productive positioning of the initiation site. In summary, this work defines a pathway in the proteasome that engages substrates by using avid low affinity binding sites, and places them in the neighborhood of deubiquitinating enzyme Ubp6 and the ATPase ring.

Methods summary

Detailed information for yeast strain construction, plate assays, protein turnover assays, expression and purification of recombinant and native proteins, ubiquitin conjugate preparation, native gel electrophoresis and mobility-shift assays, protein degradation and deubiquitination assays, hydrogen deuterium exchange, mass spectrometry, and NMR spectroscopy is provided in the SM.

Supplementary Materials

Materials and Methods

Figs. S1 to S34

Tables S1 to S5

References (5073)

References and Notes

Acknowledgments: For advice, assistance, or comments on the manuscript, we thank D. Chandler-Militello, M. Gill, J. Hanna, A. Kajava, R. King, Y. Lu, Y. Li, M. Pahre, and J. Roelofs. For gifts of reagents we thank D. Clarke, M. Glickman, F. He, Y. Ye, D. Komander, C. Larsen, J. Li, U. Nowicka, S. Sadis, and W. Tansey. This research was funded by grants from the National Institutes of Health (R37-GM043601 to D.F., CA136472 to K.J.W., and R01-GM101135 to J.R.E.), by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research to K.J.W., and by a research collaboration with the Waters Corporation (J.R.E). Atomic coordinates for the Rpn1 T1 site, Rpn1 T1:ubiquitin, and Rpn1 T1:K48 diubiquitin are available through the Protein Data Bank with accession codes 2n3t, 2n3u, and 2n3v/2n3w, respectively.

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