UBL domain of Usp14 and other proteins stimulatesproteasome activities and protein degradation in cellsHyoung Tae Kima and Alfred L. Goldberga,1

aDepartment of Cell Biology, Harvard Medical School, Boston, MA 02115

Contributed by Alfred L. Goldberg, October 30, 2018 (sent for review May 30, 2018; reviewed by George DeMartino and Andreas Martin)

The best-known function of ubiquitin-like (UBL) domains inproteins is to enable their binding to 26S proteasomes. Theproteasome-associated deubiquitinating enzyme Usp14/UBP6 con-tains an N-terminal UBL domain and is an important regulator ofproteasomal activity. Usp14 by itself represses multiple proteaso-mal activities but, upon binding a ubiquitin chain, Usp14 stimulatesthese activities and promotes ubiquitin-conjugate degradation. Here,we demonstrate that Usp14’s UBL domain alone mimics this activa-tion of proteasomes by ubiquitin chains. Addition of this UBL domainto purified 26S proteasomes stimulated the same activities inhibitedby Usp14: peptide entry and hydrolysis, protein-dependent ATP hy-drolysis, deubiquitination by Rpn11, and the degradation of ubiquiti-nated and nonubiquitinated proteins. Thus, the binding of Usp14’sUBL (apparently to Rpn1’s T2 site) seems to mediate the activationof proteasomes by ubiquitinated substrates. However, the stimula-tion of these various activities was greater in proteasomes lackingUsp14 than in wild-type particles and thus is a general response thatdoes not involve some displacement of Usp14. Furthermore, the UBLdomains from hHR23 and hPLIC1/UBQLN1 also stimulated peptidehydrolysis, and the expression of hHR23A’s UBL domain in HeLa cellsstimulated overall protein degradation. Therefore, many UBL-containing proteins that bind to proteasomes may also enhance allo-sterically its proteolytic activity.

proteasome activation | UBL domain | Usp14/Ubp6 | hHR23/Rad23 |hPLIC/ubiquilin

In mammalian cells, 26S proteasomes are the major site forprotein degradation (1). Most proteins digested by protea-

somes are first tagged with ubiquitin (Ub) chains. The 26S pro-teasome is composed of the 20S proteolytic core particle and oneor two 19S regulatory particles (PA700) (2). The cylindrical 20Sproteasome is a hollow four-ring particle that contains in its twocentral β-rings six proteolytic sites, two of which are chymotrypsin-like, two trypsin-like, and two caspase-like in specificity (3). Itsouter α-rings contain a central gated channel for substrate entry(4). The 19S regulatory particle performs several enzymatic andnonenzymatic functions required for the degradation of ubiquiti-nated proteins, including binding and disassembly of the Ub chain,ATP hydrolysis, and unfolding of the substrate protein (2). Thebase of the 19S regulatory particle contains a ring of six homol-ogous AAA ATPases (5, 6). The ATPases bind, unfold, andtranslocate the polypeptide through their central channel and thenthrough a gated entry channel into the 20S particle (7, 8). Manysteps in this degradation process are linked to ordered cycles ofATP binding and hydrolysis (2).Three different deubiquitinating enzymes (DUBs) are associ-

ated with the 19S regulatory particle of higher eukaryotes: Twoare cysteine proteases, Usp14/Ubp6 (9) and Uch37/UchL5 (10,11), and the third, Rpn11/PSMD14, is a metalloprotease (12, 13).Usp14 contains an N-terminal UBL domain and a C-terminalcatalytic USP (ubiquitin-specific protease) domain (14). Thedeubiquitinating activity of Usp14 is dramatically stimulated uponits association with the 19S particle, which requires the UBL do-main (15, 16). Rpn11 removes the Ub chain en bloc in an ATP-dependent manner and is essential for the degradation of ubiq-uitinated proteins (17). Thus, Rpn11 facilitates the translocation

of the polypeptide through the ATPase ring into the 20S particle(12, 18), whereas the other DUBs are thought to restrict the timesubstrates bind to the proteasome before processing (2, 8).Efficient degradation of a Ub conjugate requires a tightly in-

tegrated series of steps (2, 19) whose order and coordination arestill poorly understood. After a Ub chain reversibly binds to thereceptor subunits, the polypeptide, if it exposes a loosely foldeddomain, becomes tightly bound to the particle through an ATP-dependent reaction that commits the substrate to degradation(20). Surprisingly, the binding of a Ub conjugate to Usp14 orUch37 activates the entry of small peptides through the ATPasering and the gated channel into the 20S (21). If the ubiquitinatedprotein also contains a loosely folded domain, ATP hydrolysis isalso activated (19).This role of Usp14 in proteasome activation by Ub conjugates

was surprising, because Usp14/Ubp6 was previously shown to in-hibit the degradation of ubiquitinated proteins both through itsDUB activity and by an allosteric mechanism (22, 23). Because therapid trimming of Ub chains by Usp14 can lead to substrate re-lease without its degradation, Finley, King, and coworkers (16, 24)showed that inhibitors of Usp14’s catalytic activity (IU1) can en-hance the proteasomal degradation of certain proteins by slowingsubstrate deubiquitination. We recently demonstrated that in theabsence of ubiquitinated substrates, Usp14 allosterically inhibitsmultiple proteasomal activities, including peptide entry and hy-drolysis, ATP hydrolysis, and, surprisingly, deubiquitination byRpn11 (25). These inhibitory actions of Usp14 in the absence of a

Significance

26S proteasomes catalyze most of the protein degradation ineukaryotic cells, and their activity is precisely regulated. Uponbinding ubiquitinated proteins, proteasomes become activated.This activation is triggered by binding of the ubiquitin chain tothe proteasome-associated deubiquitinating enzyme Usp14/Ubp6. In studying this activation mechanism, we discovered thatUsp14’s ubiquitin-like (UBL) domain by itself stimulates multipleproteasome activities and thus appears to mediate the activationby ubiquitinated proteins. Many other proteins contain UBLdomains, and we show that the UBL domains of other proteinsalso stimulate the proteasomes’ degradative capacity. Thus, ac-tivation of proteasomes may represent a general new role forUBL-containing proteins. Furthermore, overexpressing the UBLdomain in cells increases overall protein breakdown, which mayhave therapeutic applications.

Author contributions: H.T.K. and A.L.G. designed research; H.T.K. performed research;H.T.K. contributed new reagents/analytic tools; H.T.K. and A.L.G. analyzed data; andH.T.K. and A.L.G. wrote the paper.

Reviewers: G.D., The University of Texas; and A.M., University of California, Berkeley.

The authors declare no conflict of interest.

Published under the PNAS license.1To whom correspondence should be addressed. Email: alfred_goldberg@hms.harvard.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1808731115/-/DCSupplemental.

Published online November 28, 2018.

E11642–E11650 | PNAS | vol. 115 | no. 50 www.pnas.org/cgi/doi/10.1073/pnas.1808731115

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substrate may help reduce wasteful ATP consumption and thedegradation of nonubiquitinated proteins (25).Recent cryoelectron microscopic studies have revealed that

the binding of Usp14 or its yeast homolog Ubp6 to the 19Scomplex induces marked changes in 19S structure (26–28). Uponbinding the transition-state inhibitor Ub-aldehyde to the active siteof Usp14/Ubp6, its C-terminal USP domain relocates near theATPase ring and Rpn11. However, without the binding of Ub-aldehyde or a Ub chain, this domain is more dynamic, and fails tointeract with the ATPases (26, 27). Presumably, these conforma-tional changes provide a mechanistic explanation of Usp14’sopposing allosteric actions, both its basal inhibition of the pro-teasome and its activation upon substrate binding (19, 21, 25).Although several roles have been suggested for the UBL

domains (29–31) present in many cell proteins, the best-documented function for the UBL domain is to provide affin-ity for the 26S proteasome, where the UBL domain can bind tothe Ub receptor subunits Rpn10, Rpn13, and Rpn1 (32–34).Among the many functions served by UBL-containing proteins isacting as shuttling factors (i.e., the UBL-UBA proteins), whichbring Ub-conjugated proteins to the proteasome, as Ub ligases(e.g., Parkin) or as proteasomal DUBs (35–37). Because of theimportance of Usp14’s UBL domain in its association with theproteasome and in the resulting stimulation of Usp14’s deubi-quitinating activity, we hypothesized that its UBL domain mightalso be important in mediating the activation of the proteasomeupon substrate binding to Usp14. Therefore, we have investi-gated whether upon binding to the 26S proteasome, Usp14’sUBL domain by itself activates these various proteasomal activi-ties. We demonstrate here that Usp14’s UBL domain allostericallyactivates the same proteasomal functions that are activated uponUb-chain binding to the proteasome and thus enhances proteindegradation by the UPS. These findings imply that Usp14’s UBLdomain mediates the activation by Ub conjugates. However, be-cause an even stronger activation was found in particles lackingUsp14, and because the UBL domains of other proteins were alsoshown to stimulate 26S activity, these findings also strongly suggesta new general role for UBL domains: that they not only promotethe binding of proteins to the proteasome, but also activate theproteasome for protein degradation.

ResultsThe UBL Domain of Usp14 Enhances Peptide Hydrolysis by 26SProteasomes. To dissect the roles of Usp14’s UBL and USP do-mains in the regulation of proteasome function, we tested whetherbinding of the UBL domain alone can influence its several enzy-matic activities. When the purified GST-tagged UBL domain fromUsp14 (residues 2 to 79) was incubated in the presence of ATPwith 26S proteasomes purified from mouse embryonic fibroblasts(MEFs) lacking Usp14 (Usp14KO), it stimulated the protea-somes’ chymotrypsin-like activity in a similar fashion to a ubiquitinchain (hexaubiquitin) in the presence of Usp14. The hexa-Ubcaused a larger stimulation with the enzymatically inactiveC114A mutant Usp14 than with WT enzyme. By contrast, addingeither WT Usp14 or the inactive C114A mutant without a sub-strate inhibited 26S peptidase activity, as reported previously(25). Thus, these regulatory effects are not related to Usp14’sDUB activity. Unlike the UBL domain, hexa-Ub then had onlylittle effect in the absence of Usp14, and this effect was probablythrough its binding to Uch37 (19) (Fig. 1A).This stimulation by the UBL domain increased with higher

concentration and was consistently about twofold larger inUsp14KO proteasomes than in WT 26S (Fig. 1B). The greaterenhancement of 26S activity in the absence of Usp14 contrastswith the activation by Ub conjugates, which in yeast 26S requiresUbp6 (21) and in mammalian proteasomes requires Usp14 orUch37 (Fig. 1A) (19). By itself, the purified UBL domain had nopeptidase activity (SI Appendix, Fig. S1A) and did not stimulate

peptide hydrolysis by 20S proteasomes without the 19S regula-tory particle present (SI Appendix, Fig. S1B). These observationsare not due to the linkage to GST, a dimeric protein, because asimilar stimulation of the proteasomes’ chymotrypsin-like activitywas observed upon addition of the monomeric His6-tagged UBLdomain (SI Appendix, Fig. S1C). In addition, the UBL increasedthe trypsin-like and especially the caspase-like activities of the26S, and these effects were also consistently larger in theUsp14KO 26S (Fig. 1 C and D). This enhanced hydrolysis by allthree types of active sites is most likely due to greater substrateentry through the ATPase ring and gated entry channel into the20S particle (i.e., “gate opening”), rather than by allosteric ac-tivation of the individual catalytic sites (4, 38).This simultaneous large enhancement of all three peptidase

activities resembles the stimulatory effects of PA28αβ and nu-cleotide binding, especially ATPγS, which enhances peptideentry into the 20S particle (39). However, the UBL domainstimulated peptide hydrolysis even further in the presence ofATPγS (Fig. 1D), which had been assumed to cause maximalgate opening and rates of peptide hydrolysis (SI Appendix, Fig.S1D). By contrast, the addition of full-length Usp14 to 26Sproteasomes reduced the stimulation of peptide hydrolysis byATPγS (25). The UBL domain in the presence of ATPγS sur-prisingly caused a much greater stimulation of the Usp14-deficient 26S than of the WT 26S (Fig. 1D). Thus, ATPγS andUsp14’s UBL domain appear to stimulate synergistically peptidehydrolysis by proteasomes, which implies that the stimulation bythe UBL domain requires the conformation of the 19S particleinduced by ATPγS (40).

Usp14’s UBL Domain Binds to the T2 Site of Rpn1 to Activate the 26S.Because the Usp14 UBL had no effect on the peptidase activityof the 20S core particles (SI Appendix, Fig. S1B), its activatingmechanism must differ from that of PA28 or that of theATPase’s C-terminal HbYX motif, which binds to 20S’s α-ring(4, 41). More likely, the UBL stimulates by binding to the samesite on Rpn1 as does full-length Usp14 (32, 42), and thus it doesnot directly affect the 20S gating mechanism. Accordingly, thestimulation of peptide hydrolysis was much smaller in the WTparticles than in the 26S lacking Usp14, probably because in theWT particle the Usp14 and the free UBL bind to the same site,which reduces the stimulation by the UBL domain (Fig. 1 C andD). Accordingly, modification of Usp14 with the covalent in-hibitor Ub-vinyl sulfone, which enhances its association with the26S particle (27), partially inhibits the stimulation of the chymotrypsin-like activity by the added UBL (SI Appendix, Fig. S2). To determine ifin activating peptide hydrolysis the UBL binds to the same site as isoccupied by Usp14, we tested whether adding full-length Usp14competitively inhibits the stimulation of gate opening by the UBL. Aspredicted, incubation of the Usp14KO 26S with increasingamounts of full-length WT Usp14 or its C114A active-site mutanttogether with the UBL domain reduced the stimulation of thechymotrypsin-like activity by the UBL (Fig. 1E). Furthermore,when Usp14KO proteasomes were first bound to a GST-UBLresin, they could be eluted by addition of increasing amounts ofthe full-length WT Usp14 or the inactive C114A mutant (Fig. 1F).This activation thus is not related to Usp14’s DUB activity, whichis located in its USP domain. The findings that Usp14 competeswith GST-UBL for binding to the proteasome and inhibits thestimulatory effect of UBL suggest that the binding of a Ub chainor Ub-aldehyde to Usp14 allosterically stimulates peptide entry(21) through its UBL domain, while its inhibitory USP domainprevents this stimulation by the UBL until a Ub chain binds to theUSP domain.Recently, it was shown that yeast Rpn1 has two separate Ub

binding sites, T1 and T2 (32). T1 is the binding site for Ub andthe UBL domain of Rad23, while T2 is the site for binding ofUbp6’s UBL domain (32) and therefore presumably mediates

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Fig. 1. By binding to 26S proteasomes, Usp14’s UBL domain stimulates peptide cleavage by all three peptidase sites, especially in proteasomes lacking Usp14.Activities of WT and Usp14KO 26S were assayed by measuring cleavage of the fluorogenic peptide-amc substrates (10 μM) in the presence of ATP or ATPγS(100 μM). Control (100%) indicates the peptidase activity of each type of proteasome (1 nM) without added proteins. (A) Addition of GST-UBL from Usp14(200 nM) by itself stimulates the chymotrypsin-like activity of 26S similar to addition of Usp14 (1 μM) plus a linear hexa-Ub chain (200 nM). Addition of WTUsp14 or the enzymatically inactive C114A mutant by themselves inhibited, but together with hexa-Ub stimulated, peptidase activity, especially with theinactive Usp14 mutant. LLVY-amc: Suc-Leu-Leu-Val-Tyr-amc as the substrate for the chymotrypsin-like activity. *P < 0.05 and **P < 0.01 by Student’s t test.Data are the means ± SD; n = 4. (B) The UBL of Usp14 stimulates the chymotrypsin-like activity of both WT and Usp14KO 26S. The UBL seems to bind withsimilar affinities (∼100 nM) to both, but causes a much larger stimulation of the Usp14KO 26S. Data are the means ± SD; n = 5. (C) All three peptidase activitiesare stimulated more by the UBL (200 nM) in the Usp14KO 26S than in the WT with ATP present. LRR-amc: Boc-Leu-Arg-Arg-amc as the substrate for thetrypsin-like activity; nLPnLD-amc: Ac-Nle-Pro-Nle-Asp-amc as the substrate for the caspase-like activity. *P < 0.05 and **P < 0.01 by Student’s t test. Data arethe means ± SD; n = 6. (D) In the presence of ATPγS, the UBL domain further enhanced (five- to eightfold) the three peptidase activities of Usp14KO 26S.Peptide hydrolysis by WT 26S was also stimulated, but to a smaller extent. *P < 0.05 and **P < 0.01 by Student’s t test. Data are the means ± SD; n = 6. (E) Full-length Usp14 inhibits the activation of peptide hydrolysis by UBL. Addition of increasing concentrations of WT Usp14 or the inactive C114A mutant decreasedthe chymotrypsin-like activity of 26S proteasomes in the presence of UBL and ATPγS. Usp14KO proteasomes (1 nM) were preincubated with the UBL (100 nM),and different concentrations of Usp14 were then added to the mixture. Data are the means ± SD; n = 4. (F) Full-length Usp14 competes with the GST-UBL forbinding to the 26S proteasome. Usp14KO 26S bound to UBL on resin was eluted by adding increasing concentrations of Usp14 (55). Proteasomes eluted werequantitated using LLVY-amc hydrolysis (20). Data are represented as means ± SD; n = 6. (G) The UBL domain from Usp14 stimulates the chymotrypsin-likeactivity of yeast WT and T1 mutant (ARR) 26S but not of the T2 mutant (AKAA) (32). Activities of the resin-bound yeast 26S were measured after adding theUBL derived from mammalian Usp14 (200 nM). Data are the means ± SD; n = 3.

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this activation. We therefore tested whether T1 or T2 may beimportant in the activation of peptide hydrolysis. Upon in-cubation with 26S proteasomes purified from yeast bearingmutations that inactivate the T1 or the T2 site (32), the UBL ofUsp14 stimulated the chymotrypsin-like activity of purified 26Sproteasomes of both WT (SY1214) and the T1-mutant strain(SY1210, rpn1-ARR), but not those from the T2-mutant strain(SY1724, rpn1-AKAA) (Fig. 1G). Although the absolute stim-ulation was much smaller in the yeast particles than mammalian26S, presumably because of our use of a mammalian UBL do-main, these findings indicate that binding to the T2 site, but notthe T1 site, is required for this stimulation by Usp14’s UBL.

Usp14’s UBL Domain Together with a Loosely Folded Protein EnhancesATP Hydrolysis. A general feature of hexameric AAA ATP-dependent proteases is that their ATPase activity is stimulatedupon binding a loosely folded protein substrate (43–45). How-ever, the stimulation of the proteasomal ATPases requires thesimultaneous binding of a Ub chain to Usp14 (or to Uch37) andan unstructured region of the polypeptide to the 19S ATPases,although the Ub chain and polypeptide need not be covalentlylinked (19). Thus, as we showed recently (25), Usp14 repressesproteasomal ATP hydrolysis, but upon binding of a Ub chain toUsp14 the ATPases are activated, provided an unfolded poly-peptide is also present (2).Therefore, we tested whether the Usp14’s UBL domain can

also stimulate the ATPase activity of the 26S as it stimulatespeptidase activities. Although the UBL alone had no effect, the

UBL with β-casein present stimulated ATP hydrolysis in MEFproteasomes (Fig. 2A) and did so to a similar extent as hexamericUb (Fig. 2A). Thus, stimulation of the 19S ATPases by the UBLdoes not require Ub-chain binding but still requires the bindingof a loosely folded protein, probably to ATPases (2). Thus, theUBL domain by itself mimics the ability of substrate-boundUsp14 to activate both peptide and ATP hydrolysis. Thesefindings further suggest that the UBL domain mediates this ac-tivation by substrate, and therefore that Usp14’s catalytic (USP)domain is probably responsible for the basal inhibition of theseactivities until it binds a Ub chain and interacts with ATPases(26–28). By contrast, in 26S lacking Usp14, the binding of caseinalone fully stimulated ATP hydrolysis (25), and the addition ofUBL had no further effect (Fig. 2A, Right). Interestingly, asimilar stimulation of the ATPases by a nonubiquitinated poly-peptide has been observed in reconstituted yeast proteasomes(27). Such behavior is clearly not seen in our preparations fromWT mammalian cells (2, 19), where the presence of Usp14makes the activation dependent on a ubiquitinated protein or aprotein plus a ubiquitin chain.

Usp14’s UBL Domain Stimulates the Degradation of a NonubiquitinatedUnfolded Protein. Because the Usp14 UBL domain stimulates gateopening and ATP hydrolysis, both of which are essential for effi-cient proteolysis (2), the UBL might also stimulate the proteaso-mal degradation of some nonubiquitinated proteins. In fact, theaddition of either hexa-Ub chains or the UBL alone to WT 26Sstimulated dramatically (up to 10-fold) the breakdown of the

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intrinsically disordered protein Sic1 (46) (Fig. 2B). However, inproteasomes lacking Usp14 (SI Appendix, Fig. S3A), the addi-tion of the UBL did not stimulate further the degradation ofSic1, probably because this process is already activated in theseparticles (25), as is the rate of ATP hydrolysis (25), which de-termines the rate of protein degradation (47). Another note-worthy feature of the Usp14-deficient proteasomes is that theactivation of ATP hydrolysis requires only the presence of aloosely folded protein and does not also require a Ub chain(Fig. 2A, Right). These findings provide further evidence thatUsp14 inhibits Ub-independent proteolysis until binding of a Ubchain reverses this inhibition, which seems to occur via Usp14’sUBL domain.

Usp14’s UBL Domain Stimulates Deubiquitination by Rpn11. Uponbinding a Ub chain, Usp14’s USP domain moves into proximitywith Rpn11 and sterically hinders Rpn11 (27), whose activity andaccessibility to substrates depend on ATP hydrolysis (18). Sur-prisingly, even though Usp14KO proteasomes lack an importantDUB, they have a much greater capacity to disassemble Ubchains than WT due to a stimulation of Rpn11’s activity (25). Tolearn if the UBL domain may also influence Rpn11 activity, westudied the effects of GST-UBL on the hydrolysis of tetra-Ubchains. The UBL stimulated the breakdown of both K48 and K63Ub chains by both the WT and Usp14KO 26S proteasomes (Fig.3A). The UBL even increased the proteasomes’ ability to hydro-lyze tetra-Ub (Fig. 3A) in the presence of ATPγS, which by itselfenhances Rpn11 activity (25). Accordingly, this stimulation oftetra-Ub breakdown by the UBL was blocked by an inhibitor ofRpn11, o-phenanthroline, but not by the inhibitor of cysteineDUBS, Ub-VS (Fig. 3B). In addition, the Usp14KO proteasomesdisassembled another DUB substrate, K63 FRET di-Ub, fasterupon UBL addition (SI Appendix, Fig. S3B). Thus, Usp14’s UBLdomain allosterically stimulates Rpn11’s catalytic activity, while itsUSP domain upon binding a Ub chain limits Rpn11 to handlingonly one Ub chain at a time by preventing Rpn11 from interactingwith a new substrate (27). Because Rpn11-mediated release of theUb chain is essential for substrate translocation into the 20S (18),this activation of Rpn11 is likely to have a major effect in en-hancing the degradation of ubiquitinated proteins.

The UBL Domain of Usp14 Stimulates Ub-Dependent Degradation.Because the Usp14 UBL domain stimulates several steps criti-cal for proteasomal degradation of ubiquitinated proteins (i.e.,Rpn11, gate opening and protein-activated ATP hydrolysis), wetested whether the UBL domain also enhances the degradationof a Ub conjugate, Ub5-DHFR (Fig. 3C), in which an N-terminalUb fusion of dihydrofolate reductase (DHFR) is conjugated to aK48-linked tetra-Ub chain (48). 26S proteasomes digest thisubiquitinated protein and disassemble its Ub chain to monomericUbs (Fig. 3C, lane 3). However, with Ub-aldehyde present to in-hibit Usp14 and Uch37, unattached tetra-Ub chains were gener-ated (Fig. 3C, lane 4). Because the Ub chain was not digestedwhen o-phenanthroline was present (Fig. 3C, lanes 5 and 6),Usp14 and Uch37 surprisingly are unable to deubiquitinate thissubstrate until Rpn11 first cleaves off the tetra-Ub from theDHFR [perhaps because of Usp14’s preference for multiplyubiquitinated proteins (24)] (illustrated in Fig. 3D). Thus, thedeubiquitination of Ub5-DHFR requires Rpn11 activity, as doesthe degradation of DHFR (Fig. 3E, lane 4). Even though WT 26Shydrolyzed Ub5-DHFR rapidly, as predicted, the addition of theUBL domain further enhanced its degradation (Fig. 3E).

The UBL Domains of Other UBL-UBA Proteins Can Also Stimulate 26SPeptidase Activity. Like Usp14/Ubp6, a variety of other proteinsalso contain a UBL domain, such as the shuttling factorshHR23A and B, which bind to Ub conjugates through their UBAdomains and also to proteasomes through their UBL domains

(SI Appendix, Fig. S4). To determine whether the UBL domainsof these other proteins can also stimulate proteasomal activities,we assayed peptide hydrolysis by Usp14KO 26S proteasomeswith or without the GST-UBL of hHR23B and hPLIC1/UBQLN1 (Fig. 4A). Like the UBL of Usp14, hHR23B’s UBLenhanced all three peptidase activities of Usp14KO 26S three- to

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Fig. 3. UBL domain stimulates both Ub-chain disassembly by Rpn11 and thedegradation of Ub5-DHFR. (A) UBL increases the breakdown of K48 andK63 tetra-Ub chains (368 nM) by MEF 26S with ATP or ATPγS present. 26Sproteasomes (5 nM) were incubated with UBL as indicated at room tem-perature for 15 min before the reaction, which was carried out at 37 °C for20 min. The generation of tri, di-, and mono-Ubs was analyzed by Westernblotting as previously reported (25). Similar data were obtained in threeindependent experiments. (B) The UBL stimulates breakdown of K48 tetra-Ub by WT 26S with ATP present, and this stimulation must involveRpn11 because it was inhibited by o-phenanthroline (OPT; 1 mM) but not byUb-vinyl sulfone (Ub-VS; 200 nM). Similar data were obtained in two in-dependent experiments. (C) 26S proteasomes disassemble the Ub chain onubiquitinated DHFR to generate monomeric Ubs. Upon inhibition ofUsp14 and Uch37 by Ub-aldehyde (300 nM), tetra-Ub chains are dis-assembled by Rpn11, and the Rpn11 inhibitor OPT (1 mM) blocked com-pletely deubiquitination of DHFR. Degradation of Ub5-DHFR (100 nM) by 26Sproteasomes (2 nM) in 20 min was assayed by Western blotting. The asteriskindicates nonspecific signal from Ub-aldehyde. (D) Illustration of why deg-radation of Ub5-DHFR is dependent on Rpn11. Neither Usp14 nor Uch37 cancatalyze disassembly of the tetra-Ub chain. Only after Rpn11 cleaves tet-raubiquitin chains en bloc off the protein can these other deubiquitinatingenzymes disassemble the chain. Therefore, degradation of Ub5-DHFR de-pends on Rpn11 activity. (E) Although WT 26S degrades Ub5-DHFR rapidly,the addition of UBL (500 nM) enhances this process further. However, in-hibition of Rpn11 activity by OPT (1 mM) completely blocks degradation.Similar data were obtained in three independent experiments.

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fivefold. The UBL of hPLIC1 also increased these activities, butto a much smaller extent (1.5- to 2-fold) than hHR23B’s UBL. Aphylogenetic analysis of UBL domains indicated that the primarysequences of UBLs from hHR23A and B and their yeast ho-molog Rad23 are evolutionarily close to those of Usp14 andUbp6 (SI Appendix, Fig. S4). By contrast, the UBL of hPLIC1 ismore closely related to Ub, which has no stimulatory effect (21).These findings strongly suggest that proteasomal activation is acommon property of the UBL domains found in many proteinsthat bind to the 26S.

Expression of the UBL Domain in HeLa Cells Stimulates ProteinDegradation. Because the UBL domain of Usp14 can stimulateseveral proteasome activities that are essential for Ub-conjugatedegradation and can promote the hydrolysis of a model ubiq-uitinated substrate, we tested whether expression of a UBL do-main in cultured cells can increase the degradation of cellularproteins generally. HeLa cells were transfected with pEGFP-UBLhHR23A (Fig. 4B), and the rates of degradation of long-livedproteins, which comprise the bulk of cell proteins, were mea-sured (Fig. 4C). To follow overall rates of proteolysis, we initiallyradiolabeled most cell proteins by exposure to [3H]phenylalaninefor 24 h and followed the subsequent degradation of radiola-beled proteins by measuring their conversion to TCA-solublematerial in the presence of a large excess of nonradioactivephenylalanine to prevent reincorporation of radiolabeled aminoacids (49). The HeLa cells expressing EGFP-UBLhHR23A de-graded endogenous proteins 60% more rapidly than control cellsexpressing EGFP (Fig. 4C). Most likely, this enhanced pro-teolysis occurred as a consequence of the enhanced proteasomeactivity, because the cells expressing EGFP-UBL also had amuch lower level of Ub conjugates (Fig. 4D). This enhancementof overall degradation is surprising, since overexpression of GST-UBL might be expected to inhibit many important UBL-dependent interactions in the cells, such as the binding of theshuttling factors to the proteasome. In fact, when HeLa cellswere transfected with a greater amount of the pEGFP-UBLhHR23A plasmid to increase further UBL levels, the degra-dation of long-lived cellular proteins was inhibited (SI Appendix,Fig. S5). These findings further indicate that binding of UBL toproteasomes enhances their capacity to degrade ubiquitinatedsubstrates in vivo, as was seen in vitro.

DiscussionUsp14’s UBL Domain Is an Allosteric Stimulator of Multiple ProteasomalActivities.Despite its catalytic and regulatory importance, Usp14 isfound on only a small fraction of 26S proteasomes in vivo, ap-parently on those particles actively involved in degradation ofubiquitinated proteins (50). When proteasomes bind ubiquitinconjugates, their association with Usp14 increases, and hydrolysisof the conjugates promotes dissociation of the Usp14. The bindingof Usp14 to the proteasome has been reported to dramaticallystimulate its catalytic activity and to require its UBL domain (16).However, addition of the UBL domain does not cause dissociationof Usp14 copurified with the proteasome. By helping remove Ubchains from the protein bound to the proteasome, Usp14/Ubp6 decreases the wasteful degradation of Ub by the proteasomeand promotes Ub recycling (51). However, Usp14 can both allo-sterically inhibit and activate multiple steps in Ub-conjugatedegradation (19, 21, 25). We showed here that Usp14’s UBLdomain by itself allosterically stimulates the same proteasomeactivities as are inhibited by Usp14 in the absence of a substrate

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Fig. 4. UBL domains of other UBL-UBA proteins, hHR23 and hPLIC1, alsostimulate peptide hydrolysis by 26S proteasomes, and expression of EGFP-UBL increases the degradation of long-lived proteins in HeLa cells. (A) WithATPγS (100 μM) and Usp14KO 26S (1 nM), the UBL domain (500 nM) ofhHR23B stimulates proteasomes’ three peptidase activities three- to fivefold,while the UBL domain of hPLIC1 stimulates them twofold or less. Peptidaseactivities were measured as in Fig. 1. *P < 0.05 and **P < 0.01 by Student’st test. Data are the means ± SD; n = 6. (B) EGFP and EGFP-UBL derived fromhHR23A were expressed in HeLa cells (48 h after transfection). (C) HeLa cellsexpressing EGFP-UBL degrade long-lived proteins faster than control cells.Proteins were labeled with [3H]phenylalanine (5 μCi/mL) for 24 h. After 1 h inchase medium (DMEM containing 2 mg/mL nonradioactive phenylalanine) toallow short-lived proteins to be degraded, the breakdown of cellular pro-teins was measured in fresh chase medium for up to 3 h. Data are themeans ± SD; n = 3. (D) The content of ubiquitin conjugates in HeLa cell ly-sates was lower after expression of EGFP-UBL than after EGFP. Forty-eighthours after transfection, equal amounts of proteins (2 μg) in lysates wereelectrophoresed and then Western blotted using anti-Ub antibody. Similarresults were obtained from two independent experiments. (E) Summary ofUsp14’s allosteric regulation of proteasomal degradation of ubiquitinatedproteins. Without a Ub-conjugate bound, Usp14 (through its USP domain)inhibits ATP hydrolysis, substrate entry into the 20S, and deubiquitination byRpn11 (25). However, upon binding Ub chains, Usp14 undergoes majorstructural transitions (2), and its UBL domain activates peptide entry into20S, Rpn11, and ATP hydrolysis, if an unfolded protein also binds. Theseactions promote efficient degradation of ubiquitinated substrates. This ac-tivated state should be maintained as long as Usp14 binds the Ub chain.These processes return to the quiescent basal state when the substrate isdegraded or is deubiquitinated and dissociates. These allosteric actions arenot dependent on Usp14’s catalytic activity, which functions to degrade the

Ub chain and thus limits the duration of the active state (25). Also shown isthe proposed activation of proteasomes by UBL-containing proteins, whichshould be independent of the presence of Usp14.

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and are activated when a Ub chain binds to Usp14: peptide hy-drolysis, ATPase activity, degradation of nonubiquitinated andubiquitinated proteins, and Rpn11 activity. The coordinated in-crease in the three peptidase activities strongly suggests that theassociation of the UBL domain enhances the entry of substratepeptides into the 20S chamber (Fig. 1), rather than causing anallosteric stimulation of the six peptidase sites. It has long beenassumed that ATPγS opens this gate maximally (39). However, theUBL domain stimulates peptide hydrolysis most effectively in thepresence of ATPγS (Fig. 1D). This nonhydrolyzed nucleotidecauses not only 20S’s gate opening by promoting the docking ofthe ATPases’ C-terminal HbYX motifs into the intersubunitpockets in the 20S’s outer ring (4), but also by triggering the re-organization of the central pore in the ATPase ring, leading to itsenlargement and coaxial alignment with the 20S gate (40). Asimilar structural reorganization occurs during degradation of aubiquitinated substrate (7). The present findings may suggest thatthis entry channel is much more accessible to substrate trans-location when a UBL domain and the nonhydrolyzable nucleotideare present together than with either alone. Such a large increase(five- to eightfold) in the substrate entry channel should be clearlyevident by cryoEM.Alternatively, this large increase in peptidase activity when a

UBL is present with ATPγS may occur through the activation ofa distinct population of the proteasomes that are otherwise notengaged in degradation. Even purified proteasome preparationsare heterogeneous [e.g., only a small fraction contain Usp14 orUbe3C (50)]. Cryoelectron microscopy of isolated neurons in-dicated that most cellular 26S proteasomes are not in a substrate-engaged conformation (52). Possibly the UBL domain in vitro orwhen expressed in vivo induces a more active conformation inthis population.During the degradation of a ubiquitinated protein, Rpn11 is

located above the ATPase channel (7) and cleaves primarilybetween the proximal Ub and the substrate to release the Ubchain (12). It thus facilitates translocation of the protein sub-strate through the ATPases into the 20S particle (18). Theseactions of Rpn11 are all stimulated by ATPγS (25) but, even inthe presence of ATPγS, Usp14’s UBL domain stimulates furtherRpn11’s capacity to disassemble Ub chains. This stimulation wasobserved in the absence of Usp14’s catalytic USP domain, whichnormally inhibits Rpn11 sterically (27) upon binding a Ub chain.These observations thus reveal an unexpected coordination be-tween the actions of these two 26S DUBs (Fig. 3A). Because thestimulation of peptide hydrolysis and of Ub-chain disassembly bythe UBL are additive with the effects of ATPγS, the UBL do-main probably promotes these activities by a mechanism distinctfrom that of nucleotide binding. However, the cryoEM analysesthus far have focused on defining the large changes that occursimilarly upon binding of ATPγS, Ub-aldehyde, or a Ub-conjugated substrate and involve steric inhibition of Rpn11 byUsp14 (7, 26, 27, 40), but they have not as yet exposed largeadditive effects, as demonstrated here by biochemical assays(Figs. 1D and 3A).Proteasome activation upon binding a Ub conjugate is a

multistep process (2) involving sequential recognition of theubiquitin chain and then the protein substrate (2, 19). After theinitial binding of the Ub chain to Usp14, the 19S ATPases be-come activated and commit the protein for degradation (2, 19).With casein present, the UBL alone was found to stimulate ATPhydrolysis by the proteasomes (Fig. 2A). Thus, the UBL providesa very similar positive allosteric signal to the ATPases as doesUsp14 upon binding a Ub chain or Ub-aldehyde (21).Like a Ub chain, the UBL does not by itself cause ATPase

activation, which still requires the presence of a polypeptide witha loosely folded domain. Most likely this domain interacts di-rectly with the proteasomal AAA ATPases to activate them, asoccurs with the homologous bacterial and archaeal ATP-

dependent proteases (2). Like these AAA ATPases, in protea-somes lacking Usp14 (unlike the wild-type particles), casein byitself can stimulate ATP hydrolysis without a Ub chain, Ub-aldehyde, or UBL domain present. Interestingly, in the yeast26S reconstituted in vitro, a nonubiquitinated protein substratecould activate ATP hydrolysis by itself (27). Such behavior doesnot correlate with and cannot account for the requirement forboth a ubiquitin chain and a loosely folded domain for proteindegradation by proteasomes in vivo (53).Together, the present observations make it very likely that the

proteasome activation upon substrate binding to Usp14 is me-diated by its UBL domain (Fig. 4E). Accordingly, adding WT orcatalytically inactive Usp14 reduces the ability of the exogenousUBL to stimulate substrate entry and to bind to the 26S (Fig. 1E),apparently by competing for the same binding site on Rpn1 (Fig.1F) (42, 54). Despite this competition for the same site, UBL doesnot act by displacing Usp14 from the particles, since incubation ofpurified 26S with a high concentration of GST-UBL fromUsp14 did not dissociate the bound Usp14 from the proteasome(SI Appendix, Fig. S6). Also, our method for purification of theproteasomes uses GST-UBL from Usp14 or hHR23B as an af-finity ligand (55) but fails to dislodge Usp14 from the protea-somes, and before and after this step about 20 to 30% of theparticles contain Usp14 (25, 50). This tight binding of inhibitoryUsp14 to the 26S can explain why the stimulation of differentactivities by the added UBL is consistently greater in proteasomeslacking Usp14 than in the WT. Consequently, the stimulation ofoverall proteolysis in cells by expression of UBL is likely to involvethese Usp14-deficient particles.We cannot exclude an alternative possibility that binding of a

Ub chain to Usp14’s inhibitory USP domain might also con-tribute to the increased ATP and peptide hydrolysis by tempo-rarily reversing the basal inhibition of these processes (27).However, based on the present evidence, we favor a singlecommon mechanism for UBL’s actions on proteasomes lackingUsp14 and those where Usp14’s UBL domain mediates the ac-tivation by ubiquitinated substrates.Most likely, the UBL domain activates substrate entry into the

20S, ATP hydrolysis, and Rpn11 by inducing conformationalchanges in Rpn1, since Usp14 interacts with Rpn1 through itsUBL domain (26, 32, 42). Indeed, Usp14’s UBL failed to en-hance peptidase activity of yeast 26S having an AKAA mutationin Rpn1’s T2 site where the UBL of Ubp6 binds (32) but acti-vated normally in the 26S mutated in the T1 binding site (Fig.1G). Because these three proteasomal functions activated by theUBL are catalyzed by subunits located at some distance fromUsp14, Rpn1 must mediate these allosteric actions since it in-teracts with both the ATPases and the UBL domain (54). Fur-thermore, the same activities are all dependent on the sixATPase subunits, which function coordinately in proteolysis andin 20S gating (56). Therefore, the Rpn1-mediated enhancementof ATPase activity may be responsible for both the activation ofsubstrate entry and Rpn11.In addition to UBL’s activating role, the full-length Usp14 has

inhibitory functions through its USP domain’s interactions withthe particle that are not shared by the UBL domain (25). Thisallosteric inhibition observed with proteasomes in vitro accountsfor the greater degradation of cell proteins in Usp14KO MEFcells than in WT (25). Interestingly, the UBL increased thedegradation by proteasomes of both Ub5-DHFR (Fig. 3E) andthe nonubiquitinated protein Sic1 (Fig. 2B). The enhancement ofoverall proteolysis by expression of EGFP-UBL in HeLa cells(Fig. 4C) most likely results from proteasome activation, since itwas accompanied by a fall in Ub-conjugate levels. This stimula-tion of proteolysis is particularly striking, because overexpressionof UBL would be expected to compete with the binding ofshuttling factors to the 26S and to inhibit proteolysis, as we ac-tually observed with higher expression levels.

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UBL Domains in Other Proteins Can Also Stimulate ProteasomalActivity. Like the UBL domain of Usp14, the UBL domains ofhHR23 and hPLIC1 also stimulated proteasomal peptidase ac-tivities, although to different extents. The UBL of hHR23B hadas high a stimulatory effect as that of Usp14, while that ofhPLIC1 stimulated to a lesser extent (Fig. 4A). Interestingly,phylogenetic analysis indicated that UBLs of hHR23A/B orRad23 are evolutionarily closer to that of Usp14 (SI Appendix,Fig. S4). But the UBL of hPLIC1 resembles more closely thestructure of Ub, which has no stimulatory effect as a freemonomer (21). So, in addition to serving as a “shuttling factor”that delivers Ub conjugates to the proteasome, hHR23 may alsoactivate proteasomes through its UBL domain, functioning in asimilar fashion as Usp14’s upon substrate binding. Such an ac-tivation of the proteasome upon substrate delivery by hHR23might be particularly important in activating the majority ofcellular 26S particles, which lack Usp14 (26, 50).These findings further suggest that stimulation of proteasomal

activities might be a common property shared by the broad rangeof proteins containing UBL domains. For example, shuttlingfactors such as hHR23 and hPLIC1 may not only help 26Sproteasomes bind Ub conjugates but also may help activate theproteasome for efficient degradation of substrates. Besides theseshuttling factors, other proteins containing a UBL domain havebeen known to serve various catalytic functions, such as Ub li-gation by Parkin (57), dephosphorylation by UBLCP1 (58), andproteolysis by Ddi2 (59). In fact, in our ongoing studies, we haveobtained clear evidence that several such UBL-containing pro-teins also have the capacity to stimulate proteasome activities.

Materials and MethodsPurification of Proteins. GST-UBL with the UBL domain derived from Usp14(amino acids 2 to 79), hHR23A (amino acids 2 to 82), hHR23B (amino acids 2 to82), or hPLIC1 (amino acids 38 to 111) was expressed in Escherichia coli andpurified with GSH-Sepharose as described previously (55). GST-fused Usp14(WT and active-site C114A mutant) was also expressed in E. coli and purifiedusing GSH-Sepharose as described (16). Resin-bound Usp14 was treated withthrombin to cut off GST from Usp14. Residual thrombin was cleared fromUsp14 using benzamidine-Sepharose. His-UIM derived from the UIM2 do-main of S5a (55), His-tagged PY-Sic1 (60), and His-tagged Usp14 UBL wasexpressed in E. coli and purified with Ni-NTA resin. Ub5-DHFR was a kind giftfrom Takeda Pharmaceuticals.

Purification of 26S Proteasomes. WT and Usp14KO mouse embryonic fibro-blasts (16) grown in DMEM (supplemented with 10% FBS and 1% penicillin/streptomycin) were pooled to affinity purify 26S proteasomes using themethod previously described (61). Briefly, cell lysates prepared with sonica-tion (15 s six times at 18 W) were spun for 1 h at 100,000 × g. The solubleextracts were incubated at 4 °C with GST-UBL derived from hHR23B and acorresponding amount of GSH-Sepharose. The slurry containing 26S pro-teasomes bound to GST-UBL was poured into an empty column and washed,followed by incubation with His-UIM. The eluate was collected and in-cubated with Ni-NTA agarose for 20 min at 4 °C. The Ni-NTA–bound His-UIMwas spun out by centrifugation at 3,000 × g, and the remaining supernatantcontained purified 26S proteasomes. Protein concentration was determinedusing Bradford reagent. Molarity of 26S proteasome particles was calculatedbased on a molecular mass of a doubly capped 26S particle of 2.5 MDa.

Yeast 26S proteasomes were purified as previously described (32) withmodification from SY1214 (Mata ProA-TEV-Rpt1::HIS3 rpn13-pru::natMXrpn10-uim::kanMX), SY1210 (MATa ProA-TEV-Rpt1::HIS3 rpn1-ARR::TRP1 rpn13-pru::natMX rpn10-uim::kanMX), and SY1724 (MATa ProA-TEV-Rpt1::HIS3 rpn1-AKAA::TRP1 rpn13-pru::natMX rpn10-uim::kanMX). 26S

proteasomes were purified using IgG resin (Life Technologies) and wereused for peptidase assays without elution.

Antibodies. Polyclonal rabbit anti-Ub (A-100) was from Boston Biochem, andanti-Sic1 (sc-50441) was from Santa Cruz Biotechnology. Monoclonal mouseanti-DHFR (sc-74594) was from Santa Cruz Biotechnology. HRP-conjugatedsecondary antibodies were from Promega.

Immunoblot Analysis. Samples for the immunoblots were run on 4 to 12% Bis-Tris gels (WG1403BOX10; Life Technologies) with Mes buffer (NP0002).Proteins were analyzed following SDS/PAGE and transferred onto 0.45-μmPVDF membranes (Whatman). Immunoblots were blocked and incubatedwith appropriate primary and secondary antibodies. Membranes were de-veloped with enhanced chemiluminescence reagent (Immobilon WesternHRP substrate and Luminol reagent WBKLS0500; Millipore) onto X-ray film.The ImageJ program (NIH; https://imagej.nih.gov/ij) was utilized to quantifythe signal from the film.

Proteasome Activity Assays. As indicated, proteasomes were generally in-cubated with the UBL domain, casein, linear hexa-Ub chains, or DUB inhib-itors at room temperature for 15 min before the start of reactions. Peptidehydrolysis by MEF 26S proteasomes was measured with 10 μM LRR-amc (Boc-Leu-Arg-Arg-amc), LLVY-amc (Suc-Leu-Leu-Val-Tyr-amc), or nLPnLD-amc (Ac-Nle-Pro-Nle-Asp-amc) (Bachem) (λex 380 nm; λem 460 nm) and 1 nM 26Sproteasomes at 37 °C. Proteasomal activities were calculated from 30 to60 min after the start of the reaction. The reaction mixture was composed of50 mM Tris (pH 7.6), 100 mM KCl, 0.1 mM ATP or ATPγS, 0.5 mM MgCl2,1 mM DTT, and 25 ng/μL BSA (Sigma). Degradation of PY-Sic1 (100 nM) by26S proteasomes (2 nM) was carried out in the presence of 50 mM Tris·HCl(pH 7.6), 5 mM MgCl2, 1 mM ATP, 1 mM DTT, and 10 μg/mL BSA (Sigma) forthe indicated time (0 to 4 h) at 37 °C and measured by Western blotting withanti-Sic1 antibody. Degradation of Ub5-DHFR by 26S proteasomes was car-ried out under the same conditions for 20 min at 37 °C and measured byWestern blotting with anti-DHFR antibody. ATP hydrolysis by proteasomeswas measured using the malachite green assay (62). Deubiquitination by 26Sparticles (5 nM) was assayed with tetra-Ub chains (368 nM) in 25 mM Hepesbuffer (pH 7.6) containing 100 mM KCl, 5 mM MgCl2, 1 mM DTT, and 1 mMATP or ATPγS at 37 °C for 20 min. After the reaction, the products wereanalyzed by Western blotting with anti-Ub antibodies.

Usp14 Competition Assay. 26S proteasomes from MEF cells were incubatedwith GST-UBL derived from Usp14 and GSH-Sepharose. Unbound proteinswere removed using washing buffer (50mM Tris, pH 7.6, 100 mMKCl, 0.1 mMATP or ATPγS, 0.5 mM MgCl2, 1 mM DTT, and 25 ng/μL BSA). Proteasomeswere eluted with this same buffer containing increasing concentrations ofUsp14 (WT or C114A mutant). Eluted fractions were used to measurepeptidase activity.

Degradation of Long-Lived Cellular Proteins. The overall rates of degradationof long-lived protein in HeLa cells were determined, as described previously,after transfection with pEGFP-C1 (control) or pEGFP-UBLhHR23A (25). hHR23A’sUBL (amino acids 2 to 82) was subcloned into pEGFP-C1 vector to constructpEGFP-UBLhHR23A.

Phylogenetic Tree Analysis. Amino acid sequences of UBL domains of Usp14,Ubp6, hHR23A/B, Rad23, Dsk2, hPLIC1, Parkin, as well that of Ub werealigned, and their phylogenetic tree was generated with ClustalW2 (https://www.ebi.ac.uk/tools/msa/clustalw2/). The resulting Newick-format treedata were visualized with Phylodendron (iubio.bio.indiana.edu/treeapp/treeprint-form.html).

ACKNOWLEDGMENTS. We are grateful to Megan LaChance for her valuableassistance in preparing the manuscript, Dan Finley and Byung Hoon Lee forproviding cells and reagents, and Takeda Pharmaceuticals for providing Ub5-DHFR. This work was supported by grants from the National Institute ofGeneral Medical Science (R01 GM51923) and Target ALS Foundation.

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E11650 | www.pnas.org/cgi/doi/10.1073/pnas.1808731115 Kim and Goldberg

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Correction

BIOCHEMISTRYCorrection for “UBL domain of Usp14 and other proteinsstimulates proteasome activities and protein degradation incells,” by Hyoung Tae Kim and Alfred L. Goldberg, which wasfirst published November 28, 2018; 10.1073/pnas.1808731115(Proc Natl Acad Sci USA 115:E11642–E11650).The authors note that the following statement should be

added to the Acknowledgments: “This work was also supportedby Project ALS, Cure Alzheimer’s Foundation, and the MuscularDystrophy Association of America.”

Published under the PNAS license.

Published online January 14, 2019.

www.pnas.org/cgi/doi/10.1073/pnas.1822051116

www.pnas.org PNAS | January 22, 2019 | vol. 116 | no. 4 | 1459

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ECTION