The Lysine 48 and Lysine 63 Ubiquitin Conjugates AreProcessed Differently by the 26 S Proteasome*□S

Received for publication, August 5, 2009, and in revised form, October 9, 2009 Published, JBC Papers in Press, October 26, 2009, DOI 10.1074/jbc.M109.052928

Andrew D. Jacobson‡1,2, Nan-Yan Zhang‡1, Ping Xu§, Ke-Jun Han‡, Seth Noone‡, Junmin Peng§,and Chang-Wei Liu‡3

From the ‡Department of Biochemistry and Molecular Genetics, University of Colorado Denver School of Medicine,Aurora, Colorado 80045 and the §Department of Human Genetics, Center for Neurodegenerative Diseases, Emory University,Atlanta, Georgia 30322

The role of Lys-63 ubiquitin chains in targeting proteins forproteasomal degradation is still obscure. We systematicallycompared proteasomal processing of Lys-63 ubiquitin chainswith that of the canonical proteolytic signal, Lys-48 ubiquitinchains. Quantitative mass spectrometric analysis of ubiquitinchains in HeLa cells determines that the levels of Lys-63 ubiq-uitin chains are insensitive to short-time proteasome inhibition.Also, the Lys-48/Lys-63 ratio in the 26 S proteasome-boundfraction is 1.7-fold more than that in the cell lysates, likelybecause some cellular Lys-63 ubiquitin conjugates are seques-tered by Lys-63 chain-specific binding proteins. In vitro, Lys-48and Lys-63 ubiquitin chains bind the 26 S proteasome compara-bly, whereas Lys-63 chains are deubiquitinated 6-fold fasterthan Lys-48 chains. Also, Lys-63 tetraubiquitin-conjugatedUbcH10 is rapidly deubiquitinated into the monoubiquitinatedform, whereas Lys-48 tetraubiquitin targets UbcH10 for de-gradation. Furthermore, we found that both the ubiquitinaldehyde- and 1,10-phenanthroline-sensitive deubiquitinatingactivities of the 26 S proteasome contribute to Lys-48- and Lys-63-linkage deubiquitination, albeit the inhibitory extents aredifferent. Together, our findings suggest that compared withLys-48 chains, cellular Lys-63 chains have less proteasomalaccessibility, and proteasome-bound Lys-63 chains are morerapidly deubiquitinated, which could cause inefficient degrada-tion of Lys-63 conjugates.

Protein ubiquitination is a posttranslational modificationcatalyzed by a cascade of enzymatic reactions involving a ubiq-uitin (Ub)4-activating enzyme (E1), a Ub-conjugating enzyme(E2), and a Ub ligase (E3) (1). Ub is conjugated onto protein

substrates by formation of an isopeptide bond between the car-boxyl group of the C-terminal glycine residue of Ub and the�-amino group of a lysine residue in the substrate. Furthermore,a polyubiquitin (polyUb) chain is formed by conjugating thecarboxyl group of the C-terminal glycine residue of Ub to the�-amino group of one of the seven internal lysines in the pre-ceding Ub. In addition, linear polyUbs are linked by amidebonds formed between the C-terminal glycine residue of Uband the N-terminal methionine residue of a following Ub (2).Thus, at least eight different polyUb linkages exist in cells.Protein ubiquitination plays diverse roles in regulating cellu-

lar activities. Monoubiquitination does not support degrada-tion, but it is involved in regulatingmembrane trafficking, genetranscription, DNA repair, and DNA replication (3). As forpolyubiquitination, it seems that different linkages have dis-tinct functions; polyUbs linked through Lys-48 are the primarytargeting signals for proteasomal degradation (1), whereaspolyUbs linked through Lys-63 recruit other binding partnersand execute many functions including kinase activation (4),protein synthesis (5), DNA repair (6, 7), and chromosome seg-regation (8). The functions of other polyUbs have also beensuggested such as the involvement of the Lys-6 linkage in reg-ulating DNA repair (9). Interestingly, the Ub linkage on a mod-ified substrate can be switched in response to different func-tions. For instance, in tumor necrosis factor �-stimulatedactivation of the NF�B gene (10), a Lys-63 chain on receptorinteracting protein (RIP) functions as a signaling element torecruit the I�B kinase complex, which phosphorylates I�B� (aninhibitor protein of NF�B) and triggers its Ub-dependent deg-radation. During the stimulation, A20 deubiquitinates Lys-63-linked RIP and then assembles Lys-48 polyUbs on RIP, promot-ing RIP degradation (10). In addition to Lys-48 polyUbs, arecent proteomic study found that Ub chains linked by Lys-6,-11, -27, -29, or -33 could also serve as proteolytic signals (11).Lys-11 polyUbs were found to mediate degradation of proteinsinvolved in endoplasmic reticulum-associated degradation, cellcycle progression, and other functions (11, 12), whereas Lys-29polyUbs may promote Ub fusion degradation (13).The role of Lys-63 polyUbs in targeting proteins for protea-

somal degradation is still unclear. Some studies suggested thatLys-63 polyUbs are competent proteolytic signals. For example,in vitro studies have shown that Lys-63 polyUbs are able totarget degradation of several proteins including Sic1, cyclin B1,dihydrofolate reductase, and troponin I (14–17). In Saccharo-myces cerevisiae, partial degradation of the transcription factor

* This work was supported, in whole or in part, by National Institutes of HealthGrant AG025688 (to J. P.). This work was also supported by grants from theAmerican Heart Association, a March of Dimes Basil O’Connor Award, theAmerican Cancer Society, and the Cancer League of Colorado (to C.-W. L.).

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Figs. 1–9.

1 Both authors contributed equally to this work.2 Supported by National Institutes of Health Pre-doctoral Training in Molecu-

lar Biology Grant T32-GM08730.3 To whom correspondence should be addressed: Dept. of Biochemistry and

Molecular Genetics, University of Colorado Denver School of Medicine,12801 E. 17th Ave., PO Box 6511, Mail Stop 8101, Aurora, CO 80045. E-mail:changwei.liu@ucdenver.edu.

4 The abbreviations used are: Ub, ubiquitin; polyUb, polyubiquitin; Amc, ami-no-4-methylcourmarin; RIP, receptor interacting protein; Ubal, ubiquitinaldehyde; HA, hemagglutinin; NEMO, NF-�B essential modulator.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 51, pp. 35485–35494, December 18, 2009© 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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Mga2, which releases theN-terminal p90 activator domain, canbe processed by overexpression of the UbLys-48R mutant thatpromotes the formation of Lys-63 ubiquitinated Mga2 (14).Also, inhibition of the proteasome byMG132 in S. cerevisiae ormammalian cells causes an increase of both the Lys-48 andLys-63 Ub conjugates as detected by mass spectrometric anal-ysis (14). However, to our knowledge, physiological substratesthat exclusively depend on the Lys-63 linkage for degradationhave not been identified. In contrast to the findings that suggesta role of Lys-63 polyUbs in targeting proteolysis, Xu et al. (11)proposed that the Lys-63 polyUbs are not proteolytic signals inS. cerevisiae based on quantitative proteomic studies. They alsosuggest that all other Ub linkages can support degradation andhave partially redundant functions in proteolysis (11).In this study we systematically compared proteasomal proc-

essing of Lys-63 polyUbs with that of the primary proteolyticsignal of Lys-48 polyUbs in the aspects of binding/recognition,deubiquitination, and targeting for degradation. Our resultssuggest that cellular Lys-63 Ub chains have less proteasomalaccessibility than Lys-48 chains, likely because some cellularLys-63 ubiquitin conjugates are sequestered by Lys-63 chain-specific binding proteins, such as NEMO. In vitro Lys-63and Lys-48 Ub chains bind the 26 S proteasome comparably,but Lys-63 chains are deubiquitinated 6-fold more rapidly thanLys-48 chains. Both the ubiquitin aldehyde (Ubal)- and 1,10-phenanthroline-sensitive deubiquitinating activities of the 26 Sproteasome contribute to Lys-48- and Lys-63-linkage deubiq-uitination, albeit their inhibitory extents are different. More-over, we found that rapid deubiquitination of Lys-63 chainscould cause inefficient degradation of their conjugates.

EXPERIMENTAL PROCEDURES

Reagents, Plasmids, Recombinant Protein Purification, andUbiquitination of UbcH10—See supplemental “ExperimentalProcedures.”Proteasomal Degradation and Deubiquitination Assays—

The bovine 26 S proteasome and PA700 were purified accord-ing to methods established by DeMartino and co-workers (18,19). Proteasomal degradation and deubiquitination were per-formed at 30 °C in degradation buffer (20 mM Tris, pH 7.2, 20mM NaCl, 5 mM MgCl2, 2 mM ATP, 1 mM �-mercaptoethanol,and 5% glycerol). Reaction mixtures usually contained 13.5 nM26 S proteasome and 100 nM polyubiquitinated UbcH10 orother substrates as specified in the legends to Figs. 2, 3, and 6.Samples were withdrawn at each designated time point andadded into 5� SDS sample buffer to stop the reaction immedi-ately. Usually samples of time 0 represented a reaction of about15 s except in Fig. 5,C andD, and Fig. 6Cwhere samples at time0 were prepared by adding the substrates directly into 1� SDSsample buffer with the 26 S proteasome. For reactions contain-ing epoxomicin (100�M),Ubal (2.5�M) or 1,10-phenanthroline(5 mM), the 26 S proteasome was preincubated with the corre-sponding inhibitors for 10 min before the supplementation ofsubstrates.Size-exclusion Spin ColumnAssay—12.5 nM 26 S proteasome

was preincubatedwith 2.5�MUbal and 5mM1,10-phenanthro-line for 10min at 30 °C in degradation buffer. 80 nM Lys-48 Ub4or Lys-63 Ub4 was then mixed with the preinhibited protea-

some and incubated for 2min at room temperature. 60-�l mix-tures or Ub4 alone were loaded intoMicro Bio-Spin P-30 chro-matography columns (Bio-Rad) and centrifuged according tothe manufacturer’s instruction. The flow-through was eluteddirectly into a 1.5-ml micro tube with 20 �l of 5� SDS samplebuffer. 30-�l samples were resolved by SDS-PAGE for immu-noblotting assays. To determine the binding between the 26 Sproteasome (13.5 nM) andUb4 (Lys-48 or Lys-63)-UbcH10 (100nM), we used homemade Sephadex G-100 spin columns (exclu-sion limit is 80 kDa). After centrifugation, all resultingmixtures(75 �l) were concentrated to about 30 �l by heating. Proteinsare resolved in SDS-PAGE and immunoblotted with an anti-UbcH10 antibody. Similar assays were also used to determinethe interaction between the 26 S (SW) proteasome or PA700and Usp14.Purification of the 26 S Proteasome-bound Ubiquitinated

Proteins—We obtained the pQCXIP viral expression plasmidthat expresses S13/Rpn11-HTBH or the HTBH tag alone fromDr. L. Huang at the University of California at Irvine. TheHTBH tag includes a His6 tag, tobacco etch virus protease site,in vivo biotinylation sequence, and another His6 tag. StableHeLa cell lines that express S13-HTBH or the HTBH tag wereestablished according to a published method by using HEK29310A1 packaging cells (20). To purify the 26 S proteasome, HeLacells were grown in Dulbecco’s modified Eagle’s medium con-taining 10% fetal bovine serum and 1% penicillin/streptomycin.At 90% confluency, three 10-cmplates of cells were treatedwith30 �M MG132 or 0.3% DMSO for 45 min, then washed twicewith phosphate-buffered saline before harvest. Cells were lysedwith lysis buffer (20mMTris, pH 7.2, 50mMNaCl, 10% glycerol,2 mM ATP, 5 mM MgCl2, 2 mM �-mercaptoethanol, 10 mM

iodoacetamide, 2 mM 1,10-phenathroline, and the proteaseinhibitor mixture (Roche Applied Science)). The lysates werecleared by centrifugation, and the supernatants were incubatedwith 50 �l of streptavidin-agarose for 2 h at 4 °C. The resinswere then washed three times with the lysis buffer followed bytwo additional washes with the lysis buffer without iodoacet-amide. Finally, the streptavidin-bound 26 S proteasome wasreleased by incubation with 50 ng/�l tobacco etch virus over-night at 4 °C.Quantitative Mass Spectroscopic Analysis—The whole cell

lysates and the purified 26 S proteasome described above wereresolved in SDS-PAGE. The gel regions containing the vastmajority of ubiquitinated proteins (�70 kDa), judged by immu-noblotting, were excised followed by in-gel trypsin digestion.Because trypsin cleaves ubiquitin to a small GG tag onmodifiedlysine residues, the abundance of polyUb linkages is repre-sented by the level of the GG peptides. Stable isotope-labeledGG peptides were chemically synthesized, quantified, andadded into the samples as internal standards (21, 22). The sam-ples were fractionated by reverse-phase liquid chromatographycoupled with a hybrid mass spectrometer (LTQ-Orbitrap;Thermo Fisher Scientific). The instrument was operated in themode of selective reaction monitoring to increase sensitivity.The nativeGGpeptides and the labeled internal standardswereeluted together and separated by mass spectrometry due tomass difference, enabling relative quantification.

Differential Proteasomal Processing of Lys-48 and -63 Ubiquitin Chains

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RESULTS

Neither the Cellular Level nor the 26 S Proteasome-boundLevel of Lys-63 PolyUbs Increased in Response to Short-timeProteasome Inhibition—If a specific Ub linkage targets proteinsfor proteasomal degradation, its cellular level and the 26 S pro-teasome-bound fraction should elevate in response to short-time proteasome inhibition. To test this idea, we first generatedstable HeLa cell lines that express S13/Rpn11-HTBH or theHTBH tag alone according to a previous report (20) (data notshown). This allowed us to rapidly purify the 26 S proteasomeusing affinity purification. We next examined whether treatingthese stable HeLa cell lines with 30 �M MG132 for 45 minwould increase cellular Ub conjugates. We found that the highmolecular weight Ub conjugates in the whole cell lysates signif-icantly increased as determined with immunoblotting of Ub(Fig. 1A). Quantitative mass spectrometry (21) that uses heavyisotope-labeled Ub linkage peptides as internal standards iden-tified Lys-11-, Lys-48-, and Lys-63-linked polyUbs in both theDMSO- (control) andMG132-treated whole cell lysates. Inter-estingly, the relative amounts of the Lys-11 and the Lys-48 link-ages increased 1.8- and 2.3-fold, respectively, upon MG132treatment (Fig. 1B). In contrast, the amount of the Lys-63 link-ages did not change (Fig. 1B). The other polyUb linkages wereundetectable in the whole cell lysates. Thus, the cellular level of

the Lys-63 linkages does not elevatein response to short-time protea-some inhibition, whereas the Lys-11and Lys-48 linkages promptlyincrease.Next, we examined the amount of

26 S proteasome-bound polyUblinkages to evaluate which Ub link-ages target substrates to the 26 Sproteasome. To this endwe purifiedthe 26 S proteasome from the estab-lished stable HeLa cells by utiliza-tion of the HTBH tag according to aprevious report (20). To protect thecellular polyUbs from deubiquitina-tion, the whole cell lysates weretreated with inhibitors of deubiq-uitinating enzymes (iodoacetamideand 1,10-phenanthroline). Coomas-sie-stained SDS-PAGE and immu-noblotting confirmed that the 26 Sproteasomewas purified fromHeLacells expressing S13-HTBH, but notfrom the HTBH tag alone (Fig. 1, Cand D). Immunoblotting of thepurified 26 S proteasome with ananti-Ub antibody found that thepurified 26 S proteasome containedsignificant amounts of highmolecu-lar weight Ub conjugates, withmorein the MG132-treated preparation(Fig. 1E). Next, we used mass spec-trometry to quantify the protea-some-bound polyUb linkages. After

enriching on the proteasome, the Lys-6, -11, -27, -29, -48, and-63 linkages were detectable, and their relative amounts werequantified (Fig. 1F). MG132 treatment increased the 26 S pro-teasome-bound Ub conjugates of the Lys-6-, -11-, -27-, -29-,and -48-linked at a range of 1.7–2.5-fold, whereas the Lys-63-linked only had a slight increase (Fig. 1F). Thus, the Lys-63linkage is the only detectable form in the 26 S proteasome-bound fraction that does not significantly increase in responseto short-time proteasome inhibition. Moreover, the amount ofLys-48 polyUbs bound to the 26 S proteasome is 6- and 12.7-fold more than the Lys-63 polyUbs in the DMSO- andMG132-treated preparations, respectively (Fig. 1G). This could explainwhy immunostaining of HeLa cells with Ub linkage-specificantibodies found that Lys-48 polyUbs colocalize with the 26 Sproteasome, whereas the Lys-63 polyUbs do not (23). Further-more, the Lys-48/Lys-63 ratios in the 26 S proteasome-boundfraction are 1.7-fold higher than those in the whole cell lysatesin both DMSO- and MG132-treated preparations (Fig. 1G).Thus, cellular Lys-48 polyUbs appear to havemore proteasomeaccessibility than the Lys-63 polyUbs.Both the Lys-48 and Lys-63 PolyUbs Bind the 26 S Protea-

some Comparably, but the Lys-63 PolyUbs Are RapidlyDeubiquitinated—In the DMSO-treated HeLa cells, 11 and68% of the 26 S proteasome-bound Ub conjugates are the Lys-

Figure 1. Neither the cellular level nor the 26 S proteasome-bound fraction of Lys-63 Ub conjugatesincreases in response to short-time proteasome inhibition in HeLa cells. A, Ub conjugates in the whole celllysates increase in response to a short time of MG132 treatment. HeLa cells stably expressing the HTBH tag orS13-HTBH were treated with 30 �M MG132 for 45 min. The whole cell lysates were immunoblotted against Uband �-actin. B, quantification of Lys-11-, Lys-48-, and Lys-63-linked polyUbs in the whole cell lysates by quan-titative mass spectrometry is shown. The data from MG132-treated samples were normalized to DMSO-treatedones. Data are represented as the mean � S.E. C, shown is SDS-PAGE of the purified 26 S proteasome from HeLacells using the HTBH tag. TEV, tobacco etch virus protease. D, shown is an immunoblot of the HTBH tag-purified26 S proteasome with antibodies against the proteasome subunits including S6a, S7, and the whole 20 Sproteasome. E, shown is an immunoblot of the HTBH tag-purified 26 S proteasome with an anti-Ub antibody.F, shown is quantification of the 26 S proteasome-bound polyUb linkages as shown in E by quantitative massspectroscopy. The data from MG132-treated samples were normalized to DMSO-treated ones. Data are repre-sented as the mean � S.E. G, the 26 S proteasome-bound fractions have higher Lys-48/Lys-63 ratios than thewhole cell lysates (WCL) in both DMSO- and MG132-treated preparations. The percentage of Lys-48 or Lys-63-linked Ub in the total detectable forms was calculated based on mean values from quantitative mass spec-trometry and the percentage ratios of Lys-48 versus Lys-63 are shown.

Differential Proteasomal Processing of Lys-48 and -63 Ubiquitin Chains

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63- and Lys-48-linked forms, respectively (data not shown).This indicates that both Ub linkages can bind the 26 S protea-some. We, therefore, evaluated the ability of the 26 S protea-some to process both Ub linkages from the aspects of proteaso-mal binding, deubiquitination, and degradation of conjugates.To obtain large quantities of the 26 S proteasome for biochem-ical characterization, we purified the 26 S proteasome frombovine red blood cells according to our previous report (18)(supplemental Fig. 1). Mass spectroscopy and immunoblottingdetermined that the purified 26 S proteasome contained threedeubiquitinating enzymes: Uch37, Usp14, and S13 (Rpn11)(supplemental Fig. 1, C–F and data not shown). Notably, puri-fied PA700 (19) contained both Uch37 and S13 but no Usp14(supplemental Fig. 1C). Usp14 is a proteasome-associating pro-tein, and its association is salt concentration-sensitive (24). It islikely that Usp14 dissociates from PA700 during the anionexchange purification step. To examine the ability of the mam-malian 26 S proteasome to bind Lys-48 and Lys-63 polyUbs, weperformed a size-exclusion spin column assay. We found thatboth Lys-48 and Lys-63 Ub4 had comparable binding efficiencyto the 26 S proteasome (Fig. 2A). In the spin column assay,Lys-63 or Lys-48 Ub4 are trapped inside the P-30 spin column(40-kDa exclusion limit) after centrifugation (lane 2 in Fig. 2A).Coelution of Ub4 with the 26 S proteasome in the flow-throughindicates a direct binding between them (lane 3 in Fig. 2A). It isnoteworthy that the 26 S proteasome was incubated with bothUbal and 1,10-phenanthroline to inhibit its deubiquitinatingactivities in these binding assays. Given the fact that bothLys-48 and Lys-63Ub4 efficiently bind the 26 S proteasome, the

two deubiquitinating inhibitorslikely have no effect on binding ofpolyUbs to the 26 S proteasome.We next evaluated 26 S protea-

some-mediated deubiquitination ofLys-48 or Lys-63 Ub4. Surprisingly,we found that Lys-63 Ub4 was deu-biquitinated about 6-fold more rap-idly thanLys-48Ub4 (Fig. 2B and sup-plemental Fig. 2). Coomassie-stained SDS-PAGE showed thatboth Lys-48 and Lys-63 Ub4 wereeventually deubiquitinated intomonomeric Ub (supplemental Fig.2). Themore rapid deubiquitinationof Lys-63 Ub4 correlated with itshigherVmax value as compared withLys-48 Ub4, whereas both chainshad similar Km values (Fig. 2C andsupplemental Fig. 3). Together,these results indicate that the 26 Sproteasome binds both Lys-48 andLys-63 polyUbs comparably, but itdeubiquitinates Lys-63 polyUbsmuch more rapidly.Both the Ubal- and 1,10-Phenan-

throline-sensitive DeubiquitinatingActivities Contribute to Deubiquiti-nation of Lys-48 and Lys-63 PolyUbs—

We next aimed to determine which of the deubiquitinatingactivities of the 26 S proteasome is responsible for deubiquiti-nation of Lys-48 or Lys-63 polyUbs. The 26 S proteasome con-tains two cystine-protease deubiquitinating enzymes (Uch37andUsp14) that are sensitive toUbal inhibition. It also containsa Zn2�-dependent metalloprotease (S13/Rpn11) that is inhib-ited by the metal chelator 1,10-phenanthroline. Uch37 wasshown to have a Ub chain trimming activity that initiates cleav-age from the distal site (25). S13 has a proximal deubiquitinat-ing activity that cleaves an entire polyUb from the substrates(26). Ubal had a concentration-dependent inhibitory effect on26 S proteasome-catalyzed deubiquitination (supplemental Fig.4). Accordingly, we used 2.5 �M Ubal to block the activity ofUch37/Usp14 and 5 mM 1,10-phenanthroline to inhibit theactivity of S13. For Lys-48 Ub4, at a reaction time point whenmore than 85% of Ub4 was deubiquitinated if no Ubal or 1,10-phenanthroline was added, adding Ubal exhibited nearly com-plete inhibition of both the 26 S proteasome- and PA700-me-diated deubiquitination, whereas 1,10-phenanthroline had amoremild inhibitory effect (upper panels in Fig. 3,A andB). ForLys-63 Ub4, neither Ubal nor 1,10-phenanthroline alone com-pletely inhibited deubiquitination, with Ubal having a strongereffect (lower panels in Fig. 3, A and B). In contrast, deubiquiti-nation of Lys-63 Ub4 was abolished by a combination of thesetwo inhibitors (lanes 10 and 11 in Fig. 3B). Thus, both the Ubal-and 1,10-phenanthroline-sensitive deubiquitinating enzymescontribute to deubiquitination of Lys-48 and Lys-63 polyUbs,but they have different inhibitory extents.

Figure 2. Lys-48- and Lys-63-Ub4 bind the 26 S proteasome comparably, but Lys-63-Ub4 is deubiquiti-nated much more rapidly. A, Lys-48- and Lys-63-Ub4 have comparable binding efficiency to the 26 S protea-some. Binding was assayed by using size-exclusion spin columns. Proteins in the flow-through after centrifu-gation were immunoblotted with either an anti-Ub or anti-S7 antibody. The Ub immunoblots weredensitometrically quantified, and the corresponding values were shown under the blots. B, Lys-63 Ub4 isdeubiquitinated much faster than Lys-48 Ub4 by the 26 S proteasome. Reactions contained 13.5 nM of purified26 S proteasome and 400 nM of either Lys-48 or Lys-63 Ub4. The right panel shows the densitometric quantifi-cation of the immunoblots. Deubiquitination of 50% of Lys-48 and Lys-63 Ub4 took �19.5 and 3.6 min, respec-tively. C, the rapid deubiquitination of Lys-63 Ub4 is due to its higher Vmax in comparison to Lys-48 Ub4.Reactions contained 4 nM 26 S proteasome and various concentrations of Ub4 chains (supplemental Fig. 3).Plots of velocity versus Ub4 concentration were shown, and velocity mean values of two independent experi-ments were fit into the Michaelis-Menten equation for parameters Km and Vmax.

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The 26 S Proteasome Catalyzes an Usp14-dependent deISGy-lation Activity—To investigate whether both of the thiol pro-teases (Uch37 and Usp14) of the proteasome contribute to theUbal-sensitive deubiquitination, we prepared Usp14-depleted26 S proteasome by taking advantage of the fact that its resi-dence on the 26 S proteasome is salt concentration-sensitive(24). To remove Usp14, we incubated our purified 26 S protea-some with 160 mM NaCl. Dissociated salt-sensitive bindingproteins were further separated by gel filtration (data notshown). We found that, compared with the purified intact 26 Sproteasome, the salt-treated 26 S proteasome contained 9%Usp14 and 66% Uch37 (Fig. 4A). Other intrinsic proteasomalsubunits including the S13 subunit remained intact (Fig. 4Aand supplemental Fig. 5A). Thereafter, the salt-washed 26 Sproteasome was referred to as the 26 S (SW) proteasome.Importantly, salt treatment did not disrupt proteasome integ-rity as both the regularly purified 26 S and the 26 S (SW) pro-teasomes consisted of �50% double-capped and 50% single-capped proteasomes (Fig. 4B). Also, salt treatment did notaffect the peptidase activity of the proteasomes (values listedunder the gel in Fig. 4B and supplemental Fig. 5B). Next, weexamined whether adding back recombinant Usp14 increasesthe deubiquitinating activity of the 26 S (SW) proteasome. Thesize exclusion spin column assay demonstrated that the recom-binantUsp14 bound the 26 S (SW) proteasome and PA700 (Fig.4C).Moreover, the 26 S and PA700-boundUsp14was activatedat least partially as probed by Ub vinyl sulfone (Fig. 4D). How-ever, binding of Usp14 did not promote the 26 S (SW) protea-some or PA700 to deubiquitinate Ub-Amc and Lys-48Ub4 (Fig.4E and supplemental Fig. 5, C and D). Consistent with thesefindings, the loss of the deubiquitination activity of the 26 S(SW) proteasome (39%) when using Ub-Amc as the substrate isproportional to the loss of the Uch37 subunit (34%) but notUsp14 (91%) (supplemental Fig. 5D). Uch37 associates with the26 S proteasome through interacting with Adrm1. Moreover,Adrm1 activates Uch37 deubiquitinating activity (27–29). In

contrast to Usp14, adding backpurified Adrm1/Uch37 to the 26 S(SW) proteasome significantlystimulated deubiquitination of Ub-Amc (supplemental Fig. 5D), al-though no effect was observed fordeubiquitination of Lys-48 orLys-63 Ub4 (data not shown). Thedeubiquitination discrepancy be-tween Ub-Amc and polyUbs isunclear and under investigation. Arecent study found that protea-some-bound Usp14 is reactive tothe catalytic site probes of both Ub-vinylmethyl ester and ISG15-vinylsulfone (30). ISG15 is an Ub-likemodifier found only in vertebrates,and its expression is induced by typeI interferons and viral or bacterialinfection. We, therefore, examinedif Usp14 catalyzes deISGylation.PA700 did not catalyze deISGyla-

tion of ISG15-Amc. Adding back recombinant Usp14 to PA700stimulated deISGylation of ISG15-Amc (Fig. 4F). Similarly,adding back recombinant Usp14 stimulated the 26 S (SW) pro-teasome deISGylation activity (Fig. 4F). Thus, Usp14 catalyzesthe deISGylation activity of the 26 S proteasome.Rapid Deubiquitination Causes Inefficient Degradation of

Some Lys-63 PolyUb Substrates—Because Lys-48 and Lys-63polyUbs have obviously different deubiquitination rates cata-lyzed by the 26 S proteasome, we next compared the ability ofLys-48 and Lys-63Ub4 to target proteins for degradation. To dothis, we conjugated Lys-48 or Lys-63 Ub4 to the physiologicalsubstrate, UbcH10 (31), using immunoprecipitated Xenopusanaphase-promoting complex/cyclosome as the E3 ligase (18).The size exclusion spin column assay demonstrated that bothLys-48 and Lys-63 Ub4-UbcH10 efficiently bound to the 26 Sproteasome (Fig. 5A). Consistent with our earlier report (18),Lys-48 Ub4-UbcH10was efficiently degraded by the purified 26S proteasome as judged by the fact that UbcH10 only accumu-lated in the reaction containing the proteasome inhibitor,epoxomicin (comparing lanes 1 and 3 in Fig. 5B). Concomi-tantly,more Lys-48Ub4-UbcH10 remained in the reaction con-taining epoxomicin (lane 1 in Fig. 5B), suggesting that deubiq-uitination is impaired as a secondary consequence of inhibitedproteolytic activity (18, 32). Surprisingly, Lys-63 Ub4-UbcH10was rapidly deubiquitinated by the purified 26 S proteasomeand mainly accumulated as monoubiquitinated UbcH10 (Ub-UbcH10) (Fig. 5C). Because monomeric Ub cannot efficientlybind the 26 S proteasome, rapid deubiquitination intoUb-UbcH10 could cause the substrate to dissociate from the 26S proteasome without degradation. The rapid deubiquitinationof Lys-63 Ub4-UbcH10 was inhibited by the addition of bothUbal and 1,10-phenanthroline (lane 5 in Fig. 5C). Consistentwith the results from previous deubiquitination inhibitionassays (Fig. 3),Ubal had a stronger effect than 1,10-phenanthro-line on inhibition of the deubiquitination of Lys-63 Ub4-UbcH10 (lanes 6 and 7 in Fig. 5C). Notably, about 12% of Lys-63

Figure 3. Both the Ubal- and the 1,10-phenanthroline-sensitive deubiquitinating activities of the 26 Sproteasome contribute to Lys-48- and Lys-63-linkage deubiquitination. A, shown is deubiquitination of 2�g of Lys-48 or Lys-63 Ub4 by 13.5 nM purified 26 S proteasome in the absence or in the presence of 2.5 �M Ubal,5 mM 1,10-phenanthroline, or both. The reaction times for Lys-48 and Lys-63 Ub4 were 120 and 20 min, respec-tively. B, the reactions were similar to A except that 60 nM PA700 was used. The reaction times for Lys-48 andLys-63 Ub4 were 180 and 20 min, respectively.

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Ub4-UbcH10 was degraded as determined by densitometricanalysis (comparing lanes 4 and 8 in Fig. 5C). Together, theseresults show that rapid deubiquitination of polyUbs could causeinefficient degradation of some of its conjugates.Because the majority of Lys-63 Ub4-UbcH10 was rapidly

deubiquitinated into Ub-UbcH10 without degradation, wehypothesized that the 26 S (SW) proteasome, which hasdecreased deubiquitination activity, would retain Lys-63Ub4-UbcH10 on the proteasome long enough to promotedegradation. This idea turns out to be true as we found thatthe 26 S (SW) proteasome efficiently bound Lys-63 Ub4-UbcH10 (supplemental Fig. 6) and catalyzed efficient degra-dation of Lys-63 Ub4-UbcH10 (Fig. 5D). Degradation wasabolished by adding either Ubal or 1,10-phenanthroline(lanes 5 and 6 in Fig. 5D), indicating that both of the deubiq-uitination activities are required for mediating degradation-coupled deubiquitination. Interestingly, degradation ofLys-63 Ub4-UbcH10 by the 26 S (SW) proteasome occurredeven more rapidly than the degradation of Lys-48 Ub4-UbcH10 by either the 26 S (SW) (supplemental Fig. 7) or theregular 26 S proteasome (compare Fig. 5D to 5B), indicatingthat deubiquitination could be the rate-limiting step in deg-radation of some proteins.

NEMO Protects Lys-63-linked PolyUbs from ProteasomalDeubiquitination in Vitro—The above studies indicate thatboth Lys-48 and Lys-63 polyUbs bind the 26 S proteasomeequally well in vitro (Figs. 2A and 5A), whereas Lys-48 polyUbsare found to be more abundant at the proteasome in vivo (Fig.1G). Because Lys-63 polyUbs often form a complex with theirbinding partners when performing non-proteolytic functions,we speculated that Lys-63 polyUb-interacting proteins seques-ter Lys-63 polyUbs and limit their accessibility to the 26 S pro-teasome or other deubiquitinating enzymes. In this regard, intumor necrosis factor �-stimulated activation of the NF�Bgene, I�B kinase � (IKK�) (NEMO), the non-catalytic subunitof the IKK kinase complex, binds the Lys-63 polyUb chain(s) onRIP (33), and this interaction is required for stabilization of RIP(33). This stabilization effect is likelymediated by blocking deu-biquitination of Lys-63 polyubiquitinated RIP by A20 (33).Accordingly, we hypothesized that NEMO is capable of pro-tecting Lys-63 polyUbs from deubiquitination by the 26 S pro-teasome and other deubiquitinating enzymes such as A20. Totest this hypothesis, we synthesized Lys-63 polyUb mixtures,Ubn�6. Glutathione S-transferase-NEMO pulldown experi-ments demonstrated that NEMOpreferred to bind long Lys-63chains (Fig. 6A). Consistent with our hypothesis, preincubation

Figure 4. Adding back Usp14 to the 26 S proteasome stimulates proteasome deISGylation activity but not deubiquitination activity. A, the 26 S (SW)proteasome has 9% Usp14 and 66% Uch37 as compared with the regularly purified 26 S proteasome. The 26 S or 26 S (SW) proteasome was immunoblottedwith different antibodies. Immunoblots were densitometrically quantitated to compare the quantities of Usp14, Uch37, and other subunits in these twopreparations. B, the 26 S (SW) proteasome has similar structural integrity and peptidase activity as the regularly purified 26 S proteasome. 3 �g of the 26 S or26 S (SW) proteasome was separated in a 4% native PAGE, and the in-gel activity was assayed by using succinyl-LLVY-Amc as the fluorogenic substrate. Thechymotrypsin-like activity against succinyl-LLVY-Amc (the values shown under the gel) was quantitated by a solution assay. D-26S and S-26S indicate thedouble-capped and the single-capped 26 S proteasome, respectively. a.u., arbitrary units. C, recombinant Usp14 binds the 26 S (SW) proteasome and PA700.The binding was assayed with Sephadex G-100 spin columns, similar to that in Fig. 2A. D, proteasome-bound Usp14 is partially activated. 26 S proteasome (15nM), 26 S (SW) proteasome (15 nM), PA700 (15 nM), Usp14 (45 nM), or their combinations were incubated with ubiquitin vinyl sulfone. Covalent conjugation ofubiquitin vinyl sulfone on Usp14 was assayed by immunoblotting with an anti-Usp14 antibody. E, binding of Usp14 does not stimulate the deubiquitinatingactivity of PA700 and the 26 S (SW) proteasome. The reactions contained 15 nM PA700 or the 26 S (SW) proteasome in the absence or in the presence of 45 nM

Usp14. The deubiquitinating activity was assayed with Ub-Amc as the substrate. F, Usp14 catalyzes the 26 S proteasome deISGylation activity. Reactions weresimilar to E except using ISG15-Amc as the substrate.

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of Lys-63 Ubn�6 with NEMO blocked 26 S proteasome-medi-ated deubiquitination, and this effect was more pronounced onlonger polyUbs (Fig. 6B). We next conjugated Lys-63 Ubn�6 toUbcH10 using immunoprecipitated Xenopus anaphase-pro-moting complex/cyclosome as the E3 Ub ligase. Lys-63 Ubn�6-UbcH10 was rapidly deubiquitinated into Ub-UbcH10 by the26 S proteasome without obvious degradation (comparinglanes 2 and 3 in Fig. 6C), consistent with the result obtainedfrom Lys-63 Ub4-UbcH10 (Fig. 5C). Preincubation of Ubn�6-UbcH10 with NEMO blocked proteasome-mediated deubiq-uitination (lanes 4–6 in Fig. 6C). A longer time course experi-ment showed that inhibition of the deubiquitination of Lys-63Ubn�6-UbcH10 by NEMO was extremely effective (supple-mental Fig. 8). Furthermore, the inhibitory effect was due to thespecific interaction between NEMO and Lys-63 polyUbsbecause a single residue substitution (L329P) in NEMO thatdisrupts the interaction between NEMO and Lys-63 polyUbs(33) abrogated the inhibitory effect on deubiquitination ofLys-63 Ubn�6-UbcH10 (lanes 7–9 in Fig. 6C). Moreover, wefound thatNEMOprotected Lys-63Ubn�6-UbcH10 fromA20-mediated deubiquitination (supplemental Fig. 9). Together,these results demonstrate that Lys-63 polyUb-interacting pro-teins can protect Lys-63 polyUbs from deubiquitination by the26 S proteasome and other deubiquitinating enzymes.

NEMO Protects Its Bound LinearPolyUbs from Deubiquitination inVivo—Next, we sought to examinewhether NEMO protects its boundpolyUbs from deubiquitination invivo. Linear Ub chains were recentlydiscovered to regulate activation ofthe NF�B pathway by modificationof NEMO (34). Structural studiesrevealed that NEMO binds lineardi-Ub through its CC2-LZ (alsocalled UBAN) domain (35, 36).We, therefore, examined whetherNEMO can stabilize linear Ub6 invivo. HEK293 cells were transfectedwith HA-Ub6 and FLAG-NEMO,FLAG-NEMO (L329P), or a combi-nation of both. Immunoblotting thewhole cell lysates with an anti-HAantibody found no obvious accumu-lation of HA-Ub6 in cells when itwas transfected alone (lane 2 inFig. 6D). In contrast, HA-Ub6 andits conjugates accumulated whencotransfected with NEMO but notNEMO (L329P) (lanes 5 and 6 inFig. 6D). These results suggest thatNEMO protects linear Ub6 fromdeubiquitination or degradation incells. To further examine whetherthe protection is from binding ofHA-Ub6 with NEMO, we immu-noprecipitated FLAG-NEMO orFLAG-NEMO (L329P) and found

that NEMO indeed precipitated HA-Ub6 and its conjugates(Fig. 6E). These results suggest that NEMO is able to protect itsbound polyUbs from deubiquitination in cells.

DISCUSSION

Lys-48 and Lys-63 Ub Chains Have Different ProteasomalAccessibility, Deubiquitination Rates, and Abilities to TargetProteins for Proteolysis—In this study we systematically com-pared Lys-63 polyUbs with Lys-48 polyUbs in proteasomalbinding, deubiquitination, and in targeting proteins for degra-dation. In vitro, Lys-48-linked and Lys-63-linked tetraubiquitinbound the 26 S proteasome equally well (Fig. 2A). In contrast,quantitative mass spectrometry determined that the 26 S pro-teasome-bound Ub chains had a higher Lys-48/Lys-63 ratiothan that in the whole cell lysates (Fig. 1G), indicating that cel-lular Lys-63 Ub chains have less proteasomal accessibility thanLys-48 chains. Also, we found that a Lys-63 chain-specific bind-ing protein, NEMO, protected Lys-63 or linear Ub chains fromdeubiquitination by the 26 S proteasome and/or deubiquitinat-ing enzymes (Fig. 6). Thus, some cellular Lys-63 Ub chainscould be sequestered by their binding partners. This protectingeffect might explain why our mass spectrometric analysesdetermined that Lys-63 polyUbs have less proteasomal accessi-bility than Lys-48 polyUbs in cells.

Figure 5. Lys-63 Ub conjugates are rapidly deubiquitinated by the 26 S proteasome without efficientdegradation. A, both Lys-48 and Lys-63 Ub4-UbcH10 efficiently bind the 26 S proteasome as determined bythe spin column assay. B, the degradation reaction contained 100 nM Lys-48 Ub4-UbcH10 and 13.5 nM 26 Sproteasome. The lane denoted epox indicates that the 26 S proteasome was preinhibited with epoxomicin.C, Lys-63 Ub4-UbcH10 is rapidly deubiquitinated by 26 S proteasome without efficient degradation. Reactionswere analogous to B. Lanes denoted with Ubal or phen indicate that the deubiquitinating activities of the26 S proteasome were preinhibited with 2.5 �M Ubal or 5 mM 1,10-phenanthroline or both. D, the 26 S (SW)proteasome degrades Lys-63 Ub4-UbcH10. The reactions were analogous to C except the 26 S (SW) protea-some was used.

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In response to a short time of proteasome inhibition, Lys-63polyUbs are the only detectable Ub linkage in HeLa cells thatdid not increase in the cellular level or the 26 S proteasome-bound fraction. In contrast, other detectable linkages includingthe Lys-6, -11, -27, -29, and -48 increased promptly. Under asevere proteasome inhibition condition (10 �M MG132 for15 h) we found that the cellular levels of all detectable Ub link-ages (Lys-11, -48, and -63) increased (data not shown), consist-ent with two recent studies in yeast and mammalian cells (37,38). The increase of the cellular level of Lys-63 polyUbs in thelater time of proteasome inhibition would suggest that mostLys-63 chains are not used as signals for proteasomal degrada-tion; otherwise, they would have promptly increased after pro-teasome inhibition in a similar manner as the Lys-48 polyUbs.The delayed accumulation of Lys-63 polyUbs in response toproteasome inhibition might come from a secondary effect ofan impaired Ub-proteasome pathway (37). For instance, Lys-63polyUbs are likely protected from deubiquitination/degrada-tion by binding to their partners. Therefore, impaired degrada-tion of their binding partners under severe proteasome inhibi-tion could result in the accumulation ofmore Lys-63 polyUbs incells. Certainly, a small portion of proteasome-bound Lys-63Ub conjugates could be degraded (see below); thus, severe pro-teasome inhibition would eventually result in an increase ofLys-63 polyUbs as well.Interestingly, we found that the 26 S proteasome deubiquiti-

nates Lys-48 and Lys-63 Ub chains differently. Although bothchains were bound similarly by the 26 S proteasome, Lys-63-linked Ub4 was deubiquitinated six times more rapidly thanLys-48 Ub4. When conjugated to UbcH10, Lys-48 Ub4 effi-

ciently targeted UbcH10 for degra-dation, whereas only 12% of Lys-63Ub4-UbcH10 was degraded. Themajority of Lys-63 Ub4-UbcH10was rapidly deubiquitinated intoUb-UbcH10 without degradation(Fig. 5), possibly because low pro-teasomal binding affinity of mono-meric Ub causes dissociatingUb-UbcH10 from the 26 S protea-some. Rapid deubiquitination ofLys-63 polyUbs compared withLys-48 polyUbs might result fromthe difference in topology of thesechains. The isopeptide bonds inLys-63 polyUbs are exposed in anopen conformation, whereas theyare buried in Lys-48 polyUbs (39).The open conformation mightmake Lys-63 polyUbs more accessi-ble to the deubiquitinating enzymesof the 26 S proteasome than Lys-48-linked. Certainly, we cannot excludethe possibility that the different pro-teasomal binding geometries, if any,among the different Ub linkagesmight also cause varied accessibilityto the deubiquitinating enzymes.

Accordingly, the topologies of theUb linkagesmight determinetheir rates of deubiquitination by the 26 S proteasome, confer-ring a layer of substrate selectivity for proteasomal degradation.Degradation of polyubiquitinated proteins requires highly

coordinated actions including substrate binding, deubiquitina-tion, unfolding, translocation, peptide hydrolysis, and ATP hy-drolysis (18). Disturbing this process by disrupting any one ofthese actions could be detrimental to proteasomal degradation.In the case of Lys-63 polyUb conjugates, rapid deubiquitinationcould cause the conjugates to be released from the proteasomebefore being unfolded for translocation and degradation. In thisregard we would expect that Lys-63 polyUbs could target deg-radation of unfolded proteins much more efficiently than thatof stably folded ones. Using this same line of reasoning, reduc-ing the deubiquitination activity of the 26 S proteasome wouldfacilitate the degradation of Lys-63 polyUb conjugates. Weshow this to be true using a 26 S proteasome preparation thathad decreased deubiquitinating activity (Fig. 5D). Therefore, itis not surprising that Lys-63 polyUbs were found to target sev-eral proteins for degradation in vitro in cases where the sub-strates are not well folded (14), the deubiquitinating activity ofthe 26 S proteasome is partially inhibited by Ubal (15), or deu-biquitination activity is reduced by depletion of the deubiquiti-nation enzymes (32). In addition to targeting proteins to theproteasome, Lys-63 Ub chains play a role in endosomal sortingand could target proteins to lysosomal degradation (40–42), aprocess that is also regulated by endosome-residing deubiquiti-nating enzymes (40).Both the Ubal- and the 1,10-Phenanthroline-sensitive Deu-

biquitinating Activities of the 26 S Proteasome Contribute to

Figure 6. Lys-63 polyUbs are protected from deubiquitination by their binding partners. A, NEMO prefersbinding to long Lys-63 polyUbs. The Lys-63 polyUb mixtures contained 400 nM Ubn�6 and 400 nM Lys-63 Ub4.B, NEMO protects Lys-63 Ubn�6 from proteasomal deubiquitination. Appropriate amounts of NEMO werepreincubated with 300 nM Ubn�6 for 5 min before the supplementation of 13.5 nM purified 26 S proteasome toinitiate deubiquitination. C, NEMO protects Lys-63 Ubn�6-UbcH10 from proteasomal deubiquitination. Reac-tions were analogous to B except that 100 nM Lys-63 Ubn�6-UbcH10 was used. epox, epoxomicin. D, NEMOprotects HA-Ub6 from deubiquitination in cells. HEK293T cells were transfected with HA-Ub6, FLAG-NEMO,FLAG-NEMO (L329P), or a combination. The whole cell lysates were immunoblotted with an anti-HA or anti-FLAG antibody. E, the whole cell lysates in D were immunoprecipitated (IP) with an anti-FLAG antibody, and theprecipitates were immunoblotted (IB) with an anti-HA or anti-FLAG antibody.

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Lys-48- and Lys-63-linkage Deubiquitination—We found thattheUbal- and the 1,10-phenanthroline-sensitive deubiquitinat-ing activities of the 26 S proteasome have different inhibitoryeffects on Lys-48 and Lys-63 polyUbs. Simultaneous inhibitionof both the Ubal- and the 1,10-phenanthroline-sensitive activ-ities were required for complete inhibition of Lys-63 chain deu-biquitination. In contrast, Ubal alone exhibited nearly completeinhibition of Lys-48 chain deubiquitination, whereas 1,10-phe-nanthroline had a more mild inhibitory effect. To distinguishthe role of the two Ubal-sensitive enzymes (Uch37 and Usp14),we found that add-back of Uch37/Adrm1 to the 26 S (SW)proteasomewas able to stimulate deubiquitination. In contrast,add-back of Usp14 to the 26 S (SW) proteasome or PA700 didnot stimulate deubiquitination of any tested substrates (Fig. 4and supplemental Fig. 5). This may imply that Usp14 is not amajor deubiquitinating enzyme of themammalian proteasome.However, we cannot exclude the possibilities that Usp14 mayhave specific activity against other untested Ub linkages; thatUsp14 is activated by an unknown protein that is not present inour in vitro system and/or Usp14 is redundant when coexistingwith Uch37. Additionally, Usp14 did stimulate a modest deIS-Gylation activity at the proteasome and may very well be anauthentic deISGylating enzyme, but further investigation isrequired.A recent study reported that neither N-ethylmaleimide (a

cysteine modifier that inhibits thiol proteases) nor Ubal blockbovine PA700 or 26 S (unspecified source)-catalyzed deubiq-uitination of Lys-63 Ub2, whereas1,10-phenanthroline does(43). Thus, the authors proposed that S13 is responsible forLys-63 chain deubiquitination of the 26 S proteasome. It seemsunlikely that the different conclusions originate from differentproteasome sources because we purified PA700 and the 26 Sproteasome from bovine red blood cells as well. Differentexperimental setup might explain why the previous study didnot observe the activity of Uch37 in catalyzing deubiquitinationof Lys-63 polyUbs. First, we used 2.5 �M Ubal to inhibit Uch37activity in this study, whereas the other study used 0.5 �MUbal(43). A concentration of 0.5 �M Ubal cannot efficiently blockthe chain-trimming activity of the 26 S proteasome (supple-mental Fig. 4). Second, we used Lys-63 Ub4 to evaluate protea-somal deubiquitination activities, whereas Lys-63Ub2was usedin the other study (43). It is possible that deubiquitination ofLys-63 polyUbs by Uch37 depends on efficient proteasomalbinding, which requires a minimal chain length of four Ubs(44). Unfortunately, we were not able to evaluate the effect ofN-ethylmaleimide treatment on deubiquitination because ourpurified 26 S proteasome was disassembled when incubatedwith 2 mM N-ethylmaleimide (data not shown). Presumably,N-ethylmaleimide modifies cysteine residues in some subunitsthat are essential for maintaining proteasome integrity.Uch37 and S13 belong to two different deubiquitinating

enzyme families; Uch37 is a thiol protease, and S13 is a Zn2�-dependent metalloprotease and a member of the JAMM/MPN� deubiquitinating family. Interestingly, the deubiquiti-nating activities of both Uch37 and S13 are activated whenintegrated into the 26 S complex. The JAMM/MPN� familymembers, including AMSH and Brcc36, have been shown tohave specificity for Lys-63 polyUbs (43, 45). Not surprisingly,

S13 catalyzes deubiquitination of Lys-63 polyUbs (43). In con-trast to the specificity of the JAMN/MPN� deubiquitinatingenzymes for Lys-63-linked chains, the thiol-utilizing deubiq-uitinating enzymes have diverse deubiquitination specificities.For example, Usp2, Usp5, and Usp15 can deubiquitinate boththe Lys-48 and Lys-63 polyUbs (46), whereas CYLD onlycleaves Lys-63 polyUbs (46). The data from our study revealsthat Uch37 belongs to a thiol-dependent deubiquitinatingenzyme group that cleaves both the Lys-48 and Lys-63 linkages.

Acknowledgments—We thank Z. J. Chen, R. E. Cohen, G. N. De-Martino, L. Huang, X. D. Liu, A. Ma, and J. D. Maller for providingvaluable reagents.

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and Chang-Wei LiuAndrew D. Jacobson, Nan-Yan Zhang, Ping Xu, Ke-Jun Han, Seth Noone, Junmin Peng

the 26 S ProteasomeThe Lysine 48 and Lysine 63 Ubiquitin Conjugates Are Processed Differently by

doi: 10.1074/jbc.M109.052928 originally published online October 26, 20092009, 284:35485-35494.J. Biol. Chem. 

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