A Mechanism for ProteinMonoubiquitinationDependent on a trans-ActingUbiquitin-binding Domain*

Received for publication, January 11, 2013, and in revised form, April 30, 2013Published, JBC Papers in Press, May 3, 2013, DOI 10.1074/jbc.C113.452250

Antonio Herrador‡1, Sébastien Léon§,Rosine Haguenauer-Tsapis§, and Olivier Vincent‡2

From the ‡Instituto de Investigaciones Biomédicas, Consejo Superior deInvestigaciones Cientıficas-Universidad Autonoma de Madrid (CSIC-UAM),28029 Madrid, Spain and the §Institut Jacques Monod, Centre National dela Recherche Scientifique, UMR 7592, Université Paris Diderot, SorbonneParis Cité, 75205 Paris, France

Background: The mechanisms by which ubiquitin chainlength is regulated remain largely unknown.Results: A ubiquitin-binding domain (UBD) acts in transto direct substrate monoubiquitination in vitro.Conclusion:UBD binding to the first conjugated ubiqui-tin prevents further polyubiquitination.Significance: We report a new mechanism to controlubiquitin chain elongation.

The length of the ubiquitin chain on a substrate dictates var-ious functional outcomes, yet little is known about its regulationin vivo. The yeast arrestin-related protein Rim8/Art9 is mono-ubiquitinated in vivo by the Rsp5 ubiquitin ligase. This alsorequires Vps23, a protein that displays an ubiquitin-E2 variant(UEV) domain. Here, we report that binding of the UEV domainto Rim8 interferes with ubiquitin chain elongation and directsRim8 monoubiquitination. We propose that Vps23 UEV com-peteswithRsp5HECTN-lobe for binding to the first conjugatedubiquitin, thereby preventing polyubiquitination. These find-ings reveal a novel mechanism to control ubiquitin chain lengthon substrates in vivo.

Ubiquitin ligases (E3) of the HECT (homologous to theE6-AP C terminus) family are enzymes that transfer ubiquitinto a substrate through an E3-ubiquitin thioester intermediate(1). Yeast Rsp5 is a member of the Nedd4 family of HECT E3ubiquitin ligases, characterized by a common domain architec-ture, with a C-terminal catalytic HECT domain, an N-terminalphospholipid-binding C2 domain, and several WW domainsresponsible for substrate recognition via PY (PXY or PPXY)motifs (2). Rsp5 substrates containing such motifs are prefer-entially modified with long Lys-63-linked polyubiquitin chains

in vitro, likely following a simple sequential-addition mecha-nism (3). However, some of these substrates are monoubiquiti-nated in vivo (4, 5), suggesting that additionalmechanisms con-trol ubiquitin chain length in the cell. It has been proposed thatUbp2, a deubiquitinating enzyme that antagonizes Rsp5 activ-ity, promotes the monoubiquitination of certain targets bytrimming Lys-63-linked ubiquitin chains (6, 7). Additionally, aprocess termed “coupled monoubiquitination” is responsiblefor the monoubiquitination of ubiquitin-binding domain(UBD)3-containing proteins by Nedd4/Rsp5 (8–10). Theseproteins use their UBD, instead of PY motifs, to interact with aself-ubiquitinated E3 ligase. The proposed model suggests thatmonoubiquitinated substrates cannot be further polyubiquiti-nated because the UBD folds back on the conjugated ubiquitinattached in cis, thus disrupting the association of the substratewith the ubiquitinated E3 enzyme. Nevertheless, it remains tobe clarified how Rsp5 monoubiquitinates proteins that do nothave a UBD and use a PY motif for E3 recognition.We previously found that Rsp5 binds to a PY motif in the

arrestin-related protein Rim8/Art9 and catalyzes its mono-ubiquitination in vivo (11). Rim8 is a component of the ambientpH-signaling pathway and acts as an adaptor that mediates theassociation of the putative pH sensor Rim21 with the endo-somal sorting complex required for transport (ESCRT). Dataindicate that the monoubiquitinated lysine residue in Rim8contributes to binding to the ESCRT-I subunit Vps23 (11).Intriguingly, Rim8 monoubiquitination requires Vps23 and isstrongly induced upon overexpression of the Vps23 ubiquitin-binding UEV domain. In this study, we demonstrate, both invitro and in vivo, that binding of Vps23 UEV to Rim8 favors itsmonoubiquitination by inhibiting its polyubiquitination. Wesuggest a model in which interaction of Vps23 UEV with thefirst conjugated ubiquitin prevents ubiquitin binding to Rsp5HECT N-lobe and impairs further polyubiquitination.

EXPERIMENTAL PROCEDURES

Strains and Genetic Methods—Saccharomyces cerevisiaestrains were: SUB280 (Mata ubi1-�1::TRP1 ubi2-�2:ura3,ubi3-�ub-2 ubi4-�2::LEU2 (pUB39-Ub) (pUB100) lys2-801leu2-3112 ura3-52 his3-�200 trp1-1(am)) and SUB413 (Mataubi1-�1::TRP1 ubi2-�2:ura3, ubi3-�ub-2 ubi4-�2::LEU2(pUB39-UbK63R) (pUB100) lys2-801 leu2-3112 ura3-52 his3-�200 trp1-1(am)), and the isogenic vps23::KanMX6derivatives,SUB280-vps23� and SUB413-vps23� (12, 13). Standardgenetic methods were followed, and yeast cultures were grownin synthetic dextrose medium lacking appropriate amino acidsto maintain selection for plasmids (14).Plasmids—Plasmid for the expression of GST-Vps23UEV

andHA-Rim8 were described in Ref. 11. The sequences encod-ing Rsp5, Mms2, Vps23, and Vps23 UEV domain (codons1–161) were amplified from genomic DNA of S. cerevisiae and

* This work was supported by grants from the Ministerio de Ciencia e Inno-vacion (BFU2008-02005; to O. V.) and the Association pour la Recherche surle Cancer (ARC, Grant SFI20101201844; to S. L.).

1 Recipient of a JAE fellowship from the Consejo Superior de InvestigacionesCientıficas (CSIC).

2 To whom correspondence should be addressed: Instituto de Investigacio-nes Biomédicas CSIC-UAM, Arturo Duperier 4, 28029 Madrid, Spain. Tel.:34-915854495; Fax: 34-915854401; E-mail: ovincent@iib.uam.es.

3 The abbreviations used are: UBD, ubiquitin-binding domain; Ub, ubiquitin;UEV, ubiquitin-E2 variant; ESCRT, endosomal sorting complex required fortransport; K0, lysine-less.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 23, pp. 16206 –16211, June 7, 2013© 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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cloned into a pET19b (Novagen)-derived expression plasmid.The genes encoding Rim8 or the C-terminal fragment Rim8CT(codons 461–542) were designed to encode the cAMP-depen-dent kinase recognition site (RRASV) at the 5� end and werecloned into pGEX6p1 (GEHealthcare). Vps23mutation result-ing in a S55A,D56A,G57A (�SDG)4 triple substitution andRim8 mutations resulting in K507R, K513R, K521R, or K527Rsubstitutions and in the V505A,P506A,K507A,Y508A (�PKY)or E533A,S534A,D535A,P536A (�SDP) quadruple substitu-tions were obtained by mutagenic PCR.Protein Expression and Purification—GST- and His-tagged

expression plasmids were transformed into Escherichia coliBL21, and bacterial cells were collected after a 2–3-h inductionof protein expression in the presence of 0.1 mM isopropyl-D-1-thiolgalactopyranoside. Cell pellets were resuspended in STEbuffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 150 mM NaCl)containing 1% Triton X-100, 5 mM DTT, and Complete prote-ase inhibitor mixture (Roche Applied Science) and lysed with aFrench press. His-tagged Rsp5, Ubc4,Mms2, Vps23, andVps23UEV domain were purified using Talon metal affinity resin(Clontech) and quantified usingCoomassie Brilliant Blue stain-ing with BSA standards. GST fusions to Vps23 UEV and tofull-length or truncated Rim8 and mutant derivatives wereaffinity-purified on glutathione-SepharoseTM 4B resin (GEHealthcare). For labeling of proteinswith 32P,GST fusionswereincubated with cAMP-dependent protein kinase (Promega)and adenosine 5�-[�-32P]triphosphate (PerkinElmer Life Sci-ences) on glutathione beads for 40 min at room temperature.In Vitro Ubiquitination Assays—32P-labeled GST fusions,

bound to glutathione-Sepharose, were ubiquitinated in vitro forthe indicated times in the presence of 10mMTris-HCl (pH 7.5),50 mM NaCl, 5 mM MgCl2, 0.1 mM dithiothreitol, 50 �g/mlubiquitin or, where indicated, lysine-less (K0) ubiquitin (Bos-ton Biochem), 4.5 �g/ml E1 (Uba1, Boston Biochem), 6.25�g/ml E2 (Ubc4), 6.25�g/ml E3 (Rsp5), and 2.5mMATP (40-�ltotal volume). For pulldown experiments, in vitro ubiquitina-tion assays were performed with twice as much enzyme for 180min.When indicated, ubiquitination assays were carried out inthe presence of purifiedVps23, wild type ormutant Vps23UEVdomain, or Mms2 UEV protein. Ubiquitination reactions wereinitiated by adding ATP and terminated by washing the beadswith PreScission protease cleavage buffer (50mMTris-HCl (pH7), 150 mMNaCl, 1 mM EDTA, 1 mM dithiothreitol). After GSTremoval by treatment with PreScission protease (GE Health-care) for 16 h at 4 °C, free substrates, retaining the 32P label,were analyzed by SDS-PAGE and autoradiography.Pulldown Assays—Sepharose beads loaded with GST-

Vps23UEV were incubated with in vitro ubiquitinated proteinsfor 1 h at 4 °C in 0.5ml of STEbufferwith 2%TritonX-100. Beads(bound fraction)were spundown, andproteins in the supernatant(unbound) were TCA-precipitated. Following extensive washingof the beads with the same buffer, bound, unbound, and inputmaterials were boiled in sample buffer, separated by 12% SDS-PAGE, and detected by autoradiography.

Immunoblot Analysis—Yeast strains transformedwith pHA-Rim8 were grown to mid-log phase in synthetic dextrosemedium. Protein extracts were prepared by alkaline lysis asreported previously (11), and 1� 107 cell equivalents were ana-lyzed by 7.5% SDS-PAGE and immunoblotting with anti-HA(3F10, Roche Applied Science). Antibodies were detected withECL Plus reagents (Amersham Biosciences).

RESULTS

Vps23UEVDirects Rim8Monoubiquitination in Vitro—Pre-vious in vivo studies showed that monoubiquitination of Rim8by the E3 ubiquitin ligase Rsp5 is strongly enhanced by overex-pression of the Vps23 ubiquitin-binding UEV domain (11). Togain insight into the mechanisms that underlie this process, weset up an in vitro ubiquitination assay to test whether Vps23UEV affects Rim8 ubiquitination by Rsp5 in vitro. Strikingly,although Rim8 was efficiently polyubiquitinated by Rsp5 invitro (Fig. 1A), the amount of monoubiquitinated Rim8 wassignificantly increased upon the addition of Vps23 UEV.Rim8 contains two arrestin domains and a C-terminal region

containing the in vivo ubiquitination site (Lys-521) as well as anRsp5-binding PKY motif (Fig. 1B) (11). To further analyze theeffect of Vps23 UEV on Rim8 ubiquitination, and because invitro ubiquitination can occur at multiple sites and cause diffi-culties in the analysis of reaction products, we used as substratea Rim8 C-terminal fragment (Rim8CT(1K)) containing Lys/Arg substitutions that leave only Lys-521 intact (Fig. 1B). Addi-tionally, and to avoid potential ubiquitination of the terminalamino group of the substrate (3), ubiquitination assays wereperformed with a GST fusion to Rim8CT(1K), radiolabeledwith 32P at a cAMP-dependent kinase recognition site(RRASV), and the GST tag was removed by protease cleavageafter the ubiquitination reaction was terminated (Fig. 1B).Rim8CT(1K) was polyubiquitinated in vitro and was only mod-ified at Lys-521 because no ubiquitinated products weredetected with the lysine-less derivative Rim8CT(0K) carryingthe K521R substitution (Fig. 1C). In agreement with previous invivo data (11), in vitro ubiquitination was not observed with theRim8CT(1K)�PKYmutant derivative, in which the Rsp5-bind-ing PKY motif has been substituted with alanines (Fig. 1C).Strikingly, the addition of Vps23 or the Vps23 UEV domain tothe single-lysine substrate Rim8CT(1K) inhibited the forma-tion of polyubiquitinated species, yielding a substrate ligated toonly one ubiquitin (Fig. 1, D and E). This effect was only seenabove a certain concentration ofVps23UEV (2�g/ml) (Fig. 1E),near the approximate stoichiometric equivalence with Rsp5(UEV � 1.2 �g/ml). In contrast, increased Rim8CT(1K)monoubiquitination was not observed with equivalent or evenlarger amounts of the UEV domain containing protein Mms2(Fig. 1F), thus indicating that this effect is not simply due to thepresence of a ubiquitin-binding domain in the reaction mix-ture. To determine whether the effect of the Vps23 UEV domainon Rim8CT(1K) ubiquitination was only due to an inhibition ofmultiubiquitin chain assembly or also to an increase in the rateof the initial ubiquitination reaction, we performed a time courseof ubiquitinationofRim8CT(1K)withK0ubiquitin,which cannotformubiquitin chains. In these conditions, even a large amount ofVps23UEV (8�g/ml) had no effect on Rim8monoubiquitination

4 The following multiple mutants designations were used throughout:�SDG, S55A,D56A,G57A; �PKY, V505A,P506A,K507A,Y508A; �SDP,E533A,S534A,D535A,P536A.

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(Fig. 1G), thus indicating that the Vps23 UEV domain does notaffect the conjugation of the first ubiquitin.Synergistic Binding of Vps23 UEV to Ubiquitin and an SDP

Motif—Previous work indicated that the ubiquitin moietyadded toRim8 contributes, togetherwith an SDPmotif near theubiquitinated residue, to binding to the Vps23 UEV domain(Fig. 2A) (11). To test whether these two binding sites act syn-ergistically, we carried out a pulldown assay with GST-Vps23UEV as bait and in vitro ubiquitinated Rim8CT(1K) or itsmutant derivative Rim8CT(1K)�SDP, in which the SDP motifhas been substituted with alanines (Fig. 2B). We used stringentconditions (2%Triton) to detect only the strongest interactions.Under these conditions, binding of Vps23 UEV to either ubiq-uitin or the SDP motif was not sufficient to produce a stablecomplex because neither the nonubiquitinated form ofRim8CT(1K) nor the ubiquitinated form of Rim8CT(1K)�SDPwas retained by Vps23 UEV (Fig. 2B, lanes 2 and 5). In contrast,a strong interaction was detected between Vps23 UEV and themonoubiquitinated or polyubiquitinated forms of Rim8CT(1K)(Fig. 2B, lane 2), thus indicating that simultaneous binding ofthe monoubiquitinated residue and the SDP motif to Vps23UEV strengthens the overall binding.

Molecular Determinants for Rim8 Monoubiquitination—Tofurther characterize the mechanisms underlying Rim8 mono-ubiquitination in vitro, we tested whether Vps23 UEV bindingto ubiquitin and SDP motif is required for the inhibition ofRim8 polyubiquitination. Fig. 2C shows a time course of ubiq-uitination of Rim8CT(1K) or Rim8CT(1K)�SDP, in theabsence or presence of either Vps23 UEV or the Vps23UEV�SDG mutant derivative carrying an S55A,D56A,G57Asubstitution in the �-hairpin tongue (15), which impairs ubiq-uitin binding without affecting binding to the SDP motif (11).Inhibition of Rim8 polyubiquitination was strictly dependenton binding of Vps23 UEV to both ubiquitin and SDP motifbecause the accumulation of monoubiquitinated Rim8CT(1K)upon the addition of Vps23UEV (Fig. 2C, compare panel bwithpanel a) was not observed with the Rim8CT(1K)�SDP orVps23 UEV�SDG mutant proteins (Fig. 2C, panels c and d).These findings, togetherwith previous in vivodata (11), supportthe idea that binding of the UEV domain to both the SDPmotifand the monoubiquitinated residue prevents its further polyu-biquitination by Rsp5.Although the Rim8 Lys-521 residue is the physiological site

of ubiquitination (11), we were able to detect in vitro ubiquiti-

FIGURE 1. The Vps23 UEV domain prevents Rim8 polyubiquitination in vitro. A, Rim8 was ubiquitinated for 120 min, in the absence or presence (20 �g/ml)of Vps23 UEV. Lane 1, 10% of reaction mixture without ATP. B, schematic representation of a GST fusion to the single-lysine Rim8 C-terminal fragmentRim8CT(1K) (see “Results” for details). C, ubiquitination assays were performed for 90 min with Rim8CT(1K) or the mutant derivatives Rim8CT(0K) andRim8CT(1K)�PKY. D–F, Rim8CT(1K) was ubiquitinated for 120 min in the absence or presence (20 �g/ml) of Vps23 (D) or increased amounts of Vps23 UEV (E) andMms2 UEV protein (F). Coomassie Brilliant Blue staining of Vps23 (1.2 �g) (D) and Vps23 UEV (E) or Mms2 UEV protein (0.8 �g) (F) is shown on the left. G, timecourse of ubiquitination of Rim8CT(1K) with K0 ubiquitin, in the absence or presence (8 �g/ml) of Vps23 UEV. Ub(1) or Ub(n) indicate migration positions ofsubstrate modified with 1 or n (�1) ubiquitin molecules, respectively. In panels A and C–G, molecular size markers are indicated on the left of the gels.

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nation, albeit at a lower efficiency, of a Rim8 C-terminal frag-ment (Rim8CT(3K)) lacking Lys-521 but containing the otherthree lysine residues present at the C terminus of the protein(Fig. 2D, lane 3). However, monoubiquitination of this sub-strate, in contrast to that of Rim8CT(1K), was not increasedupon the addition of the Vps23 UEV domain (Fig. 2D), thussuggesting that the precise location of the Lys-521 residue playsan important role in this process.Vps23 Prevents Rim8 Polyubiquitination in Vivo—Previous

in vivo studies showed that monoubiquitinated Rim8 is unde-tectable in a vps23� mutant (11). In light of our present results,it is likely thatmonoubiquitinated Rim8 is further polyubiquiti-nated and possibly degraded in the absence of Vps23. Thisassumption predicts that inhibition of multiubiquitin chainassembly in a vps23� mutant should restore normal levels ofmonoubiquitinated Rim8. Because Rsp5 preferentially cata-lyzes the formation of Lys-63-linked chains in vivo (6, 16), weexamined Rim8 monoubiquitination in a vps23� mutantexpressing theUbK63Rpointmutant as the sole source of ubiq-uitin. Fig. 3A shows that expression of UbK63R in a vps23�mutant restores near normal levels of monoubiquitinatedRim8, which migrates as a doublet due to protein phosphory-lation.5 We noted that levels of monoubiquitinated Rim8 werereproducibly higher in wild type cells expressing UbK63R (Fig. 3A, compare lane 2 with lane 4), which may be due to the for-

mation of ubiquitin chains linked through lysine residues otherthan Lys-63 in the absence of Vps23. In summary, these results5 A. Herrador and O. Vincent, unpublished observations.

FIGURE 2. Vps23 modulates Rim8 Lys-521 ubiquitination through binding to both ubiquitin and the Rim8 SDP motif. A, schematic diagram of theinteractions (double arrows) between the PKY and SDP motifs at the Rim8 C terminus, ubiquitin (Ub), and the Rsp5 WW and Vps23 UEV domains. Ubiquitinationof Rim8 Lys-521 by Rsp5 is indicated (dashed arrow). B, binding of GST-Vps23UEV to in vitro ubiquitinated Rim8CT(1K) or the mutant derivativeRim8CT(1K)�SDP. Right: Coomassie Blue staining of GST-Vps23 UEV (50% of reaction mixture). C, time course of ubiquitination of Rim8CT(1K) (panels a, b, andd) or Rim8CT(1K)�SDP (panel c), in the absence or presence (8 �g/ml) of Vps23 UEV (panels b and c) or the mutant derivative Vps23UEV�SDG (panel d). Lowerpanel, Coomassie Blue staining of Vps23 UEV and Vps23UEV�SDG (775 ng). D, ubiquitination assays were performed for 90 min in the absence or presence (16�g/ml) of Vps23 UEV, with Rim8CT(1K) or Rim8CT(3K). Ub(1) or Ub(n) indicate migration positions of substrate modified with 1 or n (�1) ubiquitin molecules.In panels B–D, molecular size markers are indicated on the left of the gels.

FIGURE 3. Inhibition of Rim8 polyubiquitination by Vps23 in vivo. A, West-ern blot of hemagglutinin (HA)-tagged Rim8 in yeast transformants express-ing wild type or K63R mutant ubiquitin (Ub) or in vps23� mutant derivatives. B,models for Vps23-dependent mono- versus polyubiquitination of Rim8 by Rsp5(see “Discussion” for details). C, Vps23 UEV-ubiquitin complex as taken from theProtein Data Bank entry 1UZX (15). The UEV domain is shown in blue, and ubiq-uitin is displayed in gray. Ubiquitin Lys-63 residue is highlighted in red, andselected residues at the UEV-ubiquitin interface (Ser-55-Asp-56-Gly-57 in the UEV�-hairpin tongue and the Leu-8-Ile-44-Val-70 hydrophobic patch in ubiquitin) arecolored in green. The image was generated with PyMOL (21).

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indicate that the absence of detectable levels of monoubiquiti-nated Rim8 in a vps23� mutant is mainly due to its polyubiq-uitination via Lys-63 linkage, thus supporting our in vitro datathat the Vps23 UEV domain prevents multiubiquitin chainassembly. The lack of detection of polyubiquitin laddering inthe vps23�mutantmay simply be caused by the low abundanceof ubiquitinated Rim8, which is barely detectable as a singleband in the wild type strain, or may result from either the deg-radation or the concurrent deubiquitination of the polyubiq-uitinated protein.

DISCUSSION

The results presented here address the molecular mecha-nism through which the ubiquitin-binding UEV domain ofVps23 directs the activity of the Rsp5 ubiquitin ligase towardthe formation of monoubiquitinated Rim8. Our in vitro and invivo data support the idea that Vps23 UEV promotes Rim8monoubiquitination by preventing its further polyubiquitina-tion. We propose the following models (Fig. 3B) for the mech-anism of action of the Vps23 UEV domain. In the first step,Rsp5 binds to the PKY motif at Rim8 C terminus and catalyzesthe conjugation of a single ubiquitin to the Lys-521 residue. Inthe absence of Vps23 (Fig. 3B, top), the chain is elongated byconjugation of an additional ubiquitin molecule via a Lys-63linkage. Previouswork showed that ubiquitin chain extension isdependent on an ubiquitin-binding site in the N-lobe of theNedd4/Rsp5 HECT domain (Fig. 3B, top), which would orientthe first ubiquitin to allow the addition of the next one andmayalso contribute to the interaction of the ubiquitinated substratewith the E3 (17, 18). Therefore, twomodels can be envisaged toexplain how Vps23 prevents the second conjugation reaction.In the first model (Fig. 3B), interaction of the Vps23 UEVdomain with ubiquitin could prevent further ubiquitination byrestricting Lys-63 accessibility. However, previous findings donot support this hypothesis because Lys-63 is fully exposed intheVps23UEV-ubiquitin complex (Fig. 3C) (15) and SDP bind-ing is not expected to produce any conformational changes inthe UEV domain (19). Additionally, we showed that Rim8polyubiquitination does not impair the ubiquitin-dependentinteraction with Vps23 UEV in pulldown assays (Fig. 2B), thusstrongly suggesting that the acceptor lysine residue in ubiquitinis located outside of the UEV-interacting surface.We thereforefavor a second model (Fig. 3B) in which competition of theVps23 UEV domain with the Rsp5 N-lobe for binding to thefirst conjugated ubiquitin prevents its correct positioning andimpairs further polyubiquitination. Additionally, the inabilityof Rsp5 to bind ubiquitin may weaken its interaction with themonoubiquitinated substrate. The most compelling evidencefor this competition model is that both the UEV domain andthe Rsp5 N-lobe, which bind ubiquitin with low affinity, recog-nize the same hydrophobic patch of ubiquitin (15, 17). Thus,these two interactions are mutually exclusive, and binding ofVps23 UEV to the first conjugated ubiquitin should precludeubiquitin chain extension (Fig. 3B). In this context, the Rsp5/Vps23 UEV stoichiometry could play an important role, as sug-gested by our in vitro data that Vps23 UEV prevents ubiquitinchain elongation above a minimum threshold value near theequimolar Rsp5/UEV concentration. Additionally, the relative

position of the SDP motif with respect to the ubiquitinatedlysine residue in Rim8 should also be critical in positioning theUEV domain to favor its interaction with the first conjugatedubiquitin. This observation is consistent with our findings thatVps23 UEV does not prevent the polyubiquitination of lysineresidues other than Lys-521 in vitro (Fig. 2D) and with the highlevel of conservation of the distance between Lys-521 and theSDP motif in Rim8 homologs in other fungi (11).In summary, our findings reveal a new mechanism by which

the Rsp5 ubiquitin ligase, together with the UBD-containingprotein Vps23, promotes the monoubiquitination of theESCRT-I adaptor Rim8. In the process referred to as coupledmonoubiquitination, some UBD-containing substrates ofNedd4/Rsp5 undergo monoubiquitination in a UBD-depen-dent manner (8–10). Here, we extend these findings and showthat a UBD can also promote protein monoubiquitinationwhen acting in trans, thus opening the possibility that othermonoubiquitinated substrates of Rsp5, which bind to UBD-containing proteins, are regulated in a similar fashion. Interest-ingly, the Vps23mammalian homolog, Tsg101, appears to pro-mote the monoubiquitination of the apoptosis-antagonizingtranscription factor by preventing its further polyubiquitina-tion (20). Further work will be required to determine whetherTsg101 plays a similar role as Vps23 in the regulation of ubiq-uitin chain elongation.

Acknowledgments—We thank Dan Finley (Harvard Medical School,Boston, MA) for the SUB strains, Catherine Dargemont (InstitutJacques-Monod (IJM), Paris, France) for the His6-Ubc4 plasmid, andBatool Ossareh-Nazari (IJM) and Damien Abreu for initial help insetting up the in vitro ubiquitination assays.

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REPORT: A UBD Acts in trans to Promote Monoubiquitination

JUNE 7, 2013 • VOLUME 288 • NUMBER 23 JOURNAL OF BIOLOGICAL CHEMISTRY 16211

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Antonio Herrador, Sébastien Léon, Rosine Haguenauer-Tsapis and Olivier VincentUbiquitin-binding Domain

-ActingtransA Mechanism for Protein Monoubiquitination Dependent on a

doi: 10.1074/jbc.C113.452250 originally published online May 3, 20132013, 288:16206-16211.J. Biol. Chem. 

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