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ExpressionIComplex Increases Cell Surface MHC Class

Ubiquitin−E2−Stabilization of an E3 Ligase

Lidia M. Duncan, James A. Nathan and Paul J. Lehner

http://www.jimmunol.org/content/184/12/6978doi: 10.4049/jimmunol.0904154May 2010;

2010; 184:6978-6985; Prepublished online 12J Immunol 

Referenceshttp://www.jimmunol.org/content/184/12/6978.full#ref-list-1

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The Journal of Immunology

Stabilization of an E3 Ligase–E2–Ubiquitin ComplexIncreases Cell Surface MHC Class I Expression

Lidia M. Duncan, James A. Nathan, and Paul J. Lehner

The Kaposi’s sarcoma-associated herpesvirus-encoded ubiquitin E3 ligase K3 ubiquitinates cell-surface MHC class I molecules

(MHC I), causing the internalization and degradation of MHC I via the endolysosomal pathway. K3 recruits the cellular E2

ubiquitin-conjugating enzyme Ubc13 to generate lysine-63–linked polyubiquitin chains on MHC I, leading to the clathrin-

mediated endocytosis and lysosomal degradation of MHC I. In this study, we identify a ubiquitin isoleucine-44-alanine mutant

(I44A) that inhibits K3-mediated downregulation ofMHC I by preventingMHC I polyubiqitination. This E3-specific inhibition by

I44A prevents dissociation of the MHC I–K3–Ubc13–ubiquitin complex, allows the in vivo visualization of a transient substrate–

E3–E2–ubiquitin complex interaction, and highlights a potential substrate hierarchy between the different MHC I alleles down-

regulated by K3. The I44A mutant also increases cell-surface MHC I expression in control cells in the absence of K3, predicting

the presence of an endogenous E3 ubiquitin ligase required for cell-surfaceMHC I regulation. The Journal of Immunology, 2010,

184: 6978–6985.

Major histocompatability complex class I (MHC I)molecules bind peptides derived from endogenous andviral proteins for display at the cell surface to CTLs (1).

Peptides destined for MHC I molecules are generated pre-dominantly by the proteasome and delivered to the endoplasmicreticulum (ER) via the TAP transporter for loading onto MHC Imolecules (1). The ER is therefore a major site of MHC I regu-lation, and the binding of high-affinity peptide causes MHC I todissociate from the peptide loading complex and traffic through thesecretory pathway to the cell surface. How MHC I molecules areregulated at the cell surface is less clear, but involves clathrin- anddynamin-independent, Arf6-mediated MHC I endocytosis and re-cycling (2, 3).As part of their immune evasion strategy, many viruses encode

one or more gene products that downregulate cell-surface MHC Iand help evade CTL-mediated recognition of viral peptides. Theidentification of an increasing number of viral genes emphasizesthe importance of the MHC I Ag presentation pathway in hostdefense and also provides valuable tools for analysis of thispathway. A role for ubiquitin in the regulation of cell-surface MHCI was suggested following the identification of the K3 gene familythat ubiquitinates MHC I and other critical immunoreceptors (4).We and others (5–7) showed that the K3 and K5 viral geneproducts from Kaposi’s sarcoma-associated herpesvirus (KSHV)downregulate cell-surface MHC I molecules. K3 is a viral ubiq-uitin RING-CH E3 ligase, which associates with MHC I at the

plasma membrane, leading to the lysine-63–linked ubiquitination,internalization, and endolysosomal degradation of MHC I mole-cules (8). Lysine-63–linked polyubiquitin chains are recognized toplay an increasingly important role in the endocytosis and sortingof plasma membrane proteins, such as the epidermal growth factorreceptor (9), and there is some evidence for endogenous, ubiq-uitin-dependent regulation of cell-surface MHC molecules. Themembrane-associated RING-CH (MARCH) E3 ligases are thecellular orthologs of the K3 family, and MARCH1 and MARCH8regulate cell-surface MHC class II expression in dendritic cells ina ubiquitin-dependent manner (10). Exogenous expression of therelated MARCH4 and MARCH9 causes downregulation of cell-surface MHC I, but evidence for a physiological role of thesecellular ligases in MHC I regulation is lacking (11). However, theviral ligases are likely to have appropriated a cellular route ofMHC I disposal rather than invented a new pathway, as highlyconserved lysine residues are identified in the cytosolic tail ofMHC I molecules.All ubiquitination reactions begin with a highly conserved

catalytic cascade in which ubiquitin is activated by one of the twoubiquitin-activating E1 enzymes and transferred to the active sitecysteine of one of ∼40 ubiquitin-conjugating enzymes (E2). Thespecificity of the ubiquitin reaction is conferred by the E3 ligase,which includes proteins of the RING and HECT families. Thelarger group of RING ligases does not itself bind ubiquitin, butassociates with the ubiquitin-charged E2 and facilitates transfer ofthe activated ubiquitin from the E2 to a lysine residue on the targetprotein. Thus, the RING family of E3s is not itself catalyticallyactive, but acts as a molecular scaffold to recruit specific E2s viatheir RING domain and helps orientate the E2 for ubiquitintransfer onto the substrate (12). Beyond monoubiquitination,ubiquitin can also form polyubiquitin conjugates via one or moreof its seven lysine residues and its N-terminal. The interactionbetween the E3 ligase and its physiologically relevant E2-conju-gating enzyme is critical to the outcome of the ubiquitin reaction.This interaction determines both the pattern of ubiquitin chainsthat are generated and by inference the biological outcome. Al-though E3–E2 interactions have been characterized in vitro, thereare few studies on these interactions in vivo, mainly due to the lowinteraction affinity between the two binding partners.

Department of Medicine, Cambridge Institute for Medical Research, Addenbrooke’sHospital, University of Cambridge, Cambridge, United Kingdom

Received for publication December 28, 2009. Accepted for publication April 10,2010.

This work was supported by the Wellcome Trust.

Address correspondence and reprint requests to Dr. Paul J. Lehner, University ofCambridge, Addenbrooke’s Hospital, Hills Road, Lab 5.19, Cambridge CB2 0XY,U.K. E-mail address: pjl30@cam.ac.uk

Abbreviations used in this paper: b2m, b2-microglobulin; ER, endoplasmic reticulum;I44A, isoleucine-44-alanine; K63R, lysine-63-arginine; KSHV, Kaposi’s sarcoma-associated herpesvirus; MARCH, membrane-associated RING-CH; MHC I, MHCclass I; UEV, ubiquitin E2 variant; wtUb, wild-type ubiquitin.

Copyright� 2010 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/10/$16.00

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Despite the importance of defining E2–E3 interactions, eluci-dating physiologically relevant E2–E3 pairings in vivo remainsa major challenge. Active E2–E3 pairs do not stably associate dueto weak binding (low micromolar range), a prerequisite to thedynamic catalytic process of the polyubiquitination reaction (13).Because the E1 and E3 enzyme bind the ubiquitin E2 enzyme atthe same site, E2 loading with ubiquitin is an iterative process thatrequires dissociation from the cognate E3, reloading with ubiq-uitin, and subsequent reassociation (14). Much progress has beenmade toward the identification of potential E3–E2 pairs by in vitroenzyme pairing and yeast-two-hybrid approaches (15, 16), often incombination with structural studies to confirm binding (13).However, these approaches also have their limitations and mayresult in enzymatically nonfunctional E3–E2 pairs in vitro. Forexample, the Brca1-Bard1 E3 heterodimer stably binds UbcH7in vitro, yet the complex is inactive for ubiquitin transfer (17, 18).Although the combination of structural and computational meth-ods provides critical insight into the E3–E2 binding interfaces(19), in vivo data are required to understand the physiologicaleffect of E3–E2 pairs on their substrates.Lysine-48–linked chains typically signal proteasomal degrada-

tion, whereas the conjugation of lysine-63–linked polyubiquitinchains is associated with DNA repair, kinase signaling pathways,and receptor regulation (20). A role for lysine-63–linked poly-ubiquitination in receptor regulation was first demonstrated for theyeast plasma membrane protein uracil permease (21) and inmammalian cells for the downregulation of cell-surface MHCclass I molecules by the K3 viral E3 ligase (8). K3 recruits theUbc13 E2 enzyme, which is required for the generation of lysine-63–linked polyubiquitin chains on MHC I molecules, leading totheir internalization and sorting through the endosomal pathwayfor lysosomal degradation. The endogenous downregulation anddegradation of an increasing number of unrelated cell surfacereceptors, including the IFN-aR (22), the nerve growth factorreceptor (23) the aquaporin receptor (24), and the prolactin re-ceptor, involves a similar process (25).The internalization of lysine-63 polyubiquitinated MHC I

requires ubiquitin-binding proteins including the epsin endocyticadaptor, which contains three ubiquitin-interaction motifs. Themajority of ubiquitin-interaction motifs bind ubiquitin via its hy-drophobic patch, centered on the critical Ile44 residue (26). Toinvestigate how this hydrophobic patch affects the K3-mediateddownregulation of MHC I, we made an isoleucine-44-alanine(I44A) mutant in the identical vector to the previously used wild-type and lysine-63-arginine (K63R) ubiquitin mutant. A majoradvantage of these ubiquitin mutants is that they encode a C-ter-minal GFP, which is cotranslationally cleaved such that the ex-pression level of exogenous ubiquitin is proportional to the levelof GFP. In this study, we show that the I44A mutant inducesa marked increase in cell-surface MHC I levels in HeLa-K3 aswell as HeLa cells. This is due to a loss of ubiquitination andstabilization of the MHC I–K3–Ubc13 interaction, allowing vi-sualization of this substrate–E3–E2 complex in vivo. This differ-ential effect of I44A on HLA-A and HLA-B allotypes identifies apotential hierarchy of substrate binding.

Materials and MethodsAbs

The following Abs were used: mAb HC10 (anti-class I H chain), mAb w6/32 (anti-MHC I), mAb 4E (anti–HLA-B), mAb M2 (anti-FLAG) (Sigma-Aldrich, St. Louis, MO), mAb (anti-Ubc13) (Affinity, Nottingham, U.K.),mAb BB7.2 (anti–HLA-A2) (Serotec, Oxford, U.K.), mAb DT9 (HLA-C-specific) (a kind gift from Ashley Moffett, Department of Pathology,University of Cambridge, Cambridge, U.K.), 3D12 (eBioscience, San

Diego, CA), mAb PUH0037 (anti–HLA-A28) (One Lambda, Canoga Park,CA), anti-IgG2b isotype-control (eBioscience), APC anti-human CD54(BD Biosciences, San Jose, CA), mAb AF8 (anticalnexin) (a kind gift fromM. Brenner, Harvard Medical School, Boston, MA), and mAb anti-6Histag (Qiagen, Valencia, CA). Fluorescent secondary Abs were from Mo-lecular Probes (Eugene, OR) and HRP-conjugated secondary Abs fromJackson ImmunoResearch Laboratories (West Grove, PA).

Constructs and cell lines

The constructs and cell lines used were as described (8) except: pCDNA3ZZ-Flag-wtK3 was generated from pCDNA3 Flag-wtK3 by insertion ofthe ZZ tag upstream of the N-terminal Flag tag. The HeLa-ZZ-K3 cell linewas created by transfecting the pCDNA3 ZZ-Flag-wtK3 construct into theparent cell line and selecting with neomycin. The cells were grown inRPMI 1640 supplemented with 10% FCS. The GFP-tagged wild-type andmutant ubiquitin constructs (wtUb and UbK63R) were used as described(27) (a kind gift of Doug Gray, Ottawa Hospital Research Institute, Uni-versity of Ottawa, Ottawa, Ontario, Canada). The UbI44A construct wasgenerated by site-directed mutagenesis of the above 6His-wtUb-GFP-pDG268 using the QuickChange kit (Agilent Technologies, Stockport,U.K.). The sense oligonucleotide used for I44A mutation was UbI44A59-ATCAGCAGAGACTGGCCTTTGCTGGCAAGCAG-39.

Ttransient transfections

HeLa, HeLa-K3, and HeLa-K5 cells were transfected at 60% confluencyin six-well plates with 2 mg vector per well using the TransIT-HeLa-MONSTER transfection Kit (Mirus Bio, Madison, WI). The 293 cells weresimilarly transfected using 293 Transit reagent (Mirus Bio). Transfectedcells were analyzed by flow cytometry 48 h following transfection.

Radiolabeling and immunoprecipitations

Cells were starved for 20 min in methionine, cysteine-free medium, labeledwith [35S]methionine and [35S]cysteine for 10 min, and chased in mediacontaining excess cold methionine and cysteine for the indicated timeperiods. Immunoprecipitations were performed as described (7). Sampleswere analyzed by SDS-PAGE (10% or 12%), processed for autoradiog-raphy with a Cyclone scanner (Packard Instrument, Meriden, CT) andanalyzed with OptiQuant software (PerkinElmer, Waltham, MA). Visual-ization of ubiquitin bands required autoradiography for 7–21 d. Flow cy-tometric analysis was performed as described (7) with the mAbs w6/32 andanti-mouse secondary Ab as indicated in the figure legends.

Immunofluorescence and confocal microscopy

Cells were plated on coverslips and incubated with w6/32 class I mAb for15 min. Soluble cellular proteins cells were removed and cells treated with0.01% saponin in cytosolic buffer (25 mM HEPES-KOH [pH 7.4], 25 mMKCl, 2.5 mM magnesium acetate, 5 mM EGTA, 150 mM K-glutamate) for5 s prior to fixation with 4% paraformaldehyde. Cells were then blockedwith 0.2%BSA in PBS and probed with the desired Ab. The coverslips werewashed with 0.2% BSA in PBS prior to mounting on slides. Slides werevisualized with the LSM510 META confocal microscope (Zeiss, Ober-kochen, Germany) and analyzed using AxioVision software (Zeiss).

Immunoblot analysis

Cells were lysed in 1% Triton X-100 in TBS plus PMSF and iodoacetamideon ice for 30 min. The postnuclear supernatants were diluted into 13Laemli buffer (6 DTT) and heated at 70˚C for 10 min prior to 10% SDS-PAGE. Samples were transferred to polyvinylidene difluoride (Millipore,Bedford, MA), the membranes were blocked overnight at 4˚C, incubatedwith primary for 1 h at room temperature or overnight at 4˚C, and thenincubated with 1:10,000 dilutions of HRP-conjugated secondary Abs asnoted (Jackson ImmunoResearch Laboratories). Membranes were de-veloped in West Pico Extended Chemiluminescent substrate (ThermoFisher Scientific, Waltham, MA).

ZZ pulldown assays and immunoblotting

HeLa-ZZ-Flag-K3 and HeLa control cells (6 I44A) were lysed on ice for30 min at 106 cells/ml in either TBS/1% Triton x-100 (7) or RIPA buffer(50 mM Tris [pH 8], 0.5% sodium deoxycholate, 150 mM NaCl, 1.0% w/vIGEPAL CA-630, and 0.1% SDS), both containing 0.5 mM PMSF (Sigma-Aldrich) and 5 mM iodoacetamide (Sigma-Aldrich). The postnuclear su-pernatants were incubated with IgG sepharose for 2 h at 4˚C, and pelletswere washed three times with the corresponding lysis buffers diluted 1:2prior to SDS-PAGE and immunoblotting as above.

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Live cell sorting

Live cells were labeled with w6/32 mAb at room temperature for 15 min,washed twice with PBS/2% FCS, labeled with anti-mouse Cy5 secondaryAb, and washed again as above prior to cell sorting by MoFlo Cell Sorter(Beckman Coulter, Miami, FL) for three fractions with no (2), low (+), andhigh GFP (++) intensity combined with MHC I staining.

ResultsMutant I44A ubiquitin inhibits the K3 viral E3 ligase and predictsa role for ubiquitination in the steady state regulation of MHC I

The viral K3 ubiquitin E3 ligase downregulates cell-surface MHC I(Fig. 1A). We previously showed K3 generates lysine-63–linkedpolyubiquitin chains that signal the internalization and trafficking ofMHC I through the endolysosomal pathway. Overexpression ofa ubiquitin mutant (K63R), which is unable to make lysine-63–linked polyubiquitin chains and competes with endogenously ex-pressed ubiquitin, shows a dose-dependent increase in MHC I,concomitant with the increase in GFP levels (Fig. 1A). However,a very different phenotype is observed with the I44A mutant. Incontrast to the gradual increase in cell-surface MHC I seen withK63R, there is a sudden, stepwise increase in cell-surface MHC I,and even low levels of I44A expression completely rescue MHC Iback to the cell surface. This dramatic phenotype suggested a blockrather than the graduated inhibition seen with the K63R mutant(Fig. 1A). Because the resulting cell-surface MHC I levels are evenhigher than in normal HeLa cells, we examined how the ubiquitinmutants affectedMHC I on control HeLa cells. In these cells, neitherwtUb nor mutant K63R ubiquitin affected cell-surface MHC I.However, in the presence of the I44A mutant, cell-surface MHC Ialso increased, showing that the effect is not restricted to K3-expressing cells (Fig. 1B). The same phenotype was visualized byimmunofluorescence confocal microscopy (Fig. 1C). Expressionof the I44A ubiquitin mutant induced a dramatic increase in cell-surface MHC I in K3-expressing as well as control HeLa cells.

Inhibition by the I44A ubiquitin mutant is MHC I specific anddoes not affect all E3 ligases

KSHV also encodes K5, another viral E3 ligase that shares ∼40%homology with K3 and downregulates cell-surface MHC I as wellas additional cell-surface proteins including the ICAM-1 adhesionmolecule (28, 29). To determine the specificity of the I44Aubiquitin mutant, we examined its effect on cell-surface MHC Iexpression in K5-expressing HeLa cells. In contrast to the I44A-induced increase in cell-surface MHC I expression in Hela-K3 andHeLa cells, even high levels of I44A had little effect on the K5-mediated downregulation of MHC I in HeLa-K5 cells, implyingthat I44A-mediated inhibition is selective (Fig. 2A). Furtherspecificity was determined by examining the effect of I44A onICAM-1 expression. The I44A mutant had no effect on cell-surface ICAM-1 expression in HeLa or Hela-K3 cells or on thelow ICAM-1 levels seen in HeLa-K5 cells, suggesting that theI44A mutant is both E3 and target specific (Fig. 2B).

The I44A ubiquitin mutant differentially affects cell-surfaceHLA-A, HLA-B, and HLA-C alleles

To determine whether I44A expression affects cell-surface MHC Iin other cell types, we analyzed 293 cells transfected with the I44A

FIGURE 1. The 144A ubiquitin mutant increases cell-surface MHC I in

HeLa-K3 and HeLa cells. A and B, Cytofluorometric analysis of MHC I

expression in HeLa-K3 or HeLa cells following transient transfection with

GFP-tagged wtUb, K63R-ubiquitin (UbK63R), or Ile44Ala-ubiquitin

(UbI44A). Cells were stained with mAb w6/32. C, Wild-type HeLa and

HeLa-K3 cells were transfected with His-UbI44A-GFP (first two panels)

and stained with w6/32 mAb for MHC I (third two panels) and DAPI as

a nuclear stain (second two panels) (scale bar, 10 mm).

FIGURE 2. The I44A ubiquitin mutant is K3 and MHC I specific. A,

I44A expression does not affect cell-surface MHC I in HeLa-K5 cells.

Cytofluorometric analysis of MHC I expression in HeLa-K3 or HeLa-K5

cells following transient transfection with GFP-tagged UbI44A or wtUb as

control. Cells were labeled as in Fig. 1A. B, I44A expression does not

effect cell-surface ICAM-1 levels. Cytofluorometric analysis of ICAM-1

expression in HeLa, HeLa-K5, and HeLa-K3 cells following transient

transfection with GFP-tagged UbI44A or wtUb as control. Cells were la-

beled with APC-conjugated anti–ICAM-1 mAb.

6980 IN VIVO DETECTION OF MHC I–E3–E2–UBIQUITIN COMPLEX

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mutant. Contrary to the increasedMHC I seen in Hela-K3 andHeLacells, I44A expression did not increase total cell-surface MHC Ilevels in 293 cells (Fig. 3A). Because 293 cells are HLA-A2 posi-tive, we used the HLA-A2–specific mAb BB7.2, together with thepan HLA-B–specific mAb 4E, to determine the effect of I44A onspecific MHC I allotypes. A clear differential effect of I44A on theHLA-A and -B allotypes is seen (Fig. 3A). I44A expression causeda marked increase in cell-surface HLA-A2 and a decrease in theHLA-B allele, showing that examination of cell-surface MHC Iexpression with the w6/32 mAb was masking allotype-specificeffects. Having observed this striking allele-specific difference in293 cells, HeLa-K3 and HeLa cells were similarly examined fora differential MHC I allelic phenotype (Fig. 3B, 3C). HLA-typingof our HeLa cells found them to be haploid (HLA-A28, HLA-B70,HLA-C12), andwewere therefore able todetermine the effect of I44Aon the single HLA-A, HLA-B, HLA-C, and HLA-E allotypes usingthe HLA-A28–, 4E- (pan-HLA-B), DT9- (HLA-C), and 3D12 (HLA-E)-specific mAbs, respectively. Similar to the effect seen in 293 cells,in both HeLa-K3 (Fig. 3B) and HeLa cells (Fig. 3C), HLA-A28 wasincreased at the cell surface, whereas expression of the HLA-B alleledecreased. Although little HLA-C or HLA-E is present at the surfaceof HeLa-K3 cells, HLA-C and HLA-E can be detected at the cellsurface of HeLa cells and, like the HLA-B allele, was also decreasedfollowing I44A expression. Thus, the HLA allele-specific effect ap-pears to be conserved across Hela-K3, HeLa, and 293 cells, with anincrease in the HLA-A allele and a decrease in the HLA-B allele, aswell as a decrease in HLA-C and HLA-E in HeLa cells.To further examine the differential allelic effect of I44A onMHC I

molecules, we performed MHC I pulse-chase analyses on radio-labeledHeLa-K3 andHeLa cells, initially immunoprecipitatingwith

the w6/32 Ab. We confirmed our previous finding (7) that MHC Imolecules are degraded within 3 h in the presence of K3 (Fig. 4A).This degradation was prevented in the I44A-expressing cells (Fig.4A), as would be predicted from the flow cytometry data (Fig. 1A).The flow cytometry data (Fig. 3B) had shown that in the pres-

ence of the I44A mutant, only the HLA-A allele (A28) is de-tectable at the cell surface. To differentiate the effect of I44A onthe HLA-A and HLA-B alleles, we therefore ran the SDS-PAGEfrom the total MHC I (w6/32) mAb immunoprecipitations fora longer time period to allow improved discrimination of theHLA-A and -B allotypes (Fig. 4B). In the absence of I44A, bothHLA-A and HLA-B allotypes are readily detectable at the start ofthe chase (Fig. 4B, top panel). In contrast, in the presence of I44A,whereas the HLA-A allele (A28) is visualized on the w6/32 im-munoprecipitations, no HLA-B is detected. This apparent loss ofHLA-B was confirmed by direct immunoprecipitation of theHLA-B allele with the 4E mAb, which again shows the markedreduction in HLA-B protein in the presence of I44A (Fig. 4C).Addition of a proteasome inhibitor in this experiment failed torescue HLA-B (data not shown). In summary, the I44A ubiquitinmutant increases expression levels of the HLA-A allele, at theexpense of the HLA-B allele, which is not detectable.

The I44A ubiquitin mutant prevents K3-mediatedpolyubiquitination of MHC I

Expression of the I44A mutant in HeLa-K3 cells increased cell-surface MHC I and by pulse-chase analysis was shown to prevent

FIGURE 3. The I44A ubiquitin mutant is MHC I allotype specific. A–C,

I44A expression leads to upregulation of the cell-surface HLA-A allotype

and decreased expression of HLA-B, HLA-C, and HLA-E. Cytofluorometric

analysis of MHC I expression in 293 (A), HeLa-K3 (B), or HeLa (C) cells

following transient transfection with GFP-tagged UbI44A. Cells were la-

beled with w6/32 Ab (total MHC I), anti–HLA-A2 mAb, anti–HLA-A28

mAb, anti–HLA-B mAb, anti–HLA-C, or anti–HLA-E mAb as indicated.

FIGURE 4. The I44A ubiquitin mutant prevents K3-mediated degrada-

tion of MHC I and promotes HLA-A at the expense of HLA-B expression.

A, K3-induced degradation of MHC I is inhibited by the I44A ubiquitin

mutant. Cell-sorted HeLa-K3 cells (6 I44A) were pulsed for 10 min with

[35S]methionine and [35S]cysteine and chased for the times indicated. Triton

X-100 lysates were immunoprecipitated with the anti-MHC I w6/32 mAb

and protein A-sepharose beads for 2 h and treated with Endo H to follow

maturation of MHC I. Samples were analyzed by 10% SDS-PAGE and

autoradiography. B, The I44A mutant delays MHC I maturation and pro-

motes expression of HLA-A28 at the expense of HLA-B. Cell sorted HeLa

cells (6 I44A) were pulse labeled, immunoprecipitated with the anti-MHC

I w6/32 mAb as in A, and analyzed by 12% SDS-PAGE. C, In the presence

of I44A, expression of the HLA-B allele is dramatically reduced. To

compare expression levels of the HLA-A and -B allele in the presence of

I44A, HeLa cells (6 I44A) were pulse chased for 15 min, immunopre-

cipitated with the anti-MHC I w6/32 mAb, anti–HLA-A28 mAb, and the

anti–HLA-B mAb 4E, and analyzed as in A.

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MHC I degradation. We therefore wanted to determine the effect ofthe I44A mutant on K3-mediated MHC I ubiquitination. Wepreviously showed that immunoprecipitation of radiolabeled MHCI molecules from K3-expressing cells allows the visualization ofhigher m.w. ubiquitinated MHC I species (7). MHC I moleculeswere therefore immunoprecipitated from radiolabeled HeLa-K3cells with the w6/32 mAb. In the presence of the I44A ubiquitinmutant, polyubiquitinated MHC I species were reduced (Fig. 5A).K3-associated, ubiquitinated MHC I molecules may also be visu-alized in radiolabeled cells by immunoprecipitating the K3 com-plex and reprecipitating MHC I-associated proteins (7). To furtherdetermine the extent that I44A inhibited K3-mediated ubiquitina-tion, we radiolabeled two sorted cell populations (Fig. 5B). Hela-K3cells were transfected with the I44A mutant and sorted for theGFP-positive (I44A-expressing, MHC I-high population) andGFP-negative (untransfected, MHC I-low population) (Fig. 5B, leftpanel). Following radiolabeling, FLAG-K3 was immunopre-cipitated and the resulting complex reprecipitated with theMHC IHchain-specific mAb HC10, using HeLa cells as a control. Althoughpolyubiquitinated MHC I was detected in Hela-K3 cells, theseubiquitinatedMHC I species were completely lost in the presence ofI44A (Fig. 5B, right upper panel). Furthermore, examination of theloading controls for the primary FLAG-K3 immunoprecipitation

(10% of input) revealed the presence of associatedMHC I H chains,not normally seen at this low level of K3 protein loading, suggestingan accumulation of MHC I on K3 in the presence of I44A anda potential block in the K3/MHC I complex (Fig. 5B, right lowerpanel).Because the I44A ubiquitin mutant blocked K3-mediated MHC I

ubiquitination, it was important to demonstrate that the I44A mutantcould be incorporated into polyubiquitin chains and does not in-terfere with all polyubiquitin chain formation. This was alreadysuggested by our observation that K5-mediated downregulation ofMHC I is not blocked by the I44A mutant (Fig. 2A). We confirmedthat HeLa-K5 cells transfected with I44A show normal poly-ubiquitinated MHC I chain formation (Fig. 5C), showing that pol-yubiquitin chain generation is not necessarily inhibited by the I44Amutant. To ensure that the I44A mutant does not interfere with theproteasomal pathway, total cell lysates from HeLa cells transientlytransfected with the I44A mutant were examined for the formationof His-tagged polyubiquitin chains. High m.w. polyubiquitinatedproteins were readily visualized with the His-specific Ab andshowed a marked accumulation in the presence of proteasome in-hibitors (Fig. 5D). Therefore, despite our findings that I44A blocksK3-mediated ubiquitination, in the presence of K5, the I44A mutantis readily incorporated into polyubiquitin chains, and I44A is alsoincorporated into polyubiquitin chains destined for proteasome-mediated degradation. Polyubiquitin chain generation is not gen-erally inhibited by the I44A mutant.

The I44A ubiquitin mutant traps the otherwise transientMHC I–E2–E3 ligase complex

In the presence of the I44A mutant, K3-mediated MHC I ubiq-uitination is inhibited. RING E3 ligases, such as K3, do notthemselves directly bind ubiquitin, but recruit a ubiquitin-chargedE2 enzyme that promotes transfer of the ubiquitin to the substrate,in this case the MHC I H chain. As our previous studies have shownthat K3 recruits the Ubc13 E2 enzyme to generate lysine-63–linkedubiquitin chains on the MHC I H chain (8), we reasoned that theI44A-induced ubiquitin block may involve the E2 Ubc13. Toexamine the effect of I44A on Ubc13, we sorted I44A-transfectedHeLa-K3 cells into three populations (Fig. 6A, upper panel) rep-resenting: 1) untransfected GFP-negative, I44A-negative (2),MHC I-low cells; 2) GFP-intermediate, I44A intermediate (+),MHC I-low cells; and 3) GFP-high, I44A-high (++), MHC I-highcells. This allowed a direct comparison of cells expressingequivalent levels of GFP from the wild-type versus the I44Amutant-expressing population. Detergent cell lysates from thesepopulations were analyzed for the presence of Ubc13 by immu-noblotting (Fig. 6A, lower panel) under nonreducing (Fig. 6A,lower left panel) and reducing conditions (Fig. 6A, lower rightpanel). Ubc13 is predominantly detected at its predicted 16 kDamolecular mass, and under nonreducing conditions, the ubiquitin-charged Ubc13 at 24 kDa and the slightly larger His-tagged Ubc13are readily visualized and disappear on reduction. In Hela-K3 cellsexpressing high yet comparable levels of exogenous wtUb andI44A ubiquitin, significantly more His-tagged UbI44A was de-tected on Ubc13 than exogenous wtUb (Fig. 6A, lower panel, lane3 versus lane 6), suggesting that the normal catalytic removal ofI44A from Ubc13 is inhibited. In Hela-K3 cells expressing highlevels of I44A, an additional 120-kDa band is also detected undernonreducing conditions and disappears upon reduction. Therefore,in the presence of I44A, an SDS-stable, reducible high m.w.complex containing the Ubc13 E2 enzyme is visualized.To identify additional components present in this 120-kDa

complex, epitope-tagged K3 was immunoprecipitated from Hela-K3 cells containing I44A under different detergent conditions.

FIGURE 5. The I44A ubiquitin mutant prevents the K3- but not K5-

mediated polyubiquitination of MHC I. A, HeLa and HeLa-K3 cells (6transiently transfected I44A) were pulsed for 15 min and chased for 45 min.

Triton X-100 lysates were immunoprecipitated with the anti-MHC I w6/32

mAb and protein A-sepharose beads for 2 h and treated as in Fig. 4A. B,

HeLa-K3 cells transfected with I44A were cell sorted for high- and low-

expressing GFP (I44A ubiquitin) populations, pulsed for 20 min in [35S]

methionine and [35S]cysteine, and chased for 30 min. Triton X-100 lysates

were immunoprecipitated with the anti-FLAG M2 mAb, the resulting

complexwas dissociated (1%SDS) and reprecipitatedwith theHC10 (class I

H chain [HC])-specific mAb, and analyzed by SDS-PAGE and autoradiog-

raphy. C, The I44A ubiquitin mutant does not affect K5-mediated poly-

ubiquitination of MHC I. HeLa-K5 cells transiently transfected with I44A

were immunoprecipitated as in A. D, Polyubiquitination and proteasomal

degradation is normal in the presence of I44A. HeLa cell lysates (6 I44A)

were immunoblotted for 6His-I44A-Ub in the presence or absence of the

MG132 inhibitor. Calnexin was used as loading control.

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Immunoprecipitated K3 and associated proteins were then visu-alized by immunoblotting. Following epitope-tagged K3 immu-noprecipitation, high molecular mass bands at 120-kDa (Fig. 6B)were again detected and shown to contain K3 (Fig. 6Bi), MHC I Hchain (Fig. 6Bii), Ubc13 (Fig. 6Biii), and ubiquitin (Fig. 6Biv).Upon addition of DTT to the sample preparation buffer, the highm.w. complex was again lost and the high molecular mass com-plex reduced into its monomeric components. Thus, the I44Aubiquitin mutant allows the in vivo visualization of a stable sub-strate–E3–E2–Ub complex.

DiscussionExpression of the I44A mutant ubiquitin caused a marked increasein cell-surface MHC I expression in HeLa-K3, HeLa, and HEK293cells. This was predominantly due to an increase in the HLA-A allotype at the expense of the HLA-B allotype. In HeLa-K3 cells,

expression of I44A correlates with a loss of MHC I ubiquitinationand stabilization of a DTT-sensitive substrate–E3–E2–Ub com-plex. The detection of K3 in association with its cognate E2(Ubc13), substrate (MHC I), and ubiquitin likely represents atransient intermediate complex, which is blocked and unable todissociate due to the I44A mutant.The key features in the I44A-mediated increase in surfaceMHC I

are the loss of MHC I ubiquitination and the appearance of theDTT-sensitive substrate–E3–E2–Ub complex. In trying to un-derstand these results, the most likely primary event is the trap-ping of the transient intermediate substrate–E3–E2–Ub complexdue to an inability to ubiquitinate the MHC I substrate, resulting inthe stabilization of the intermediary components at the cell sur-face. The resulting increase in cell-surface MHC I delays MHC Itrafficking through the secretory pathway and causes a subsequentdownregulation of the HLA-B allotype, which is likely to bea secondary event (see below).Why does the I44A ubiquitin induce trapping of the substrate–

E3–E2–Ub complex? I44A ubiquitin-charged Ubc13 clearly as-sociates with K3, but the viral ligase appears unable to dischargethe I44A ubiquitin, resulting in a stabilization of the substrate–E3–E2–Ub complex. Support for this model comes from the findingthat the substrate–E3–E2–Ub interaction is only detectable undernonreducing conditions, and addition of a reducing agent dis-sociates the complex into its constituent components. Because thethioester bond linking I44A ubiquitin to Ubc13 is the only readilyreducible bond in the complex, reduction of this thioester causesdisintegration of the high m.w. complex.More difficult to determine in vivo is why the I44A ubiquitin

cannot be discharged. The generation of lysine-63–linked poly-ubiquitin chains requires formation of a heterodimer betweenUbc13 and the inactive ubiquitin E2 variants (UEVs), MMS2 inthe nucleus or its homolog UEV1a in the cytoplasm (30, 31).Although Ubc13 forms a thioester bond with the activated ubiq-uitin, also referred to as the donor ubiquitin, UEVs bind bothUbc13 and the ubiquitin on the substrate, the acceptor ubiquitin, ina noncovalent manner (19). UEVs then orientate the acceptorubiquitin to the Ubc13 active site such that only lysine-63 isavailable for chain formation (32, 33). The UEV-acceptor ubiq-uitin interface is centered around the critical Ile44 residue (34),and mutations of this residue inhibit lysine-63–linked poly-ubiquitin chain formation in the yeast RAD6 pathway (35). Fur-thermore, the affinity of the UEV for Ubc13 may be enhanced by

FIGURE 6. The I44A ubiquitin mutant stabilizes the MHC I–E3–E2–Ub

ligase complex. A, The Ubc13 E2-conjugating enzyme is detected in

a 120-KDa reducible complex in the presence of the I44A ubiquitin mu-

tant. I44A-transfected HeLa-K3 cells were labeled for surface MHC I with

the w6/32 Ab, and cells were sorted into three populations representing

untransfected GFP-negative (I44A-negative) cells (2); GFP-intermediate,

(I44A-intermediate) MHC I low cells (+); and GFP-high, (I44A-high),

MHC I-high cells (++) prior to immunoblotting equivalent amounts of total

lysates for Ubc13 under nonreducing and reducing conditions (lower

panels). B, Ubc13, K3, MHC I, and I44A-Ub are components of the 120-

kDa complex. HeLa-ZZ-Flag-K3 cells 6 I44Awere lysed in 1% Triton X-

100/TBS or RIPA buffers and total cell lysates (Lys) were either directly

immunoblotted for K3 (anti-Flag M2 Ab), MHC I (HC10 Ab), Ubc13, and

6HisI44A (anti-His Ab) or pulldowns performed with ZZ-FLAG-K3 (PD)

and the resulting complex separated by SDS-PAGE and then probed for the

same components. Calnexin was used as loading control.

FIGURE 7. Schematic model to show trapping of the MHC I–E3–E2–

UbI44A complex. A, In the presence of wtUb, K3 recruits Ub-charged

Ubc13 for transfer of the charged donor ubiquitin from Ubc13 to the ac-

ceptor ubiquitin. B, In the presence of the I44A mutant ubiquitin, Uev1a is

unable to bind the I44 patch on the acceptor ubiquitin and orient its K63

for conjugation by Ubc13. Uev1a dissociates, and in the absence of pol-

yubiquitination, the monoubiquitin on MHC I is likely removed by

a deubiquinating enzyme. As Ubc13 is unable to transfer the donor

ubiquitin to an acceptor ubiquitin, it cannot dissociate from K3, stabilizing

the otherwise transient MHC I–E3–E2–I44A complex.

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synergistic noncovalent interactions between the UEV and theacceptor ubiquitin on the substrate (19). We speculate that theabsence of I44 is likely to prevent the binding of the UEV com-plex to the acceptor ubiquitin of the catalytic complex and thusprevents Ubc13 from transferring its charged ubiquitin to MHC I.The E3–E2–Ub complex is therefore inactive and unable topolyubiquitinate (Fig. 7). Although Ubc13 is readily detectable inthe high m.w. complex, we have been unable to detect UEV1a, thelikely UEV binding partner. The predicted molecular mass of theidentified complex components is 130 kDa, which includesubiquitin-charged Ubc13 (26 kDa), MHC I H chain (43 kDa), b2-microglobulin (b2m) (13 kDa), and K3 (48 kDa), and this corre-lates with the 120-kDa complex identified under nonreducingconditions and further suggests that UEVs are not present.The identification of K3 associated with its cognate E2 is most

unusual. The very transient nature of the interaction between E3ligases and E2-conjugating enzymes means there are few data onE3–E2 pairing in vivo. In those few examples in which high-affinity E3–E2 binding has been visualized in vivo, the E3 RINGdomain does not contribute to formation of the complex. High-affinity binding between the Ubc2 E2 enzyme and the RING-E3ligase of the N-end rule pathway, Ubr1, maps to a basic rich re-gion, rather then the proximal RING domain (36). Similarly, gp78,an ER-resident E3 ligase, recruits its cognate E2, Ube2g2 viaa specific high-affinity binding site that is distinct from the RING(37). Therefore, our knowledge on E3–E2 interactions facilitatedby the RING domain is based on in vitro studies. RING domainsare thought to either position the ubiquitin-charged E2 and facil-itate the transfer of ubiquitin to substrates or function as allostericactivators of ubiquitin-charged E2s (12, 38). Because the E2binding sites for RING fingers and the E1 enzyme overlap, an E2which discharges its ubiquitin must dissociate from its cognateligase prior to reloading with ubiquitin (14). The affinity of anubiquitin-charged E2 for its E3 is therefore likely to be higher thanthe noncharged E2, and this is indeed the case as reported for theE2–E3 HsUbc2b–E3a pair, in which the affinity of the ubiquitin-charged E2 is 8.4 times higher than the noncharged E2 (39).A surprising finding of this study is the I44A-induced, allotype-

specific increase in MHC I, with the differential effect on theHLA-Aand -Ballotypes seen inbothHeLaandHEK293cells, aswellas HLA-C and HLA-E in HeLa cells. Although K3 downregulatesbothHLA-A andHLA-B equally effectively, in the presence of I44A,only HLA-A is rescued to the cell surface, at the expense ofHLA-B,which is undetectable. This allotype-specific phenotypemayreflect an increased affinity of K3 for HLA-A over HLA-B, creatinga substrate hierarchy, though the explanation may be more complex.The tight control of cell-surface MHC I expression seen in most

cell types reflects multiple levels of regulation including tran-scriptional, posttranslational, and peptide loading steps. For ex-ample, different MHC I allotypes vary in their genomic promoterregulatory elements (40) and MHC class I H chains compete forfolding, independently of b2m and peptide (41). In the ER, peptideloading of MHC I molecules on the TAP peptide loading complexis the major site of MHC I regulation, and competition betweenMHC I alleles for peptide loading on TAP is well described (42,43). The accumulation of HLA-A molecules at the cell surface ofI44A-expressing cells is likely to induce secondary effects onMHC I loading and trafficking through the secretory pathway,particularly on the peptide-loading complex. Radiolabeled pulse-chase analysis showed that in I44A-expressing HeLa cells, HLA-A28 transport through the secretory pathway is delayed, whichmay cause a backlog of MHC I proteins and increased competitionfor binding the peptide loading complex. Our inability to detectany HLA-B allotype, even in the presence of proteasome in-

hibitors, suggests further effects of the I44A mutant, which mightalso be secondary to the accumulation of cell-surface HLA-A orunrelated to the cell-surface phenotype. These possibilities arebeing pursued.A critical control in these experiments was the lack of effect of

I44A on K5-expressing cells. Unlike the effect seen with HeLa andHeLa-K3 cells, I44A had no effect on K5’s ability to ubiquitinateand downregulate MHC I (Fig. 4). Indeed, K5 is refractory to theI44A effect. This resistance of MHC I molecules to I44A in HeLa-K5 cells serves as a useful control for the increase in cell-surfaceMHC I seen in HeLa-K3 and HeLa cells. It shows that I44Aneither blocks polyubiquitination in general nor lysine-63–linkedpolyubiquitination in particular, as the K5-mediated ubiquitinationand downregulation of cell-surface MHC I is absolutely dependenton lysine-63–linked polyubiquitin chains (44). Furthermore,HLA-B is readily detected in I44A-expressing HeLa-K5 cells,supporting our assertion that the decreased HLA-B expression inHeLa-K3 and HeLa cells is secondary to the cell-surface blockinduced by I44A. Although we do not fully understand why I44Ais able to block the K3 but not K5 viral ligase, it is likely to reflectthe recruitment of different E2s or differences in E2 binding. Despitesharing MHC I as a common substrate, there are significant differ-ences between K3 and K5 that may help explain K5’s protectionagainst I44A. K3’s primary substrates areMHC I molecules, where-as K5 efficiently downregulates a number of immunoreceptors fromthe cell surface (11). K5 has other unusual properties. It promotesubiquitination on cysteines as efficiently as lysine residues in thecytoplasmic tail of HLA-B7 (45) and generates mixed lysine-11 andlysine-63–linked polyubiquitin chains on MHC I. It may thereforerecruit cellular E2s in addition to Ubc13 to catalyze the poly-ubiquitination and downregulation of MHC I.Our data also provide insight into the regulation of MHC I cell-

surface molecules in the absence of viral ligases. The increase inMHC I expression in I44A-expressing HeLa as well as HeLa-K3cells implies the existence of an ubiquitin-mediated mechanism inthe steady-state regulation of MHC I molecules at the plasmamembrane. Assuming the same mechanism is occurring in HeLa asin HeLa-K3 cells, I44Amay block the endogenous cellular ligase inan identical way to the inhibition seen in K3-expressing cells.Regulation of cell-surface MHC I by an endogenous ligase is likely,as evolutionarily conserved lysine residues can be identified in theMHC I H chain cytosolic tail as far back as sharks. Ubiquitinationmay promote MHC I H chain internalization following peptide orb2m dissociation at the cell surface. MARCH1 and -8, orthologsof K3 and K5, regulate cell-surface MHC class II (46), andMARCH orthologs or unrelated ligases may be involved in MHC Iregulation. Although overexpression of two MARCH familymembers (MARCH4 and -9) causes MHC I downregulation (47,48), a physiological role for these ligases in MHC I regulation inHeLa cells has not yet been demonstrated.In summary, the I44A Ub mutant inhibits the K3 viral ligase,

resulting in increased cell-surface MHC I expression. This hasallowed visualization of an otherwise transient substrate–E3–E2–Ub complex in vivo, exposed an MHC I allotype hierarchy, andpredicted the presence of a cellular E3 ligase involved in thesteady state regulation of cell-surface MHC I.

AcknowledgmentsWe thank D. Gray (University of Ottawa, Ottawa, Ontario, Canada) for mu-

tant ubiquitin-GFP constructs.

DisclosuresThe authors have no financial conflicts of interest.

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References1. Peaper, D. R., and P. Cresswell. 2008. Regulation of MHC class I assembly and

peptide binding. Annu. Rev. Cell Dev. Biol. 24: 343–368.2. Radhakrishna, H., and J. G. Donaldson. 1997. ADP-ribosylation factor 6 regu-

lates a novel plasma membrane recycling pathway. J. Cell Biol. 139: 49–61.3. Naslavsky, N., R. Weigert, and J. G. Donaldson. 2004. Characterization of

a nonclathrin endocytic pathway: membrane cargo and lipid requirements. Mol.Biol. Cell 15: 3542–3552.

4. Lehner, P. J., S. Hoer, R. Dodd, and L. M. Duncan. 2005. Downregulation of cellsurface receptors by the K3 family of viral and cellular ubiquitin E3 ligases.Immunol. Rev. 207: 112–125.

5. Coscoy, L., D. J. Sanchez, and D. Ganem. 2001. A novel class of herpesvirus-encoded membrane-bound E3 ubiquitin ligases regulates endocytosis of proteinsinvolved in immune recognition. J. Cell Biol. 155: 1265–1273.

6. Ishido, S., C. Wang, B. S. Lee, G. B. Cohen, and J. U. Jung. 2000. Down-regulation of major histocompatibility complex class I molecules by Kaposi’ssarcoma-associated herpesvirus K3 and K5 proteins. J. Virol. 74: 5300–5309.

7. Hewitt, E. W., L. Duncan, D. Mufti, J. Baker, P. G. Stevenson, and P. J. Lehner.2002. Ubiquitylation of MHC class I by the K3 viral protein signals in-ternalization and TSG101-dependent degradation. EMBO J. 21: 2418–2429.

8. Duncan, L. M., S. Piper, R. B. Dodd, M. K. Saville, C. M. Sanderson, J. P. Luzio,and P. J. Lehner. 2006. Lysine-63-linked ubiquitination is required for endoly-sosomal degradation of class I molecules. EMBO J. 25: 1635–1645.

9. Huang, F., D. Kirkpatrick, X. Jiang, S. Gygi, and A. Sorkin. 2006. Differentialregulation of EGF receptor internalization and degradation by multi-ubiquitination within the kinase domain. Mol. Cell 21: 737–748.

10. Ohmura-Hoshino, M., Y. Matsuki, M. Mito-Yoshida, E. Goto, M. Aoki-Kawasumi, M. Nakayama, O. Ohara, and S. Ishido. 2009. Cutting edge: re-quirement of MARCH-I-mediated MHC II ubiquitination for the maintenance ofconventional dendritic cells. J. Immunol. 183: 6893–6897.

11. Nathan, J. A., and P. J. Lehner. 2009. The trafficking and regulation of membranereceptors by the RING-CH ubiquitin E3 ligases. Exp. Cell Res. 315: 1593–1600.

12. Lorick, K. L., J. P. Jensen, S. Fang, A. M. Ong, S. Hatakeyama, andA. M. Weissman. 1999. RING fingers mediate ubiquitin-conjugating enzyme(E2)-dependent ubiquitination. Proc. Natl. Acad. Sci. USA 96: 11364–11369.

13. Deshaies, R. J., and C. A. Joazeiro. 2009. RING domain E3 ubiquitin ligases.Annu. Rev. Biochem. 78: 399–434.

14. Eletr, Z. M., D. T. Huang, D. M. Duda, B. A. Schulman, and B. Kuhlman. 2005. E2conjugating enzymes must disengage from their E1 enzymes before E3-dependentubiquitin and ubiquitin-like transfer. Nat. Struct. Mol. Biol. 12: 933–934.

15. Markson, G., C. Kiel, R. Hyde, S. Brown, P. Charalabous, A. Bremm, J. Semple,J. Woodsmith, S. Duley, K. Salehi-Ashtiani, et al. 2009. Analysis of the humanE2 ubiquitin conjugating enzyme protein interaction network. Genome Res. 19:1905–1911.

16. vanWijk, S. J., S. J. de Vries, P. Kemmeren, A. Huang, R. Boelens, A. M. Bonvin,and H. T. Timmers. 2009. A comprehensive framework of E2-RING E3 inter-actions of the human ubiquitin-proteasome system. Mol. Syst. Biol. 5: 295.

17. Brzovic, P. S., J. R. Keeffe, H. Nishikawa, K. Miyamoto, D. Fox, 3rd,M. Fukuda, T. Ohta, and R. Klevit. 2003. Binding and recognition in the as-sembly of an active BRCA1/BARD1 ubiquitin-ligase complex. Proc. Natl. Acad.Sci. USA 100: 5646–5651.

18. Christensen, D. E., P. S. Brzovic, and R. E. Klevit. 2007. E2-BRCA1 RINGinteractions dictate synthesis of mono- or specific polyubiquitin chain linkages.Nat. Struct. Mol. Biol. 14: 941–948.

19. VanDemark, A. P., R. M. Hofmann, C. Tsui, C. M. Pickart, and C. Wolberger.2001. Molecular insights into polyubiquitin chain assembly: crystal structure ofthe Mms2/Ubc13 heterodimer. Cell 105: 711–720.

20. Ikeda, F., and I. Dikic. 2008. Atypical ubiquitin chains: new molecular signals.‘Protein Modifications: Beyond the Usual Suspects’ review series. EMBO Rep. 9:536–542.

21. Galan, J. M., and R. Haguenauer-Tsapis. 1997. Ubiquitin lys63 is involved inubiquitination of a yeast plasma membrane protein. EMBO J. 16: 5847–5854.

22. Kumar, K. G., H. Barriere, C. J. Carbone, J. Liu, G. Swaminathan, P. Xu, Y. Li,D. P. Baker, J. Peng, G. L. Lukacs, and S. Y. Fuchs. 2007. Site-specific ubiq-uitination exposes a linear motif to promote interferon-alpha receptor endocy-tosis. J. Cell Biol. 179: 935–950.

23. Geetha, T., J. Jiang, and M. W. Wooten. 2005. Lysine 63 polyubiquitination ofthe nerve growth factor receptor TrkA directs internalization and signaling. Mol.Cell 20: 301–312.

24. Kamsteeg, E. J., G. Hendriks, M. Boone, I. B. Konings, V. Oorschot, P. van derSluijs, J. Klumperman, and P. M. Deen. 2006. Short-chain ubiquitination me-diates the regulated endocytosis of the aquaporin-2 water channel. Proc. Natl.Acad. Sci. USA 103: 18344–18349.

25. Varghese, B., H. Barriere, C. J. Carbone, A. Banerjee, G. Swaminathan,A. Plotnikov, P. Xu, J. Peng, V. Goffin, G. L. Lukacs, and S. Y. Fuchs. 2008.

Polyubiquitination of prolactin receptor stimulates its internalization, post-internalization sorting, and degradation via the lysosomal pathway. Mol. Cell.Biol. 28: 5275–5287.

26. Hicke, L., H. L. Schubert, and C. P. Hill. 2005. Ubiquitin-binding domains. Nat.Rev. Mol. Cell Biol. 6: 610–621.

27. Tsirigotis, M., M. Zhang, R. K. Chiu, B. G. Wouters, and D. A. Gray. 2001.Sensitivity of mammalian cells expressing mutant ubiquitin to protein-damagingagents. J. Biol. Chem. 276: 46073–46078.

28. Coscoy, L., and D. Ganem. 2001. A viral protein that selectively downregulatesICAM-1 and B7-2 and modulates T cell costimulation. J. Clin. Invest. 107:1599–1606.

29. Ishido, S., J. K. Choi, B. S. Lee, C. Wang, M. DeMaria, R. P. Johnson,G. B. Cohen, and J. U. Jung. 2000. Inhibition of natural killer cell-mediatedcytotoxicity by Kaposi’s sarcoma-associated herpesvirus K5 protein. Immunity13: 365–374.

30. Hofmann, R. M., and C. M. Pickart. 1999. Noncanonical MMS2-encodedubiquitin-conjugating enzyme functions in assembly of novel polyubiquitinchains for DNA repair. Cell 96: 645–653.

31. Deng, L., C. Wang, E. Spencer, L. Yang, A. Braun, J. You, C. Slaughter,C. Pickart, and Z. J. Chen. 2000. Activation of the IkappaB kinase complex byTRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a uniquepolyubiquitin chain. Cell 103: 351–361.

32. McKenna, S., L. Spyracopoulos, T. Moraes, L. Pastushok, C. Ptak, W. Xiao, andM. J. Ellison. 2001. Noncovalent interaction between ubiquitin and the humanDNA repair protein Mms2 is required for Ubc13-mediated polyubiquitination. J.Biol. Chem. 276: 40120–40126.

33. McKenna, S., T. Moraes, L. Pastushok, C. Ptak, W. Xiao, L. Spyracopoulos, andM. J. Ellison. 2003. An NMR-based model of the ubiquitin-bound humanubiquitin conjugation complex Mms2.Ubc13. The structural basis for lysine 63chain catalysis. J. Biol. Chem. 278: 13151–13158.

34. Eddins, M. J., C. M. Carlile, K. M. Gomez, C. M. Pickart, and C. Wolberger.2006. Mms2-Ubc13 covalently bound to ubiquitin reveals the structural basis oflinkage-specific polyubiquitin chain formation. Nat. Struct. Mol. Biol. 13: 915–920.

35. Tsui, C., A. Raguraj, and C. M. Pickart. 2005. Ubiquitin binding site of theubiquitin E2 variant (UEV) protein Mms2 is required for DNA damage tolerancein the yeast RAD6 pathway. J. Biol. Chem. 280: 19829–19835.

36. Xie, Y., and A. Varshavsky. 1999. The E2-E3 interaction in the N-end rulepathway: the RING-H2 finger of E3 is required for the synthesis of multi-ubiquitin chain. EMBO J. 18: 6832–6844.

37. Chen, B., J. Mariano, Y. C. Tsai, A. H. Chan, M. Cohen, and A. M. Weissman.2006. The activity of a human endoplasmic reticulum-associated degradation E3,gp78, requires its Cue domain, RING finger, and an E2-binding site. Proc. Natl.Acad. Sci. USA 103: 341–346.

38. Ozkan, E., H. Yu, and J. Deisenhofer. 2005. Mechanistic insight into the allo-steric activation of a ubiquitin-conjugating enzyme by RING-type ubiquitin li-gases. Proc. Natl. Acad. Sci. USA 102: 18890–18895.

39. Siepmann, T. J., R. N. Bohnsack, Z. Tokgoz, O. V. Baboshina, and A. L. Haas.2003. Protein interactions within the N-end rule ubiquitin ligation pathway. J.Biol. Chem. 278: 9448–9457.

40. van den Elsen, P. J., T. M. Holling, H. F. Kuipers, and N. van der Stoep. 2004.Transcriptional regulation of antigen presentation. Curr. Opin. Immunol. 16: 67–75.

41. Tourdot, S., M. Nejmeddine, S. J. Powis, and K. G. Gould. 2005. Different MHCclass I heavy chains compete with each other for folding independently of beta 2-microglobulin and peptide. J. Immunol. 174: 925–933.

42. Knittler, M. R., K. Gulow, A. Seelig, and J. C. Howard. 1998. MHC class Imolecules compete in the endoplasmic reticulum for access to transporter as-sociated with antigen processing. J. Immunol. 161: 5967–5977.

43. Tussey, L. G., S. Rowland-Jones, T. S. Zheng, M. J. Androlewicz, P. Cresswell,J. A. Frelinger, and A. J. McMichael. 1995. Different MHC class I allelescompete for presentation of overlapping viral epitopes. Immunity 3: 65–77.

44. Boname, J. M., M. Thomas, H. R. Stagg, P. Xu, J. Peng, and P. J. Lehner. 2010.Efficient internalization of MHC I requires lysine-11 and lysine-63 mixedlinkage polyubiquitin chains. Traffic 11: 210–220.

45. Cadwell, K., and L. Coscoy. 2005. Ubiquitination on nonlysine residues bya viral E3 ubiquitin ligase. Science 309: 127–130.

46. Matsuki, Y., M. Ohmura-Hoshino, E. Goto, M. Aoki, M. Mito-Yoshida,M. Uematsu, T. Hasegawa, H. Koseki, O. Ohara, M. Nakayama, et al. 2007.Novel regulation of MHC class II function in B cells. EMBO J. 26: 846–854.

47. Bartee, E., M. Mansouri, B. T. Hovey Nerenberg, K. Gouveia, and K. Fruh. 2004.Downregulation of major histocompatibility complex class I by human ubiquitinligases related to viral immune evasion proteins. J. Virol. 78: 1109–1120.

48. Hoer, S., L. Smith, and P. J. Lehner. 2007. MARCH-IX mediates ubiquitinationand downregulation of ICAM-1. FEBS Lett. 581: 45–51.

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