RESEARCH ARTICLE SUMMARY◥

PROTEOSTASIS

UBE2O remodels the proteome duringterminal erythroid differentiationAnthony T. Nguyen,* Miguel A. Prado,* Paul J. Schmidt, Anoop K. Sendamarai,Joshua T. Wilson-Grady, Mingwei Min, Dean R. Campagna, Geng Tian, Yuan Shi,Verena Dederer, Mona Kawan, Nathalie Kuehnle, Joao A. Paulo, Yu Yao, Mitchell J. Weiss,Monica J. Justice, Steven P. Gygi, Mark D. Fleming,† Daniel Finley†

INTRODUCTION: The reticulocyte–red bloodcell transition is a canonical example of ter-minal differentiation. The mature red bloodcell has one of the simplest cellular proteomesknown, with hemoglobin remarkably concen-trated to ~98%of soluble protein. During retic-ulocytematuration, the proteome is remodeledthrough the programmed elimination of mostgeneric constituents of the cell, in parallel withabundant synthesis of cell type–specific pro-teins such as hemoglobin. Themechanisms thatdrive rapid turnover of soluble and normallystable proteins in terminally differentiating cellsremain largely unclear.

RATIONALE:The ubiquitin-proteasome system(UPS) was discovered in reticulocytes, whereit is highly active. However, its function in thisdevelopmental context has not been established.UBE2O is an E2 (ubiquitin-conjugating) enzymethat is co-induced with globin and expressedat elevated levels late in the erythroid lineage.We identified an anemic mouse line with a nullmutation inUbe2o, and used multiplexed quan-titative proteomics to identify candidate sub-strates of UBE2O in an unbiased and globalmanner. We found that the protein compo-sitions of mutant and wild-type reticulocytesdiffered markedly, suggesting that UBE2O-

dependent ubiquitination might target its sub-strates for degradation to effect remodeling ofthe proteome.To test whether UBE2O was sufficient for

proteome remodeling, we engineered a non-erythroid cell line to inducibly express UBE2Oabove its basal level. Upon induction, we ob-served the decline of hundreds of proteins fromthese cells, in many cases the same proteinsas those eliminated from reticulocytes. Over-expression of an active-site mutant of UBE2Odid not show these effects. Therefore, a ma-jor component of the specificity underlyingdifferentiation-linked proteome remodelingappears to be carried by UBE2O itself. Theseresults also indicate that UBE2O may func-tion as a hybrid enzyme with both E2 and E3(ubiquitin-ligating) activities. In support ofthis model, candidate substrates identifiedby proteomics were ubiquitinated by purifiedUBE2O without the assistance of additionalspecificity factors.

RESULTS: The most prominent phenotypesof theUbe2omutant are an anemia character-ized by small cells with lowhemoglobin content(microcytic hypochromic anemia), and a defectin the elimination of ribosomes, the latter beinga key aspect of reticulocytematuration.When

we added recombinant UBE2O protein to re-ticulocyte lysates fromthenullmutant, ubiquitinwas conjugatedprimarily to ribosomal proteins.Moreover, immunoblot analysis and quantita-tive proteomics revealed elevated levels ofmul-tiple ribosomal proteins inmutant reticulocytes.Sucrose gradient analysis indicated the persist-ence not only of ribosomal proteins but of ribo-somes themselves during ex vivo differentiation

ofmutant reticulocytes.Ac-cordingly, ribosomeswereeliminated upon inductionofUBE2Oinnon-erythroidcells. The elimination oforganelles from reticulo-cytes, as exemplified by

that of mitochondria, was not affected in theUbe2omutant, indicating the specificity of itseffects on programmed protein turnover.Free ribosomal proteins were ubiquitinated

by purified UBE2O, which suggests that theseproteins are true substrates of the enzyme. How-ever, UBE2O substrates are diverse in natureand not limited to ribosomal proteins. Indi-vidual domains of UBE2O bound substrateswith distinct specificities. Thus, the broad spec-ificity of UBE2O reflects the presence of mul-tiple substrate recognition domains within theenzyme.Proteasome inhibitors blocked the degra-

dation of UBE2O-dependent substrates inreticulocytes, although UBE2O does not formpolyubiquitin chains. Rather, UBE2O adds singleubiquitin groups to substrates at multiple sites.Proteasome inhibitor treatment ex vivo led todepletion of the pools of many amino acids;this result implies that the flux of ubiquitinatedsubstrates through the reticulocyte proteasomeis sufficient to supply amino acids needed forlate-stage translation of mRNA. In late eryth-ropoiesis, several ubiquitin-conjugating enzymesand ligases are induced together with Ube2owhile most components of the UPS disappear.We propose that the UPS is not simply amplifiedduring erythroid maturation, but is insteadbroadly reconfigured to promote remodelingof the proteome.

CONCLUSION: A highly specialized UPS isexpressed in the reticulocyte and is used toremodel the proteome of these cells on a globalscale. UBE2O, a hybrid E2-E3 enzyme, functionsas a major specificity factor in this process. Inreticulocytes, and perhaps in other differenti-ated cells such as in the lens, the induction ofubiquitinating factors may drive the transitionfrom a complex to a simple proteome.▪

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Nguyen et al., Science 357, 471 (2017) 4 August 2017 1 of 1

The list of author affiliations is available in the full article online.*These authors contributed equally to this work.†Corresponding author. Email: mark.fleming@childrens.harvard.edu (M.D.F.); daniel_finley@hms.harvard.edu (D.F.)Cite this article as A. T. Nguyen et al., Science 357,eaan0218 (2017). DOI: 10.1126/science.aan0218

UBE2O drives remodeling of the proteome during erythroid differentiation.The transformation of reticulocytes into erythrocytes involves the elimination of myriadproteins. UBE2O is an E2-E3 hybrid enzyme that directly recognizes and ubiquitinates proteinsthat are fated for elimination. The target protein is degraded by the proteasome; ubiquitin (Ub)is recycled. UBE2O substrates include ribosomal proteins, recognized in a free form or possiblywithin the ribosome complex.

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RESEARCH ARTICLE◥

PROTEOSTASIS

UBE2O remodels the proteome duringterminal erythroid differentiationAnthony T. Nguyen,1* Miguel A. Prado,1* Paul J. Schmidt,2 Anoop K. Sendamarai,2

Joshua T. Wilson-Grady,1 Mingwei Min,1† Dean R. Campagna,2 Geng Tian,1 Yuan Shi,1‡Verena Dederer,1§ Mona Kawan,1§ Nathalie Kuehnle,1§ Joao A. Paulo,1 Yu Yao,3

Mitchell J. Weiss,3 Monica J. Justice,4 Steven P. Gygi,1 Mark D. Fleming,2‖ Daniel Finley1‖

During terminal differentiation, the global protein complement is remodeled, asepitomized by erythrocytes, whose cytosol is ~98% globin. The erythroid proteomeundergoes a rapid transition at the reticulocyte stage; however, the mechanisms drivingprogrammed elimination of preexisting cytosolic proteins are unclear. We found thata mutation in the murine Ube2o gene, which encodes a ubiquitin-conjugating enzymeinduced during erythropoiesis, results in anemia. Proteomic analysis suggested thatUBE2O is a broad-spectrum ubiquitinating enzyme that remodels the erythroid proteome.In particular, ribosome elimination, a hallmark of reticulocyte differentiation, was defectivein Ube2o−/− mutants. UBE2O recognized ribosomal proteins and other substratesdirectly, targeting them to proteasomes for degradation. Thus, in reticulocytes, theinduction of ubiquitinating factors may drive the transition from a complex to asimple proteome.

The cytosol of mature red blood cells (RBCs) is~98% globin (1), constituting one of the sim-plest cellular proteomes known. Achievingthis unique state may require programmeddegradation of preexisting cellular proteins

in terminally differentiating erythroid precursors.Interest in this problem led to the discovery ofthe ubiquitin-proteasome system (UPS) in re-ticulocytes, where it is highly active (2, 3). Thesynthesis of certain components of the UPS, par-ticularly ubiquitin-conjugating enzyme UBE2O(also called E2-230K), is highly induced in theerythroid lineage in parallel with globin, contem-poraneously with the loss of many other UPS com-ponents (4). The gene encoding UBE2O appearsto share a GATA1-dependent mechanism of tran-scriptional induction with globin (5), leading toUBE2O expression in reticulocytes as well as theirerythroblast precursors (6–8). Thus, a program ofubiquitination that is specific to terminal differ-entiation might drive remodeling of the erythroidproteome. We assessed this model after identifyinga mutation, hem9, in the gene encoding UBE2O.

The autosomal recessive hem9 mutation wasidentified in a systematic ethylnitrosourea screenfor mouse mutants (9). Homozygous hem9 mu-tants have a form of anemia characterized bysmall cells (microcytosis), reduced concentrationsof hemoglobin (hypochromia), and elevated RBCcounts (erythrocytosis) (Table 1). Reciprocal trans-plantation studies demonstrated that these phe-notypes are intrinsic to the hematopoietic system(fig. S1). Histological characterization of peripheralblood smears (fig. S2, A and B) confirmed the phe-notype of hypochromic anemia.We mapped hem9 to a ~309-kb interval on chro-

mosome 11. A single gene in this interval, Ube2o,was expressed in erythroid precursors far aboveits basal level in other cell types (4, 10, 11). Se-quencing revealed a nonsense mutation predictedto truncate the C-terminal 168 amino acids. UBE2Oprotein was undetectable by immunoblot analysisof reticulocyte-rich blood from Ube2ohem9 animals(fig. S2C). To validate the mutation, we bred hem9mutants to two Ube2o gene-trap alleles. Com-pound heterozygotes lacked UBE2O protein andexhibited an RBC phenotype similar to that ofhem9 homozygotes (fig. S2D). Thus, hem9 is anull allele of Ube2o (hereafter Ube2o−/−).Ube2o−/− reticulocytes showed reductions in

many ubiquitin conjugate species (Fig. 1A). Giventhat hundreds of ubiquitinating factors are ex-pressed in mammals, it is unusual to find such adominant role of a single component. Unassembledand misfolded globins are preferentially ubiquiti-nated, as seen in b-thalassemia, where unpaireda-globin precipitates cause cellular damage, in-effective erythropoiesis, hemolysis, and anemia (12).Purified UBE2O ubiquitinated purified a-globin,although some ubiquitination of endogenous

a-globin persisted in the Ube2o−/−mutant (fig. S3,A and B).We tested whether UBE2O-dependent degra-

dation of excess a-globin might contribute tothe Ube2o−/− phenotype by crossing a deletionof the b-globin loci (Hbbth3 allele), which causesa b-thalassemia intermedia phenotype (13), withthe Ube2o−/− line. However, UBE2O deficiency ac-tually increased hemoglobin levels in theHbbth3/+

mutant (Table 1). Mitigation of the anemia in thedouble mutant was accompanied by increasednumbers of RBCs (Table 1), which were smallerin mean volume than controls and had diminishedhemoglobin content.UBE2O deficiency markedly reduced precipi-

tated hemoglobin aggregates that are associatedwith b-globin deficiency, and accordingly dimin-ished the accumulation of insoluble a-globin,without affecting the steady-state a/b-globin ratiofor soluble protein (fig. S4, A to D). Furthermore,double mutants showed reduced splenomegaly,a major consequence of b-thalassemia causedby increased RBC destruction and heightenedsplenic erythropoiesis. RBC life span was alsoincreased in the double mutants, which is con-sistent with a reduction in harmful unpaireda-globin chains (fig. S4E).In summary, although a-globin is a UBE2O

substrate, defective ubiquitination and degrada-tion of a-globin could not fully explain theUbe2o−/−

phenotype; instead, the loss of UBE2O amelioratedphenotypes attributable to a-globin excess. Thereduced globin levels of the mutant appear to re-flect attenuated translation of a- and b-globin, asindicated by ribosome profiling (fig. S5, A and B).In the mutant, eukaryotic initiation factor 2a(eIF2a) phosphorylation was induced, which is sup-pressive of translation and may therefore accountfor the reduction of globin expression (fig. S5C).

Ribosomal proteins are abundantlyubiquitinated by UBE2O

To identify additional substrates of UBE2O, weadded recombinant enzyme or a catalytically in-active mutant (UBE2O-C1037A; hereafter UBE2O-CA), at physiological levels, to reticulocyte lysatesfrom null animals. Prior to the reaction, endog-enous E2 activity was quenched chemically. Re-combinant UBE2O formed ubiquitin conjugatesrobustly, whereas UBE2O-CA was inactive (Fig. 1B).Newly formed conjugates were identified by liquidchromatography–tandem mass spectrometry (LC-MS/MS) analysis (table S1). Although numeroustargets were identified, including a- and b-globin(fig. S6A), the major class of targets was ribosomalproteins (RPs), comprising 87% of spectral countsof all ubiquitinated peptides that were specific towild-type UBE2O, other than those from UBE2Oitself (Fig. 1C).The two most frequently identified ubiquiti-

nated RPs were RPL29 and RPL35, which hadbeen suggested to interact with UBE2O by high-throughput affinity purification mass spectrome-try (14). Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis (15) of all105 UBE2O targets (table S1) showed a strongselection for RPs (adjusted P value = 3.65 × 10−17);

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Nguyen et al., Science 357, eaan0218 (2017) 4 August 2017 1 of 7

1Department of Cell Biology, Harvard Medical School, Boston,MA 02115, USA. 2Department of Pathology, Boston Children’sHospital and Harvard Medical School, Boston, MA 02115,USA. 3Department of Hematology, St. Jude Children’sResearch Hospital, Memphis, TN 38105, USA. 4Genetics andGenome Biology Program, Hospital for Sick Children, PeterGilgan Centre for Research and Learning, Toronto, OntarioM5G 0A4, Canada.*These authors contributed equally to this work. †Present address:Department of Chemistry and Biochemistry, University of Colorado,Boulder, CO 80309, USA. ‡Present address: Department ofMolecular and Clinical Pharmacology, University of California, LosAngeles, CA 90095, USA. §Present address: Zentrum für MolekulareBiologie der Universität Heidelberg, 69120 Heidelberg, Germany.||Corresponding author. Email: mark.fleming@childrens.harvard.edu (M.D.F.); daniel_finley@hms.harvard.edu (D.F.)

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STRING analysis (16) also showed a definitivecluster of RPs (fig. S6, B and C). When conju-gate purification was repeated under denatur-ing conditions, RPs remained the major class ofubiquitination targets (fig. S6D and table S1).

Thus, the ubiquitination pattern does not re-flect preexisting UBE2O-independent ubiquitinmodifications that cofractionated with UBE2O-dependent modifications simply because theywere present on the same ribosome.

A hallmark of the transition from the reticu-locyte to the erythrocyte stage of differentiationis the elimination of ribosomes (17). The associ-ated mechanism is poorly understood (18, 19). Weprobed reticulocyte lysates from wild-type and

Nguyen et al., Science 357, eaan0218 (2017) 4 August 2017 2 of 7

Table 1. Ube2o−/− mice have hypochromic, microcytic anemia. Erythropoietic parameters in Ube2ohem9 (Ube2o−/−) and genetic interaction between

Ube2o−/− and the b-globin–deficient Hbbth3 (th3) allele. Spleen:BW, ratio of spleen weight to body weight; RBC, red blood cells; HGB, hemoglobin; MCV,

mean cell volume; MCH, mean corpuscular hemoglobin; RETIC ABS, absolute reticulocytes; CHr, mean reticulocyte hemoglobin content. All studies wereperformed in 8-week-old female littermates, with n = 6 animals in each group. Significance was calculated by one-way ANOVA with Tukey multiple-comparisons

test. *P < 0.05 versus wild type; †P < 0.05 versus Hbb+/+ Ube2o−/−; ‡P < 0.05 versus Hbbth3/+ Ube2o+/+. Values are means ± SD. (See also fig. S3F.)

Hbb Ube2oSpleen:BW

(mg/g)

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(×106/ml)

HGB

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MCV

(fl)

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RETIC ABS

(×106/ml)

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+/+ +/+ 3.9 ± 0.2 8.50 ± 0.27 13.2 ± 0.8 58.9 ± 1.7 15.6 ± 0.5 0.22 ± 0.02 15.4 ± 0.6.. .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ..

+/+ −/− (hem9) 3.8 ± 0.2 11.59 ± 0.39* 11.6 ± 0.6* 43.3 ± 1.3* 10.0 ± 0.2* 0.44 ± 0.12 12.0 ± 0.3*.. .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ..

th3/+ +/+ 22.4 ± 1.1*† 6.86 ± 0.41*† 6.6 ± 0.2*† 48.5 ± 1.1*† 9.8 ± 0.6* 1.66 ± 0.35*† 12.9 ± 0.3*†.. .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ..

th3/+ −/− (hem9) 9.8 ± 4.0*†‡ 11.53 ± 0.86*‡ 8.3 ± 0.8*†‡ 34.0 ± 1.1*†‡ 7.1 ± 0.4*†‡ 1.19 ± 0.22*†‡ 9.7 ± 0.2*†‡.. .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ..

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RPL36ARPS19RPS30RPS9RPS6RPS25RPS15

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Total 49 152

Fig. 1. Ube2o−/− reticulocytes are deficient in eliminating ribosomalproteins. (A) Proteins from reticulocyte lysates were resolved by SDS–polyacrylamide gel electrophoresis (PAGE) and immunoblotted with anantibody to ubiquitin. Each sample is from a different mouse whosereticulocytes were induced by serial bleeding. Ub-HBA represents theubiquitinated form of a-globin. (B) Ubiquitination was reconstituted inUbe2o−/− reticulocyte lysates by means of recombinant UBE2O. Reactionswere supplemented with biotin-tagged ubiquitin and ubiquitin-activatingenzyme (UBE1) and were incubated for 45 min at 37°C. Samples wereresolved by SDS-PAGE. Proteins were electroblotted and visualized usingstreptavidin–horseradish peroxidase.W, UBE2O-WT (wild type); C, UBE2O-CA(the catalytically inactive C1037A mutant, used as a control). UBE2O-CAdid not have conjugating activity in lysates, as the only bands evident in thiscase were biotin-ubiquitin and autoubiquitinated E1. In the absence oflysate, UBE2O was autoubiquitinated, as previously reported (27). (C) UBE2Oubiquitinates RPs in reconstituted Ube2o−/− lysates. Ubiquitin conjugates(see Fig. 2A) purified by NeutrAvidin-biotin pull-down were digested on beads

with trypsin. Peptides containing diglycine-modified lysines (ubiquitination sites)were identified and mapped by LC-MS/MS. All RP ubiquitination events wereunique to the UBE2O-WTsample. Ub sites, unique ubiquitination sites identified;D spectral counts are obtained by subtracting all spectral counts collected inthe UBE2O-CA sample from those of the UBE2O-WT reaction for a givendiglycine peptide over six replicate experiments. (D) Levels of RPs from Ube2o−/−

and wild-type reticulocytes assessed by immunoblotting (a denotes anantibody). GAPDH and b-spectrin are loading controls; 100 mg of protein wasloaded per lane. (E) Volcano plot of quantitative proteomics analysis,representing the relation of the log10 of the P value adjusted using Benjamini-Hochberg correction [–log10(adj. P value)] and the log2 of the fold change[log2(Ube2o

−/−/Ube2o+/+)]. Proteins that were significantly (adj. P value <0.05) up-regulated or down-regulated at least 25% in Ube2o−/− samples areshown in red and blue, respectively. Highly significant enrichment of ribosomes(adj. P value = 3.23 × 10−6) was found by KEGG pathway enrichment analysisof all proteins up-regulated significantly and by more than 25% in Ube2o−/−

reticulocytes. Numbers of proteins per group are indicated in parentheses.

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Ube2o−/− mice with antibodies to seven RPs(Fig. 1D). In all cases, we observed elevated levelsin the mutants, consistent with a broad defect inRP elimination in Ube2o−/− mice.To search globally for proteins that may be

targets of UBE2O, we compared the proteomesof wild-type andUbe2o−/− reticulocytes by tandemmass tagging (TMT) mass spectrometry (20)(fig. S7). Of 1235 proteins quantified (Fig. 1E andtable S2), 183 were elevated in mutant reticulo-cytes. The results suggested that UBE2O, wheninduced to high levels, might execute a broad pro-gram of ubiquitination and degradation. KEGGpathway enrichment analysis yielded a highlysignificant enrichment of ribosomes (Fig. 1E).Ferritins (FTL1 and FTH1) were also elevated inUbe2o−/− reticulocytes (Fig. 1E). The increase inferritin levels appears to reflect translational con-trol, because ribosome profiling studies showedelevated levels of translating mRNA for Ftl1 andFth1 in the mutant (fig. S8A). Ribosome pausingwas prominent at iron-responsive elements withthe Ftl1 and Fth1 mRNAs in wild-type but notmutant reticulocytes (fig. S8B); this result sug-gested that Ube2o−/− reticulocytes are respondingto increased cytosolic iron or heme levels andthat reticulocyte iron deficiency is unlikely to bethe cause of the anemia. Accordingly, the heme-regulated eIF2a kinase (HRI), a sensor of irondeficiency, was not induced in Ube2o−/− mutants,nor did a null mutation in the HRI-encoding genemodify the anemic phenotype (fig. S9).

Ube2o−/− mice are deficient inribosome elimination

We next tested whether UBE2O governs the levelof ribosomes or, more narrowly, that of free RPs,which are typically unstable (21). Sucrose gradi-ent analysis showed that Ube2o−/− reticulocyteshave elevated levels of ribosomes, particularly 80Smonosomes (Fig. 2A and fig. S10). When culturedex vivo, reticulocytes mature into erythrocytesover 48 to 72 hours (19, 22), with both ribosomes

and mitochondria being eliminated. Culturedreticulocytes were analyzed by flow cytometryusing MitoTracker Deep Red, which stains viablemitochondria, and thiazole orange, which stainsRNA; in reticulocytes, the most abundant RNAis ribosomal (rRNA) (Fig. 2B). After 72 hours ofex vivo differentiation, the median fluorescenceintensity of thiazole orange staining in Ube2o−/−

reticulocytes was higher than in the wild type bya factor of 28, whereas MitoTracker staining washigher in the mutant only by a factor of 1.6. Thelack of significant UBE2O involvement in mito-chondrial elimination is consistent with previousindications that maturation-associated mitophagy,an autophagic process, is ubiquitin-independentin reticulocytes (19, 23).Polysome profiles from wild-type reticulocytes

cultured ex vivo showed that ribosome elimina-tion was nearly complete by 31 hours; by contrast,the 80S monosome peak remained prominent inUbe2o−/− reticulocytes (Fig. 2A), consistent withdelayed turnover of multiple RPs during matu-ration (fig. S11). Thus, ribosome levels are ele-vated in Ube2o−/− reticulocytes because of a defectin ribosome elimination during terminal differ-entiation. Despite the persistence of this peak,few polysomes were evident in the gradient pro-file, perhaps because of low levels of mRNA ortranslational initiation factors at this stage ofdifferentiation.

UBE2O is sufficient to drive ribosomeelimination in non-erythroid cells

Having found that UBE2O is critical for ribosomeelimination in reticulocytes, we tested whether itis sufficient for elimination in a non-erythroidcell, as might be expected if UBE2O recognizedRPs directly. We integrated the full-lengthUBE2Oopen reading frame into Flp-In T-REx 293 cellsunder a doxycycline-inducible promoter. TheUBE2O-CAmutant, integrated at the same locus,was used as a control (Fig. 3A and fig. S12, A andB). UBE2O was induced for 48 to 72 hours to

recapitulate the period of terminal erythroid mat-uration (fig. S12A). 293-E2O cells remained viable,with no indication of induced cell death (fig.S12C). The effect of UBE2O expression on theproteome was assessed by mass spectrometry,which quantified 7807 proteins (fig. S13, A and B,and table S3). Upon UBE2O induction, we iden-tified 685 proteins present at substantially lowerlevels (≤50%) than in UBE2O-CA by day 3 (Fig.3B), with a clear preferential effect on basic pro-teins (Fig. 3D). Thus, ectopic UBE2O inductionled to extensive proteome remodeling.Among proteins whose levels were reduced

more than 50% upon UBE2O induction, KEGGpathway enrichment analysis identified a highlysignificant down-regulation of RPs (Fig. 3, Band C). This was confirmed by immunoblottingfor RPL29, RPL35, and RPL23A (Fig. 3E). RPswere not degraded at comparable rates, in-consistent with what would be expected for anautophagocytic mechanism. Ribosome degrada-tion might proceed in steps, with RPs showingthe fastest degradation more likely to be directsubstrates of UBE2O. We also observed signifi-cant enrichment in ribosome biogenesis and rRNAprocessing pathways, which are nonfunctional inreticulocytes because they are enucleate (Fig. 3Band fig. S13, C to E). These effects of UBE2O arelikely direct, because multiple ribosomal and nu-cleolar proteins were ubiquitinated by recombi-nant UBE2O when added to extracts of humanembryonic kidney (HEK) 293 cells and analyzed bymass spectrometry (fig. S14, A to C, and table S1).After UBE2O induction, polysome profiles

showed a strong reduction of the 80S mono-some peak (Fig. 3F). Although reticulocytes can-not produce ribosomes de novo, a reduction ofribosome levels in 293-E2O cells could reflecteither degradation of preexisting ribosomes orinterference with ribosome synthesis. To discrim-inate between these possibilities, we suppressedthe transcription of new rRNA with a selectiveinhibitor of RNA polymerase I, CX-5461 (24, 25).Under these “chase” conditions, UBE2O drove thedestabilization of RPs after 24 to 48 hours ofinhibitor treatment, with no effect observed inthe catalytic null mutant (fig. S15). Therefore,preexisting RPs can be eliminated by UBE2O.In summary, the 293-E2O cell data recapitulate

a critical aspect of normal erythroid differenti-ation and confirm the inducible breakdown ofribosomes—complexes that are thought to behighly stable under basal conditions. Further-more, UBE2O is sufficient to drive ribosome elim-ination in non-erythroid cells in the absence ofreticulocyte-specific factors, which suggests thatthe control of erythroid ribosome turnover maybe dependent principally on UBE2O itself.

UBE2O executes a broad programof ubiquitination

On the basis of proteomic data on reticulocytesand 293-E2O cells, we selected seven potentialnonribosomal targets of UBE2O for confirma-tion by immunoblotting. We observed stronglyelevated levels of all of these proteins in mutantreticulocytes, including a chaperone for a-globin,

Nguyen et al., Science 357, eaan0218 (2017) 4 August 2017 3 of 7

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Fig. 2. Ube2o−/− reticulocytes have elevated levels of ribosomes. (A) Polysome profiles weregenerated by fractionating Ube2o−/− and wild-type reticulocyte lysates on 20 to 50% sucrosegradients. The x axis represents position in the gradient; A260, absorbance at 260 nm. Relative to thewild type, Ube2o−/− reticulocytes showed an elevated 80S monosome peak when freshly isolated(left) and after 31 hours of ex vivo differentiation (right). (B) Flow cytometry analysis after ex vivodifferentiation. Wild-type CD71+ reticulocytes showed progressive loss of both MitoTracker Deep Redand thiazole orange staining. Ube2o−/− CD71+ reticulocytes retained thiazole orange staining after72 hours of ex vivo differentiation (top); mitochondrial elimination was unaffected (bottom).

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AHSP (a-hemoglobin stabilizing protein) (Fig. 4A).AHSP was strongly stabilized in a time-coursestudy using ex vivo cultured Ube2o−/− reticulo-cytes (fig. S16A). However, elevated AHSP levels donot explain the erythroid phenotype of Ube2o−/−

mutants, because an Ahsp−/− mutation did notalleviate the Ube2o−/− phenotype (fig. S16, B andC). In summary, immunoblot analysis validatedthe proteomics data and thus suggested thatan extensive set of RPs and non-RPs is elim-inated from reticulocytes under the control ofUBE2O.Our finding that UBE2O is sufficient to direct

the degradation of its target proteins suggeststhat it may mediate direct recognition of theseproteins. However, E3 rather than E2 enzymestypically mediate substrate recognition (26). Themolecular mass of UBE2O is 143 kDa (Fig. 4B),whereas typical E2 enzymes are of 20 to 25 kDa. Ittherefore seemed likely that UBE2O may func-tion as an E3 enzyme fused to an E2 (27–29). Totest this model, we reconstituted the ubiquitina-tion of candidate UBE2O substrates with purifiedcomponents.When partially purified ribosomes were tested

by immunoblot analysis, we observed that ubiq-uitination of RPs was efficient relative to thatof histone H2B, a model substrate of UBE2O(Fig. 4C). The ubiquitin acceptor proteins in thisreaction were confirmed as RPs by LC-MS/MS–mediated ubiquitination site mapping (fig. S17and table S1). Although these proteins were addedto the assay principally in the form of ribosomecomplexes, it is possible and perhaps likely thatUBE2O prefers the free forms of these proteinsas substrates. Indeed, when tested individually,recombinant RPL35, RPL36A, and RPL37 allproved to be UBE2O substrates (Fig. 4D). Otherproteins predicted from proteomics data to beUBE2O substrates were all efficiently ubiquiti-nated, whereas calmodulin, a negative control,was not (Fig. 4D). UBE2O added multiple ubiq-uitin groups to these substrates, although almostexclusively in the form of multi-monoubiquitinmodification (fig. S18). In summary, several in vivotargets of UBE2O were also modified by thisenzyme in a purified system, which suggests thatUBE2O, in addition to being an E2 enzyme, isalso an E3—a specificity-determining ubiquiti-nation factor.In addition to its E2 (or UBC) domain, UBE2O

has four distinct evolutionarily conserved re-gions: CR1, CR2, CR3, and a predicted coiled-coil (CC) element (Fig. 4B). When truncated formsof UBE2O were assayed, the importance of thetested domains for conjugation rates varied depend-ing on the substrate tested, consistent with CR1and perhaps other conserved domains being sub-strate specificity elements (Fig. 4C). The hypothesisthat CR1 and CR2 are substrate recognition domainswas assessed more directly by testing them forinteraction with UBE2O substrates in pull-downbinding assays. Both CR1 and CR2 bound UBE2Osubstrates, although their binding specificity dif-fered (Fig. 4E). CR1 and CR2 contain acidic patches,which may be involved in their binding to basicproteins. A confirmed acidic substrate, AHSP, did

not show significant binding to CR1 or CR2 (Fig. 4E),nor was its ubiquitination dependent on thesedomains (Fig. 4C). These results indicate that UBE2Ois capable of multiple modes of substrate recog-nition. In summary, UBE2O has the expectedfeatures of an E2-E3 hybrid enzyme in whichubiquitin is charged by the UBC domain of UBE2O,to be subsequently donated to proteins that arerecognized in most cases by the CR1 and CR2domains.

Ribosomal proteins are degraded bythe proteasome

Previous studies have shown that regulated ribo-some degradation occurs via autophagy in yeast(30–33). In reticulocytes, however, inhibitors ofautophagy do not affect ribosome breakdown(19). Selective autophagy in reticulocytes is me-

diated by NIX, but this factor drives the elim-ination of mitochondria rather than ribosomes(19, 23, 34).To test whether RPs are eliminated by the

proteasome, we incubated reticulocytes with theproteasome inhibitors PS-341 and epoxomicin.After ex vivo differentiation, treated wild-typereticulocytes retained thiazole orange staining,resembling Ube2o−/− reticulocytes that had notbeen exposed to proteasome inhibitor (Fig. 5Aand fig. S19A). These data suggest that RPs aredegraded by the proteasome. In fact, treated wild-type reticulocytes retained their thiazole orangestaining more fully than did untreated Ube2o−/−

reticulocytes (Fig. 5A), suggesting the existenceof alternative pathways of ribosome eliminationthat are UBE2O-independent but proteasome-dependent. In contrast to the proteasome, p97—

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Fig. 3. UBE2O is sufficient to drive the elimination of ribosomes in HEK293-derived cells.(A) UBE2O induction by doxycycline treatment in Flp-In T-REx 293 cells was assessed byimmunoblotting with antibodies to UBE2O. Full-length human UBE2O-WTand UBE2O-CA genes weregenomically integrated in an untagged form and induced with doxycycline for 24 hours. GAPDH isa loading control; 20 mg of cell lysate was loaded per lane. (B and C) Mass spectrometry (TMT)quantification of 7808 proteins after UBE2O-WT or UBE2O-CA induction for 0, 12, 24, 48, and72 hours. (B) All 685 proteins that were down-regulated more than 50% after 72 hours of UBE2O-WTinduction, in comparison to UBE2O-CA. Coloring reflects Pearson correlation (r) to the medianpattern, from high (red) to low (black). (C) Results from KEGG pathway enrichment analysis, whichindicated highly significant enrichment (adj. P value = 2.77 × 10−6) of ribosomes, with 18 proteinsdown-regulated. (D) Isoelectric point (pI) comparison of all quantified proteins (red) and proteinsdown-regulated by more than a factor of 2 (blue), based on data from (B). pI values were obtainedfrom the proteome pI database (46). (E) After induction of UBE2O-WT or UBE2O-CA for theindicated times (48 and 72 hours), RP levels were analyzed by immunoblotting. GAPDH is a loadingcontrol; 20 mg of cell lysate was loaded per lane. All samples were from the same experiment.(F) Sucrose gradient analysis of cells overexpressing UBE2O-WT showed a reduced 80S monosomepeak after 72 hours of doxycycline treatment.

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which dissociates components of several multi-subunit complexes in a ubiquitin-dependentmanner (35)—had no detectable effect on ribo-some elimination (fig. S20).Ubiquitin depletion is evident in reticulo-

cytes treated with the highest concentrations ofproteasome inhibitors (fig. S19B). To addressthis potential caveat, we reconstituted RP deg-radation in a cell-free reticulocyte lysate sys-tem, which permitted supplementation of freeubiquitin. The degradation of RPs was reconsti-tuted by adding back UBE2O to null reticulocytelysate. RPs were not destabilized by UBE2O-CA,or under conditions of proteasome inhibitor treat-ment, while maintaining free ubiquitin levels (Fig.5B and fig. S19C). However, the stabilizing ef-fect of the inhibitors, although apparently com-plete for RPL29 and RPS23, was partial in thecase of RPL35 and RPL36A, as judged from theintensity of the major protein band (Fig. 5B andfig. S19C). For both proteins, a high–molecularweight species appeared upon proteasome in-hibition, which likely represented ubiquitinatedforms of RPL35 and RPL36A. To confirm thatproteasome inhibitors completely blocked RPdegradation, we treated the reaction with USP21to deubiquitinate high–molecular weight formsof the protein. This treatment concentrated RPL35into a single band that was constant in intensityover the time course of incubation (fig. S19D).The importance of the proteasome in remodel-

ing the proteome in reticulocytes was furthertested by metabolomic analysis. Amino acids suchas Lys and Arg, which are abundant in RPs, wereindeed depleted by proteasome inhibitors (Fig.5C). We therefore considered whether UBE2Omight cooperate with other ubiquitinating fac-tors that may be induced in late erythroid differ-entiation. Using RNA-sequencing data for erythroidcells taken from progressive stages of differentia-tion (6–8), we identified eight E2 and E3 enzymesthat are induced in late erythroid cells in parallelwith UBE2O (Fig. 5D). We hypothesize that theseenzymes work in concert to globally remodel theerythroid proteome. Expression of the vast ma-jority of ubiquitinating enzymes is extinguishedduring this period (fig. S21, A and B), so thatthe UPS is not simply amplified during ery-throid differentiation, but is instead deeply re-configured. The unique transformation of theUPS in these cells may reflect a strategy toeliminate thousands of cellular proteins fromreticulocytes, thus enabling extreme concentra-tion of globin.

Discussion

In many cases of terminal cellular differentiation,the proteome is remodeled on a global scale togenerate a highly specific cellular phenotype. Theextreme case is erythroid differentiation, wherea single species, hemoglobin, is concentrated to~98% of soluble protein (1). Accomplishing thisrapid transformation of the cellular phenotyperequires not only highly active protein synthe-sis, but also degradation of a vast set of preex-isting, otherwise relatively stable, soluble proteins.This degradation must also be selective, so as

to spare properly assembled globin and othercomponents of the mature RBC. Despite this activedegradation, most components of the ubiquitinpathway are down-regulated transcriptionallyduring erythroid differentiation. However, a smallsubset of ubiquitin-conjugating enzymes andubiquitin ligases is induced, among them UBE2O.We propose that UBE2O plays a major role inproteomic remodeling, with tens and perhapshundreds of proteins being eliminated throughits activity.

As an E2-E3 hybrid enzyme, UBE2O functionsautonomously and can thus remain active wellinto the reticulocyte stage, promoting maturationat a time when most E2 enzymes have been lost.The ability of UBE2O to promote reticulocyte mat-uration also depends on its broad substrate spec-ificity; multiple substrate recognition domainsenable the recognition of diverse substrates andmay, through avidity, allow coordinate low-affinitysubstrate interactions to be productive of ubiquiti-nation. In an accompanying paper, Yanagitani et al.

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Fig. 4. UBE2O recognizes substrates directly. (A) Immunoblotting of Ube2o−/− and wild-typereticulocyte lysates showed an elevation of putative nonribosomal UBE2O substrates. GAPDHand b-spectrin were loading controls; 100 mg of protein was loaded per lane. (B) Schematicrepresentation of domains within UBE2O. The T1 construct lacks conserved region 1 (CR1), whereasT2 lacks both CR1 and CR2. In the CA mutant, the active-site Cys1037 is substituted with Ala(indicated by asterisk). Domain assignment is described in the supplementary materials. (C) In vitroubiquitination assays were performed in a purified system with UBE1, hemagglutinin (HA)–taggedubiquitin, UBE2O-WT (W), or UBE2O-CA (C) and the indicated candidate substrates for 4 hours at37°C. Histone H2B is a model substrate of UBE2O (27). N-terminal truncations (T1 and T2) abrogatethe ubiquitination of RPs and of H2B but do not prevent modification of AHSP. After SDS-PAGE,samples were analyzed by immunoblotting with antibodies to either HA or AHSP. The left and rightpanels represent different exposures from the same gel. (D) Left: UBE2O can directly recognizeand ubiquitinate putative nonribosomal substrates DDX56, NOL12, NOP16, and PTRF, which areelevated in Ube2o−/− reticulocytes and reduced in 293-E2O cells. Right: Purified UBE2O canubiquitinate individual RPs outside of the 80S complex. Samples were analyzed by immunoblottingwith antibodies to HA after gradient SDS-PAGE. W, UBE2O-WT; C, UBE2O-CA. The molecularmasses of the tagged forms of substrates are as follows: calmodulin, 17 kDa; DDX56, 64 kDa;NOL12, 27 kDa; NOP16, 25 kDa; PTRF, 55 to 65 kDa; RPL35, 17 kDa; RPL36A, 16 kDa; RPL37, 15 kDa.(E) CR1 and CR2 provide substrate recognition with distinct specificities. The CR1 and CR2fragments were expressed in Escherichia coli in a biotin-tagged form and loaded onto streptavidinresin, using amounts sufficient to saturate the resin’s binding capacity. Purified UBE2O substrateswere mixed with resin at 20-fold excess in the presence of carrier. The resin was washed in50 mM NaCl, 20 mM Tris-HCl (pH 7.4). Bound material was eluted and 25% of the eluate wasresolved by SDS-PAGE followed by immunoblotting. The input represents 2.5% of the total sample;consequently, CR1 and CR2 are not visualized in the input lanes, although present. Rows 6 to 8are from the same experiment.

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(36) report that UBE2O is a quality-control ubiq-uitin ligase. Because quality-control ligases arecharacteristically broad in their specificity, a li-gase of this type may be pre-adapted to serve inglobal proteome remodeling when induced tohigh levels.UBE2Oprefers basic substrates, includingRPs,

and this specificity may underlie a defining fea-ture of the reticulocyte-erythrocyte transition:the elimination of ribosomes (17). Although theubiquitination of ribosomes and of free RPs haspreviously been observed (21, 37–39), our find-ings show that ubiquitination of RPs, in eithertheir free or assembled state, can be used topromote the elimination of ribosomes from cells.Indeed, ribosome ubiquitination has previouslybeen associated with the suppression of theirturnover via autophagy (33).Contrary to the longstanding paradigm that

multiubiquitin chains are required to initiatedegradation, UBE2O-dependent ubiquitination

events efficiently targeted substrates to the pro-teasome throughmulti-monoubiquitination events.Indeed, recent work from several groups sup-ports the sufficiency ofmulti-monoubiquitinationfor proteasomal degradation, depending on thesubstrate (40–42).The robust proteasome-dependent protein

turnover seen during the reticulocyte stage ofdifferentiation not only serves to eliminate pro-teins other than globin from these cells, but mayalso boost globin synthesis by supplying aminoacids for late-stage synthesis of globin. The lossof amino acid transporters during erythroidmat-uration (7, 43) may impose a strong link betweenprotein synthesis and degradation in these cells.Consistent with the proposed interconnectionbetween proteasome function and translation,UBE2O-deficient mice exhibit hypochromic ane-mia, and ribosomeoccupancy of the globin genes—themajor synthetic output—is decreased. Loss ofUBE2O accordingly ameliorates phenotypes at-

tributable to a-globin excess, as seen for amodelof b-thalassemia. UBE2O is thus a potential ther-apeutic target not only formixed-lineage leukemiaand other cancers, as recently proposed (44, 45),but also for b-thalassemia, one of the most prev-alent inherited diseases worldwide.

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Fig. 5. The ubiquitin-proteasome system in late erythroid differentiation. (A) Wild-type andUbe2o−/− reticulocytes were differentiated ex vivo at 37°C for 48 hours with proteasome inhibitors(50 nM epoxomicin and 50 nM PS-341) or DMSO vehicle control, then analyzed by fluorescence-activated cell sorter. Left: Wild-type reticulocytes treated with proteasome inhibitors versus DMSOcontrol. Right: Both wild-type and Ube2o−/− reticulocytes treated with proteasome inhibitors andDMSO. (B) Reconstitution of RP degradation in Ube2o−/− reticulocyte lysates. Ubiquitin levels weresupplemented to prevent depletion of free ubiquitin upon proteasome inhibition. Proteasomes wereinhibited by adding PS-341 (50 nM) and epoxomicin (50 nM) together. GAPDH is a loading control;100 mg of protein was loaded per lane. (C) Wild-type reticulocytes were treated with proteasomeinhibitors (50 nM epoxomicin and 50 nM PS-341) and differentiated ex vivo for 48 hours. Quantitativemetabolomic profiling of treated and untreated wild-type reticulocytes showed a depletion ofmultiple free amino acids due to proteasome inhibition, with Lys and Arg most strongly affected.Significance was calculated by two-sample t test. *P < 0.05, **P < 0.01, ***P < 0.001. (D) Anensemble of ubiquitin ligases and ubiquitin-conjugating enzymes is induced in late erythroiddifferentiation. Stages are arranged in a temporal progression. Stage 1 includes proerythroblastsand early basophilic erythroblasts; stage 2, early and late basophilic erythroblasts; stage 3,polychromatophilic and orthochromatophilic erythroblasts; stage 4, late orthochromatophilic erythro-blasts and reticulocytes. The induction of globin mRNA is shown for comparison. Raw RNA-sequencing data are taken from (6).

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ACKNOWLEDGMENTS

Special thanks to S. Elsasser for bioinformatics analysis of Ube2o,and to B. Wadas for characterizing autophagy inhibitors. Wethank M. Hegde for communicating results prior to publication.Supported by NIH grants 5R21HL116210 and 5R01HL125710,Biogen-Idec grant 6780680-01, and the Diamond Blackfan AnemiaFoundation (D.F. and M.D.F.); NIH Medical Scientist TrainingProgram grant T32GM007753 and NIH F30 grant HL124980(A.T.N.); NIH K01 grant DK098285 (J.A.P.); and NIH grantsU01 HD39372 and R01 CA115503 (M.J.J.). We thank R. King,C. Widmaier, S. de Poot, K. Y. Hung, and D. Brown for advice andassistance. Hem9 mice are available from M.J.J. under a materialtransfer agreement with Baylor College of Medicine. The datapresented in this paper are tabulated in the main paper and thesupplementary materials.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/357/6350/eaan0218/suppl/DC1Materials and MethodsSupplementary TextFigs. S1 to S21Tables S1 to S3References (47–78)

20 February 2017; accepted 8 June 201710.1126/science.aan0218

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UBE2O remodels the proteome during terminal erythroid differentiation

Monica J. Justice, Steven P. Gygi, Mark D. Fleming and Daniel FinleyWeiss,Campagna, Geng Tian, Yuan Shi, Verena Dederer, Mona Kawan, Nathalie Kuehnle, Joao A. Paulo, Yu Yao, Mitchell J.

Anthony T. Nguyen, Miguel A. Prado, Paul J. Schmidt, Anoop K. Sendamarai, Joshua T. Wilson-Grady, Mingwei Min, Dean R.

DOI: 10.1126/science.aan0218 (6350), eaan0218.357Science 

, this issue p. 472, p. eaan0218; see also p. 450Sciencepathway in the differentiation of red blood cells.

show the importance of the UBE2Oet al.tractable route to detailed mechanistic and structural dissection. Nguyen Encoding all three activities in a single factor whose function can be reconstituted in a purified system provides a separate factors for target selection (often a chaperone), ubiquitin donation (an E2), and ubiquitin conjugati on (an E3).enzyme (UBE2O) that recognizes substrates and tags them for destruction. Other quality-contr ol pathways tend to use

show that the central player in this process is an unusualet al.the Perspective by Hampton and Dargemont). Yanagitani pathway that acts on some of the most abundant protein complexes in the human body: hemoglobin and ribosomes (see''orphans'' are recognized and tagged for degradation is poorly understood. Two papers identify a protein quality-control

The degradation of excess subunits of protein complexes is a major quality-control problem for the cell. How suchRemoving orphan proteins from the system

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REFERENCES

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