Research Article

UBE2O remodels the proteome during terminal erythroid differentiation

See allHide authors and affiliations

Science  04 Aug 2017:
Vol. 357, Issue 6350, eaan0218
DOI: 10.1126/science.aan0218

You are currently viewing the abstract.

View Full Text

Log in to view the full text

Log in through your institution

Log in through your institution

Removing orphan proteins from the system

The degradation of excess subunits of protein complexes is a major quality-control problem for the cell. How such “orphans” are recognized and tagged for degradation is poorly understood. Two papers identify a protein quality-control pathway that acts on some of the most abundant protein complexes in the human body: hemoglobin and ribosomes (see the Perspective by Hampton and Dargemont). Yanagitani et al. show that the central player in this process is an unusual enzyme (UBE2O) that recognizes substrates and tags them for destruction. Other quality-contr ol pathways tend to use separate factors for target selection (often a chaperone), ubiquitin donation (an E2), and ubiquitin conjugati on (an E3). Encoding all three activities in a single factor whose function can be reconstituted in a purified system provides a tractable route to detailed mechanistic and structural dissection. Nguyen et al. show the importance of the UBE2O pathway in the differentiation of red blood cells.

Science, this issue p. 472, p. eaan0218; see also p. 450

Structured Abstract


The reticulocyte–red blood cell transition is a canonical example of terminal differentiation. The mature red blood cell has one of the simplest cellular proteomes known, with hemoglobin remarkably concentrated to ~98% of soluble protein. During reticulocyte maturation, the proteome is remodeled through the programmed elimination of most generic constituents of the cell, in parallel with abundant synthesis of cell type–specific proteins such as hemoglobin. The mechanisms that drive rapid turnover of soluble and normally stable proteins in terminally differentiating cells remain largely unclear.


The ubiquitin-proteasome system (UPS) was discovered in reticulocytes, where it is highly active. However, its function in this developmental context has not been established. UBE2O is an E2 (ubiquitin-conjugating) enzyme that is co-induced with globin and expressed at elevated levels late in the erythroid lineage. We identified an anemic mouse line with a null mutation in Ube2o, and used multiplexed quantitative proteomics to identify candidate substrates of UBE2O in an unbiased and global manner. We found that the protein compositions of mutant and wild-type reticulocytes differed markedly, suggesting that UBE2O-dependent ubiquitination might target its substrates for degradation to effect remodeling of the proteome.

To test whether UBE2O was sufficient for proteome remodeling, we engineered a non-erythroid cell line to inducibly express UBE2O above its basal level. Upon induction, we observed the decline of hundreds of proteins from these cells, in many cases the same proteins as those eliminated from reticulocytes. Overexpression of an active-site mutant of UBE2O did not show these effects. Therefore, a major component of the specificity underlying differentiation-linked proteome remodeling appears to be carried by UBE2O itself. These results also indicate that UBE2O may function as a hybrid enzyme with both E2 and E3 (ubiquitin-ligating) activities. In support of this model, candidate substrates identified by proteomics were ubiquitinated by purified UBE2O without the assistance of additional specificity factors.


The most prominent phenotypes of the Ube2o mutant are an anemia characterized by small cells with low hemoglobin content (microcytic hypochromic anemia), and a defect in the elimination of ribosomes, the latter being a key aspect of reticulocyte maturation. When we added recombinant UBE2O protein to reticulocyte lysates from the null mutant, ubiquitin was conjugated primarily to ribosomal proteins. Moreover, immunoblot analysis and quantitative proteomics revealed elevated levels of multiple ribosomal proteins in mutant reticulocytes. Sucrose gradient analysis indicated the persistence not only of ribosomal proteins but of ribosomes themselves during ex vivo differentiation of mutant reticulocytes. Accordingly, ribosomes were eliminated upon induction of UBE2O in non-erythroid cells. The elimination of organelles from reticulocytes, as exemplified by that of mitochondria, was not affected in the Ube2o mutant, indicating the specificity of its effects on programmed protein turnover.

Free ribosomal proteins were ubiquitinated by purified UBE2O, which suggests that these proteins are true substrates of the enzyme. However, UBE2O substrates are diverse in nature and not limited to ribosomal proteins. Individual domains of UBE2O bound substrates with distinct specificities. Thus, the broad specificity of UBE2O reflects the presence of multiple substrate recognition domains within the enzyme.

Proteasome inhibitors blocked the degradation of UBE2O-dependent substrates in reticulocytes, although UBE2O does not form polyubiquitin chains. Rather, UBE2O adds single ubiquitin groups to substrates at multiple sites. Proteasome inhibitor treatment ex vivo led to depletion of the pools of many amino acids; this result implies that the flux of ubiquitinated substrates through the reticulocyte proteasome is sufficient to supply amino acids needed for late-stage translation of mRNA. In late erythropoiesis, several ubiquitin-conjugating enzymes and ligases are induced together with Ube2o while most components of the UPS disappear. We propose that the UPS is not simply amplified during erythroid maturation, but is instead broadly reconfigured to promote remodeling of the proteome.


A highly specialized UPS is expressed in the reticulocyte and is used to remodel the proteome of these cells on a global scale. UBE2O, a hybrid E2-E3 enzyme, functions as a major specificity factor in this process. In reticulocytes, and perhaps in other differentiated cells such as in the lens, the induction of ubiquitinating factors may drive the transition from a complex to a simple proteome.

UBE2O drives remodeling of the proteome during erythroid differentiation.

The transformation of reticulocytes into erythrocytes involves the elimination of myriad proteins. UBE2O is an E2-E3 hybrid enzyme that directly recognizes and ubiquitinates proteins that 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 possibly within the ribosome complex.


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

View Full Text