UBE2O remodels the proteome during terminal erythroid differentiation

TLDR: Results indicate that UBE2O may function as a hybrid enzyme with both E2 and E3 (ubiquitin-ligating) activities in the reticulocyte–red blood cell transition, and that a major component of the specificity underlying differentiation-linked proteome remodeling appears to be carried by UBE1O itself.

Abstract: INTRODUCTION 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. RATIONALE 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. RESULTS 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. CONCLUSION 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.