A Novel Program of Ubiquitination Remodels the Erythroid Proteome During Terminal Differentiation

Abstract

The ubiquitin-proteasome system was initially discovered in reticulocytes, which undergo massive and rapid proteome remodeling. During terminal differentiation, hundreds of generic constituents of the cell undergo programmed elimination. However, the mechanisms that drive the turnover of normally stable proteins remain largely unknown. Two decades ago, an unusually large ubiquitin-conjugating enzyme, Ube2O, was found to be strongly and specifically upregulated in terminally differentiating reticulocytes, contemporaneously with the induction of globin. A null mutation in the murine Ube2O gene, known as hem9, resulted in a hypochromic, microcytic anemia, suggesting that Ube2O may be a major ubiquitinating factor in erythropoiesis. To understand the role of Ube2O in terminal differentiation, we first found that all major low molecular weight ubiquitin-protein conjugate bands are greatly reduced in levels in hem9 reticulocytes. When null reticulocyte lysates were treated with recombinant Ube2O, ribosomal proteins were overwhelmingly the major class of targets. Accordingly, hem9 reticulocytes have elevated ribosomal protein levels and 80S ribosomes. This phenotype of elevated ribosome abundance was accounted for by a defect in the elimination of ribosomes. Furthermore, overexpression of Ube2O was sufficient to drive ribosomal degradation in non-erythroid 293 cells. Quantitative mass spectrometry on these cells confirmed the destabilization of ribosomal proteins and indicated that Ube2O has a specific, yet broad ubiquitination program. Interestingly, the hem9 defect was phenocopied by treating wild-type reticulocytes with proteasome inhibitors. To confirm this finding, we reconstituted the degradation of ribosomal proteins in a cell-free reticulocyte lysate system. We also reconstituted the ubiquitination of purified ribosomes by recombinant Ube2O in vitro, and the degradation of several specific ribosomal proteins with purified proteasomes. Next, we found that the initiation factor eIF2α is hyperphosphorylated in hem9 reticulocytes, suggesting a global inhibition of protein synthesis. This was independent of HRI, a dominant regulator of translation in reticulocytes; instead, another eIF2α kinase, GCN2 was activated in the null mutant. Consistent with these findings, null reticulocytes were deficient in free amino acids, a phenotype that was recapitulated by proteasome inhibition. In summary, Ube2O selectively ubiquitinates ribosomal proteins and targets them to the proteasome for degradation, thus playing a central role during terminal erythroid differentiation.