PROJECT SUMMARY / ABSTRACT As cells undergo extreme forms of terminal differentiation, they are able to accumulate specific proteins to exceptionally high levels–hundreds of milligrams per ml in the case of globins and crystallins. At the same time, almost all other cellular components are eliminated. How cells can carry out such vast programs of biosynthesis and degradation simultaneously has been almost a complete mystery. We proposed in 1995 that the ubiquitin-proteasome system (UPS) may play a central role in global proteome remodeling. Using murine reticulocytes, a uniquely powerful system to study global proteome remodeling, we found that indeed UBE2O, a ubiquitin-conjugating enzyme that is strongly induced in late erythroid differentiation, mediates the elimination of ribosomes and myriad other proteins via the proteasome. We proceeded to examine other UPS components that are strongly induced in erythroid cells, and found that TRIM10 eliminates dynactin, many myosins, actin crosslinking proteins, the erythroid regulator TMCC2, and COP1 vesicles; TRIM58 eliminates dynein and centrosomal proteins; the unique ubiquitin-like protein TBCEL specifically dismantles the tubulin cytoskeleton; and UBE2H, together with the GID complex, eliminates a broad set of mRNA-binding translational regulatory proteins while also promoting the elimination of many mitochondrial proteins. Thus, these UPS components have highly distinct specificities, each driving the elimination of different parts of the cell or proteome. This work uncovers a vast new regulatory pathway that appears to be central to the maturation of the erythrocyte. It also indicates a remarkable new capacity of the UPS: to effect global and developmentally controlled proteomic remodeling. In contrast to the above-described proteins, most UPS components disappear during erythroid maturation; thus, a highly specialized variant of the UPS mediates remodeling. Focusing on TBCEL, TRIM10, and TRIM58, we will use biochemical reconstitution, crystallography, and hydrogen exchange mass spectrometry to resolve specific mechanisms of degradation and degradation signals in target proteins. Cellular studies will focus on the cytoskeleton and on translational control as highlighted by our proteomic data. As the erythroblast matures into the red blood cell, its radial, microtubule-based cytoskeleton is replaced by an acentric actin-based network. We will characterize how the cytoskeleton functions during the unusual and to date uncharacterized transition period that takes place in the reticulocyte. We will then assess the impact of programmed elimination of tubulin and other cytoskeletal proteins on this cytoskeletal transformation. Although late erythroid cells are known to be characterized by extensive translational regulation, our findings indicate a new mechanism by which diverse translational regulators are themselves controlled. We will use our mutants to determine the impact of UBE2H-dependent ubiquiti...