Abstract Immature erythroblasts undergo significant proteome remodeling events prior to their development into functional red blood cells. In the final steps of terminal erythropoiesis, ribosomes are cleared from reticulocytes by the ubiquitin-proteasome system (UPS). During this process, individual unassembled, or orphan, ribosomal subunit proteins (RPs) appear to be recognized by E3 ligases, enzymes capable of ubiquitylating proteins and targeting them for proteasomal degradation. Defects in ribosome homeostasis, including in the clearance process, can cause forms of anemia and are linked to blood cancers. Despite the need for ribosome regulation during development, little is known about the mechanisms underlying ribosome homeostasis in erythroid cells. Recently, researchers identified UBE2O as a hybrid E2/E3 enzyme that ubiquitylates orphan RPs during erythroid ribosome clearance. UBE2O recognizes some specific RPs, but it is not known which of all 80 RPs are UBE2O clients or how they are selected. The ability of orphan RP interactors, such as chaperones, to participate in the ubiquitylation process has also not been explored. Although UBE2O is the only protein known to modify RPs during ribosome clearance, other E3 ligases may be involved, as around 60% of RPs can be degraded effectively in the absence of UBE2O. However, additional ligases have not been identified. A candidate is HUWE1, the mammalian homolog of TOM1, an E3 ligase required for ubiquitylation and degradation of many orphan RPs in yeast. The ability of HUWE1 to directly modify and target orphan mammalian RPs for degradation, including those not recognized by UBE2O, has not been studied in depth. I hypothesize that distinct features of individual RPs are recognized and targeted for proteasomal degradation by unique E3 ligases during erythroid differentiation. In this proposal, I will advance the field by combining in vitro biochemistry and cell-based assays to reveal mechanistic details of mammalian orphan RP recognition, ubiquitylation, and proteasomal degradation. In my first aim, I will characterize the ubiquitylation pattern of all 80 RPs and identify interactors of orphan RPs in reticulocyte lysate. In my second aim, I will reconstitute UBE2O-mediated ubiquitylation of orphan uL14 and assess the contribution of the uL14 interactor NAP1L1 to this process. Additionally, I will identify regions of uL14 recognized by both NAP1L1 and UBE2O and assess the influence of both proteins on uL14 degradation in cell culture systems. In my third aim, I will reconstitute HUWE1-mediated ubiquitylation of orphan uS3, identify regions of uS3 recognized by HUWE1, and determine if HUWE1 targets uS3 for degradation in cells. Studying the mechanisms of client recognition and selection will provide insight into how RPs are targeted for degradation in an effective manner during red blood cell development.