PROJECT SUMMARY CULLIN-RING E3 ubiquitin ligases (CRLs) employ ~200 adaptor proteins to bind and ubiquitylate substrates, and their dynamic assembly with adaptor proteins is regulated by the neddylation cycle together with the CAND adaptor exchange factor. Several CRLs have been linked with turnover of proteins involved in aging, cell cycle control, DNA repair, transcription, and cellular signaling pathways. Despite these advances, our understanding of the CRL system is incomplete for several reasons. First, the majority of studies on CRLs have focused on the functions of a small number of well-studied adaptors. As such, we have a limited understanding of the targets and biological functions for a substantial portion of the CRL system. Second, the many studies have been performed in cancer cell lines, leaving open the roles of these proteins in a more physiological setting. Third, many adaptors likely have multiple substrates and function through structurally related degrons in diverse substrates, yet we do not understand the biochemistry of degron targeting for the majority of adaptors. Such information can broadly inform discovery of biological functions, as has been the case for a small number of CRL adaptors such as TRCP. To bridge these knowledge gaps, our work has focused on the development of genetic methods to rapidly identify CRL adaptor targets and cognate degron sequences in substrates, and on the development of proteomic approaches that allow global quantification of proteome remodeling in response to cellular, genetic, or chemical cues. In AIM 1, we build on our discovery of 10 N/C-End E3 ubiquitin ligases that recognize their targets based on sequences at their N or C-termini to systematically elucidate their targets and roles in proteome remodeling as cells undergo differentiation to germ cell lineages. Human stem cell differentiation to germ cell lineages is associated with alterations in the abundance of >30% of the proteome, and we propose that N/C-End E3s play a central role in proteome remodeling, in part via quality control dependent protein degradation. This aim also builds on our extensive characterization of CRLs in this system, and on the development of multiple proteomic methods that provide a quantitative understanding of protein turnover and remodeling during changes in cell state. In AIMs 2 and 3, we further develop an experimental platform for genetic discovery of CRL targets and degrons (including regulated phosphodegrons), as well as matching of substrates and degrons with specific CRL adaptors. We provide examples of how these two platforms have resulted in the mechanistic dissection of new pathways for turnover of key transcription factors involved in nutrient signaling and propose to analyze newly discovered substrate-E3 pairs using systematic biochemical and cell biological approaches. Our expectation is that these studies will substantially advance our understanding of the molecular determinants in CRL depend...