Project Summary/Abstract Protein quality control (QC) mechanisms exist in eukaryotic cells to monitor and clear misfolded, damaged, or aggregated proteins, and defects in QC systems are recognized to be causative in several disease states, particularly neurodegenerative disorders, cancer, cardiovascular, metabolic diseases, aging. Many protein QC systems function at the earliest stages of the life of a protein, ensuring that newly synthesized proteins reach a mature and functional state. While the ubiquitin-proteasome system (UPS) has long been recognized to play an important role in protein QC, this was generally assumed to occur after unsuccessful attempts at correctly folding newly synthesized proteins. In contrast, we made the surprising discovery that ubiquitylation of nascent polypeptides also occurs within active translation complexes, essentially marking proteins for destruction before their synthesis is even complete. Co- translation ubiquitylation (CTU) is a surprisingly robust process in human cells, with approximately 10% of nascent chains being ubiquitylated within active translation complexes. The incorporation of amino acid analogs into nascent chains, as well as Hsp70 inhibitors, leads to sharp increases in CTU, strongly suggesting that CTU reflects a protein QC pathway that targets aberrant or misfolded nascent polypeptides. Importantly, CTU is distinct from ubiquitylation of nascent chains that occurs in the context of defective and disassembled ribosome complexes (the “RQC” Ribosome Quality Control pathway, dependent on the Ltn1 ubiquitin ligase). The goals of this proposal are to characterize the CTU pathway with respect to the ubiquitin ligases, the targets, the factors that influence and potentially regulate CTU, and to assess the biological importance of CTU. The specific aims are to 1) identify the CTU ubiquitin ligases, in both yeast and human cells, through biochemical and genetic approaches, 2) characterize the preferential targets of CTU by proteomic approaches in order to determine the features of nascent polypeptides that are recognized by this QC system, and 3) determine the factors that influence and regulate CTU. This will significantly impact our understanding of the relationship between protein synthesis and protein quality control, with implications for disease states where proteostasis is particularly critical.