PROJECT SUMMARY The secretory pathway must fold and traffic one third of the proteome, while handling an exceptional dynamic range of client load. Quality control mechanisms prevent proteostasis in the secretory pathway from being overwhelmed by proteins in non-native states. One such mechanism is preemptive quality control (preQC), wherein ER stress inhibits ER translocation of nascent proteins. This process protects the ER from being overwhelmed with nascent protein during misfolded protein stress, a situation associated with diverse disease included diabetes and neurodegenerative diseases. However, this protection leads to secretory protein accumulating in the cytosol, which can be proteotoxic and lead to cell death and dysfunction. Due to methodological limitations, the substrates, causes, mechanism, and consequences of preQC are largely unknown. Hence, the physiological relevance of preQC, while expected to be significant, is not well understood. We propose to apply our recently developed assay for secretory protein mistargeting to systematically characterize preQC. We will identify which stresses induce preQC for a series of protein substrates in secretory cells from diverse human lineages. We will identify what factors govern triaging of mistargeted secretory proteins between aggregation and degradation mechanisms. Because our assay is performed in living cells, we will determine which signaling pathways mediate induction of preQC by ER stress. Finally, we will extend our assay to allow proteome-wide quantification of both basal and stress-dependent ER mistargeting. This extension will allow us to identify the factors that govern susceptibility of proteins to preQC. The outcome of the proposed research will be systematic characterization of how the cell remodels ER translocation in response to stress.