Project Summary/Abstract Protein quality control systems in the secretory pathway normally maintain protein homeostasis by refolding or destroying misfolded proteins. The accumulation and aggregation of misfolded secretory pathway proteins including transmembrane proteins and glycosylphosphatidylinositol-anchored proteins (GPI-APs) signify a breakdown in secretory pathway protein quality control, and are often associated with devastating, incurable, and fatal protein-misfolding diseases. Examples include amyloid precursor protein (APP) and prion protein (PrP) whose misfolding is associated with Alzheimer's and prion diseases, respectively. RESET is a newly discovered protein quality control pathway that handles diverse misfolded GPI-APs, including human disease mutants of PrP. Our preliminary data strongly suggests that the pool of RESET substrates extends to select transmembrane proteins, including some misfolding mutants of APP. During RESET, misfolded proteins are released by the endoplasmic reticulum (ER)-resident chaperone, calnexin, and bound by p24-family member, Tmp21, for export to the Golgi. The misfolded proteins subsequently transit the cell surface en route to lysosomes where they are destroyed. RESET contrasts with better-characterized protein quality control systems, ER associated degradation and autophagy, which precludes the entry of misfolded proteins into the secretory pathway and degrades them at the ER. The discovery of a selective ER-export pathway for misfolded proteins, RESET, reveals new and unexplored contributors to the development of associated misfolding diseases. The long-term goal of this project is to fully understand the mechanism of RESET and its role in maintaining protein homeostasis in the secretory pathway. The objective of this proposal is to deduce how calnexin and Tmp21 control the fate of misfolded proteins and to identify the determinants of specificity for this pathway by characterizing multiple RESET substrates. Our central hypothesis is that RESET is regulated by calnexin, Tmp21 and associated protein quality control factors that coordinate the export of diverse, but specific, misfolded proteins out of the ER for downstream degradation. We will apply imaging, biophysical, biochemical and cell biological approaches to test this hypothesis through three specific aims (1) Determine the mechanism by which CNX directs substrates to and regulates RESET. (2) Determine the mechanism by which Tmp21 escorts RESET substrates out of the ER. (3) Identify the determinants presented by misfolded proteins that route them for RESET. Successful completion of these proposed studies will provide critical insights into the mechanisms that underlie the newly discovered RESET pathway, providing essential but currently unavailable insights with direct implications for the development of therapeutic strategies directed towards the treatment and cures of diverse protein misfolding diseases.