Project Summary: Membrane protein (MP) biogenesis begins in the endoplasmic reticulum (ER) and is a complex process, as domains on both sides of the ER membrane, and within the membrane bilayer, must fold and assemble. MPs understudy include ABC-Transporters, P-Type ATPases, G-protein coupled receptors, and BRICHOS proteins. Missense mutations that cause misfolding and premature degradation of MPs give rise to diseases such as cystic fibrosis, hypogonadotropic hypogonadism, retinitis pigmentosa and idiopathic lung fibrosis. In order to prevent toxic accumulation, misfolded MPs are targeted for ER-associated degradation (ERAD) by E3 ubiquitin ligase complexes. The ER transmembrane Hsp40 DNAJB12 (JB12) recruits cytosolic Hsp70 to the cytoplasmic face of the ER and together these chaperones deliver the misfolded MPs to ERAD machinery. In order for a misfolded protein to be a candidate for ERAD however, it must be competent for extraction from the confines of the ER membrane and delivered to the cytosolic proteasome. Misfolded proteins with structural restraints that prevent entrance into the proteasome necessitate an alternative quality control mechanism to ensure degradation. Misfolded MPs expose surfaces in the ER lumen, membrane, and the cytosol, so the coordinated action of ERQC factors in different locations manage this challenge. Mistakes in MP protein management are fatal when rogue clients adopt a toxic shape that enable rogues to damage membranes and dominantly poison essential cell functions. Hsp70 and Hsp40s act with folding and degradation machines to triage MPs. They shield against proteotoxicity through suppressing aggregation, refolding clients, selection of clients for degradation and regulating flux though protein biosynthetic systems. A major problem to ERQC systems are the MPs that accumulate in thermodynamically stable intermediate states that are non-native and self-associate to form oligomers, amorphous aggregates, and amyloid-like fibrils. Such misfolded conformers bury surfaces that are normally recognized by ERQC factors, and their thermodynamic stability hinders the unfolding events required for their extraction from membranes and degradation in the narrow central cavity of the proteasome. Thermodynamically stable MP intermediates are resistant to ERAD and we discovered that they are degraded by a JB12 dependent ER-phagy mechanism. The overall goal of the experiments included in this proposal is to define nodes of cross-talk between the Hsp70 chaperone system, ERAD and ER-phagy that are essential for cell viability and proteome maintenance.