Protein quality control at eukaryotic membranes

Project Summary/Abstract Integral membrane protein quality control systems are present within every major eukaryotic organelle. The archetypal integral membrane protein quality control system is called Endoplasmic reticulum-associated degradation (ERAD). For ERAD, ER proteins are degraded, either because they are misfolded or because their degradation is physiologically regulated. While its primary function is to target misfolded or regulated proteins, ERAD also functions to alleviate ER stress and is exploited by bacterial and viral pathogens to gain access to the cytosol and degrade host cell immune complexes. During normal ERAD, protein targets are selected within the ER lumen and transported across the membrane to the cytosol (retrotranslocation). Once in the cytosol, the targets are polyubiquitinated, extracted/dislocated from the membrane, and degraded by the proteasome. Even with decades of genetic, biochemical, and structural data, many aspects of ERAD remain ambiguous. Currently, basic information like the degrons that target substrates to the ERAD system, the mechanics for how ERAD interacts with target proteins, and even the range of cellular pathways under the control of ERAD are mysterious. The goals of this proposal are to: 1) define the features of targets directing them to ERAD (the degrons), 2) determine how the ERAD system recognizes, and retrotranslocates, target proteins across the ER membrane, 3) understand how the membrane composition controls ERAD function, 4) dissect the mechanics of other (non- ERAD) integral membrane protein quality control systems. We will use a multifaceted approach with biochemistry, cell biology, and genetics to address these central questions in membrane-associated protein quality control. We will leverage our unique in vivo and in vitro assays (and continue to design innovative assays) to dissect the mechanisms of target degradation by eukaryotic systems. A mechanistic understanding of how these systems function (including ERAD) and the processes they regulate will establish these systems as viable therapeutic targets in medically relevant pathways of protein misfolding, protein misregulation, and pathogen hijacking.