The long-term goal of this project is to elucidate the mechanisms by which proteins in the secretory pathway are recognized and destroyed by the ubiquitin-proteasome system (UPS) in a process known as endoplasmic reticulum-associated degradation (ERAD). Substrates committed to this fate are diverse, and include secreted and integral membrane proteins that are unable to fold correctly as a consequence of mutation or inefficient assembly into oligomeric complexes. During the current funding period we have made exceptional progress in achieving our primary aims of establishing a robust and versatile functional genomics platform to map functional substrate-specific sub-networks within the mammalian ERAD system, and have exceeded these aims in developing powerful proteomic tools that have enabled us to compile a preliminary map of the mammalian ERAD interactome. The present application proposes to expand these achievements with the goal of defining the role of chaperones and glycans in luminal ER quality control surveillance and to develop a comprehensive understanding of the composition and functional organization of the ERAD system in mammals. These goals are defined three specific aims. The first specific aim will employ established biochemical, cell biological and biophysical methodology to define the mechanisms by which substrates are committed to ERAD pathway and the role of the molecular chaperone, GRP94, in this process. The second specific aim will conduct a large-scale comprehensive functional genomic screen of the entire UPS in the degradation of a set of topologically diverse ERAD substrates and will validate these components using a battery of informatic and biochemical tools. The third specific aim is to construct a comprehensive map of the mammalian ERAD network by combining in-depth mining of the ERAD proteome with gene silencing to identify network topology, and to overlay this map on the substrate-specific functional network generated in Aim 2. These studies are focused on elucidating the molecular mechanisms by which folding or assembly-defective proteins are destroyed by mammalian cells, and therefore, have direct bearing on human health.

Public Health Relevance

The proposed studies will investigate the process by which misfolded or damaged proteins in the secretory pathway are identified and destroyed. Since mutations which influence protein folding underlie most genetic diseases, understanding the cellular mechanisms that defend against such mutations is critical to our understanding of the molecular basis of genetic disease.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Study Section
Membrane Biology and Protein Processing (MBPP)
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Wehrle, Janna P
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Stanford University
Schools of Arts and Sciences
United States
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Leto, Dara E; Morgens, David W; Zhang, Lichao et al. (2018) Genome-wide CRISPR Analysis Identifies Substrate-Specific Conjugation Modules in ER-Associated Degradation. Mol Cell :
Pataki, Camille I; Rodrigues, João; Zhang, Lichao et al. (2018) Proteomic analysis of monolayer-integrated proteins on lipid droplets identifies amphipathic interfacial ?-helical membrane anchors. Proc Natl Acad Sci U S A 115:E8172-E8180
van der Goot, Annemieke T; Pearce, Margaret M P; Leto, Dara E et al. (2018) Redundant and Antagonistic Roles of XTP3B and OS9 in Decoding Glycan and Non-glycan Degrons in ER-Associated Degradation. Mol Cell 70:516-530.e6
Hwang, Jiwon; Walczak, Christopher P; Shaler, Thomas A et al. (2017) Characterization of protein complexes of the endoplasmic reticulum-associated degradation E3 ubiquitin ligase Hrd1. J Biol Chem 292:9104-9116
Schrul, Bianca; Kopito, Ron R (2016) Peroxin-dependent targeting of a lipid-droplet-destined membrane protein to ER subdomains. Nat Cell Biol 18:740-51
Olzmann, James A; Kopito, Ron R; Christianson, John C (2013) The mammalian endoplasmic reticulum-associated degradation system. Cold Spring Harb Perspect Biol 5:
Olzmann, James A; Richter, Caleb M; Kopito, Ron R (2013) Spatial regulation of UBXD8 and p97/VCP controls ATGL-mediated lipid droplet turnover. Proc Natl Acad Sci U S A 110:1345-50
Tyler, Ryan E; Pearce, Margaret M P; Shaler, Thomas A et al. (2012) Unassembled CD147 is an endogenous endoplasmic reticulum-associated degradation substrate. Mol Biol Cell 23:4668-78
Dowlatshahi, Dara P; Sandrin, Virginie; Vivona, Sandro et al. (2012) ALIX is a Lys63-specific polyubiquitin binding protein that functions in retrovirus budding. Dev Cell 23:1247-54
Greenblatt, Ethan J; Olzmann, James A; Kopito, Ron R (2012) Making the cut: intramembrane cleavage by a rhomboid protease promotes ERAD. Nat Struct Mol Biol 19:979-81

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