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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM074874-07
Application #
8260579
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Gindhart, Joseph G
Project Start
2006-05-01
Project End
2014-04-30
Budget Start
2012-05-01
Budget End
2013-04-30
Support Year
7
Fiscal Year
2012
Total Cost
$558,385
Indirect Cost
$207,503
Name
Stanford University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
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
Kaiser, Stephen E; Riley, Brigit E; Shaler, Thomas A et al. (2011) Protein standard absolute quantification (PSAQ) method for the measurement of cellular ubiquitin pools. Nat Methods 8:691-6
Christianson, John C; Olzmann, James A; Shaler, Thomas A et al. (2011) Defining human ERAD networks through an integrative mapping strategy. Nat Cell Biol 14:93-105
Greenblatt, Ethan J; Olzmann, James A; Kopito, Ron R (2011) Derlin-1 is a rhomboid pseudoprotease required for the dislocation of mutant ýý-1 antitrypsin from the endoplasmic reticulum. Nat Struct Mol Biol 18:1147-52
Olzmann, James A; Kopito, Ron R (2011) Lipid droplet formation is dispensable for endoplasmic reticulum-associated degradation. J Biol Chem 286:27872-4

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