During the previous funding period we discovered a fundamental and unanticipated structural organization for the COPII machinery directing export of nearly one-third of the proteins encoded by the eukaryotic genome from the endoplasmic reticulum (ER). Using cryoEM and single particle analysis we have shown the COPII cage composed of Sec13-31 heterotetramer cytosolic complex is a self-assembling nanoparticle that has a novel cuboctahedron geometry unprecedented in biology. Understanding the structure and function of the COPII cage and the intact Sec23-24 containing COPII coat is the focus of this competitive renewal. We propose in 3 Aims to dissect the structural, biochemical and molecular properties of COPII cage and coat that allow it to facilitate selective and efficient collection of cargo for export from the ER. We will explore the general hypothesis that the self-assembling properties of the COPII cage from its cytosolic heterotetramer precursor provides an ancient and fundamental organizing principle for cargo selection and collection into budding vesicles.
Aim 1 will develop an in depth structural description of cages and coats at low- and high-resolution to elucidate principles of self-assembly.
Aim 2 will analyze the biochemical and physiological properties that regulate the assembly and disassembly of the Sec13- 31 cage in the presence of absence of Sec23-24 adaptors in vitro and in vivo. We will explore the hypothesis that key residues controlling protein-protein interactions at the vertex of assembled cages control the extent and kinetics of assembly and disassembly of the cage and coat.
Aim 3 will use both genetic and molecular techniques to define the amino acid residues involved in cage and coat function. We will explore the hypothesis that evolutionarily conserved residues at vertex interfaces control cage assembly and cargo collection. We anticipate that knowledge of the assembly and disassembly properties of the COPII cage and coat will not only provide important insights into the basic functional properties of the ER COPII machinery, but provide a fresh view into the many misfolding diseases including Gaucher, cystic fibrosis, type II diabetes and childhood emphysema (among many others) that fail to engage the COPII machinery leading to cell and tissue dysfunction. These studies will significantly advance the field by providing insight into a ubiquitous eukaryotic coat complex responsible for defining the membrane architecture of the eukaryotic cell.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Membrane Biology and Protein Processing (MBPP)
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Ainsztein, Alexandra M
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Scripps Research Institute
La Jolla
United States
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Amaral, Margarida D; Balch, William E (2015) Hallmarks of therapeutic management of the cystic fibrosis functional landscape. J Cyst Fibros 14:687-99
Roth, Daniela Martino; Hutt, Darren M; Tong, Jiansong et al. (2014) Modulation of the maladaptive stress response to manage diseases of protein folding. PLoS Biol 12:e1001998
Hutt, Darren M; Balch, William E (2013) Expanding proteostasis by membrane trafficking networks. Cold Spring Harb Perspect Med 3:1-21
Powers, Evan T; Balch, William E (2013) Diversity in the origins of proteostasis networks--a driver for protein function in evolution. Nat Rev Mol Cell Biol 14:237-48
Pottekat, Anita; Becker, Scott; Spencer, Kathryn R et al. (2013) Insulin biosynthetic interaction network component, TMEM24, facilitates insulin reserve pool release. Cell Rep 4:921-30
Hutt, Darren M; Balch, William E (2013) Expanding proteostasis by membrane trafficking networks. Cold Spring Harb Perspect Biol 5:
Coppinger, Judith A; Hutt, Darren M; Razvi, Abbas et al. (2012) A chaperone trap contributes to the onset of cystic fibrosis. PLoS One 7:e37682
Hutt, Darren M; Roth, Daniela Martino; Chalfant, Monica A et al. (2012) FK506 binding protein 8 peptidylprolyl isomerase activity manages a late stage of cystic fibrosis transmembrane conductance regulator (CFTR) folding and stability. J Biol Chem 287:21914-25
Bouchecareilh, M; Balch, W E (2012) Proteostasis, an emerging therapeutic paradigm for managing inflammatory airway stress disease. Curr Mol Med 12:815-26
Bouchecareilh, Marion; Hutt, Darren M; Szajner, Patricia et al. (2012) Histone deacetylase inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA)-mediated correction of ýý1-antitrypsin deficiency. J Biol Chem 287:38265-78

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