Efficient and accurate protein secretion is a fundamental process that plays a pivotal role in the life of all eukaryotic cells. Fully one-third of the eukaryotic proteome is targeted to the membrane compartments that comprise the secretory pathway. These proteins must be faithfully delivered to these organelles after synthesis and folding in the endoplasmic reticulum (ER). The first step in protein delivery to downstream compartments is the selective capture of proteins into ER-derived transport vesicles, known as COPII vesicles for the cytoplasmic coat proteins that sculpt these transport carriers from the donor membrane. Cargo proteins are enriched in nascent vesicles through the action of the COPII coat subunit, Sec24p, which serves as a cargo- binding platform by providing multiple interfaces for cargo-coat interaction. Although we have gained enormous insight into this function of Sec24p through biochemical, genetic and structural analyses, we are currently lacking a full description of the diversity and flexibility of this process, which must accommodate a vast array of client proteins with distinct structures, functions, and ultimate destinations.
We aim to fully understand the molecular interactions that drive export out of the ER, and are working to determine the full spectrum of cargo proteins that bind to Sec24p to ensure efficient ER egress. We use the model organism, Saccharomyces cerevisiae, to study this fundamental process using a combination of genetic, proteomic and biochemical approaches. This research proposal consists of two specific aims. (1) To determine the full repertoire of secretory proteins that interacts with the three known cargo-binding sites on Sec24p and to define the mode of interaction of novel client cargoes. (2) To determine the nature of cargo capture or coat assembly defects associated with three novel Sec24p mutants that were isolated from a structure-based genetic screen. Using these approaches, we seek to gain detailed insight into the molecular mechanisms that underlie the diversity and flexibility of cargo capture. Ultimately, a more detailed understanding of this fundamental eukaryotic process will have important implications in the many aspects of human disease and development that are impacted by early stages of protein biogenesis and deployment within the secretory pathway. We study the detailed molecular interactions that drive the selective capture of newly synthesized secretory proteins into transport vesicles that bud from the endoplasmic reticulum. Public Health Relevance: This process represents a critical quality control decision in the cell, and has important implications for the growing number of diseases that are caused by aberrant folding, trafficking and deployment of secretory proteins, including cystic fibrosis, familial heart disease and lung disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM085089-04
Application #
8102174
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Ainsztein, Alexandra M
Project Start
2008-09-01
Project End
2012-12-31
Budget Start
2011-07-01
Budget End
2012-12-31
Support Year
4
Fiscal Year
2011
Total Cost
$295,678
Indirect Cost
Name
Columbia University (N.Y.)
Department
Biology
Type
Other Domestic Higher Education
DUNS #
049179401
City
New York
State
NY
Country
United States
Zip Code
10027
Geva, Yosef; Crissman, Jonathan; Arakel, Eric C et al. (2017) Two novel effectors of trafficking and maturation of the yeast plasma membrane H+ -ATPase. Traffic 18:672-682
Davis, Saralin; Wang, Juan; Zhu, Ming et al. (2016) Sec24 phosphorylation regulates autophagosome abundance during nutrient deprivation. Elife 5:
D'Arcangelo, Jennifer G; Crissman, Jonathan; Pagant, Silvere et al. (2015) Traffic of p24 Proteins and COPII Coat Composition Mutually Influence Membrane Scaffolding. Curr Biol 25:1296-305
Pagant, Silvere; Wu, Alexander; Edwards, Samuel et al. (2015) Sec24 is a coincidence detector that simultaneously binds two signals to drive ER export. Curr Biol 25:403-12
Wang, Juan; Davis, Saralin; Menon, Shekar et al. (2015) Ypt1/Rab1 regulates Hrr25/CK1? kinase activity in ER-Golgi traffic and macroautophagy. J Cell Biol 210:273-85
Miller, Elizabeth A; Schekman, Randy (2013) COPII - a flexible vesicle formation system. Curr Opin Cell Biol 25:420-7
D'Arcangelo, Jennifer G; Stahmer, Kyle R; Miller, Elizabeth A (2013) Vesicle-mediated export from the ER: COPII coat function and regulation. Biochim Biophys Acta 1833:2464-72
Lord, Christopher; Ferro-Novick, Susan; Miller, Elizabeth A (2013) The highly conserved COPII coat complex sorts cargo from the endoplasmic reticulum and targets it to the golgi. Cold Spring Harb Perspect Biol 5:
Miller, Elizabeth A (2013) A sustained passion for intracellular trafficking. Mol Biol Cell 24:3270-2
Stachowiak, Jeanne C; Brodsky, Frances M; Miller, Elizabeth A (2013) A cost-benefit analysis of the physical mechanisms of membrane curvature. Nat Cell Biol 15:1019-27

Showing the most recent 10 out of 20 publications