Efficient and accurate protein secretion is a fundamental process that plays a pivotal role in the ability of all eukaryotic cells to function, grow and communicate. 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). Proteins that fail to fold correctly are prevented from leaving the ER and targeted for destruction, a process known as ER quality control. We study the relationship between protein folding within the ER and the ability of nascent proteins to be captured into ER-derived transport vesicles for downstream delivery.
We aim to uncover the rules that govern whether a protein is a substrate for forward transport, a question that lies at the heart of ER quality control. We use the model organism, Saccharomyces cerevisiae, to study this problem using a combination of genetic and biochemical approaches, focusing on the biogenesis of the yeast ABC transporter, Yor1p, a drug pump that confers resistance to the toxin, oligomycin. Yor1p is broadly related to the human cystic fibrosis transmembrane conductance regulator, CFTR, which causes cystic fibrosis when mutated. Expression of disease-related misfolded variants of Yor1p have permitted the discovery of components involved in biogenesis of this family of membrane proteins through genome-wide phenotypic analysis of oligomycin resistance. This approach led us to a set of ER membrane proteins that function in regulating the fate of folded versus misfolded forms of Yor1p at distinct stages. The EMC complex seems to act very early during protein synthesis to stabilize an aberrant variant of Yor1p;the putative export factor, Erv14p, functions to promote efficient ER egress of both wild-type and mutant forms of Yor1p. The current research proposal consists of three specific aims. (1) To determine the mechanism by which the EMC complex and related components influence the biogenesis of nascent membrane proteins. (2) To further define the cellular machinery that acts in the ER during biogenesis of membrane proteins to regulate ER quality control and forward traffic. (3) To characterize the complex mechanisms that drive Erv14p- and Sec24p-dependent export from the ER. Using these approaches, we seek to gain detailed insight into the molecular mechanisms that underlie the ER quality control checkpoint. Ultimately, a more detailed understanding of this fundamental eukaryotic process will have important implications in the many aspects of human disease that are impacted by protein folding lesions that preclude deployment within the secretory pathway.

Public Health Relevance

We study the process of protein quality control in the eukaryotic endoplasmic reticulum to understand how cells make decisions about the fate of proteins to prevent the deployment of aberrant, potentially toxic molecules. This work 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 #
5R01GM078186-08
Application #
8654342
Study Section
Membrane Biology and Protein Processing Study Section (MBPP)
Program Officer
Ainsztein, Alexandra M
Project Start
2006-08-01
Project End
2016-04-30
Budget Start
2014-05-01
Budget End
2015-04-30
Support Year
8
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Columbia University (N.Y.)
Department
Biology
Type
Graduate Schools
DUNS #
City
New York
State
NY
Country
United States
Zip Code
10027
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
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
Barlowe, Charles K; Miller, Elizabeth A (2013) Secretory protein biogenesis and traffic in the early secretory pathway. Genetics 193:383-410
Miller, Elizabeth A (2013) The COPII cage sharpens its image. Nat Struct Mol Biol 20:139-40
Miller, Elizabeth A; Schekman, Randy (2013) COPII - a flexible vesicle formation system. Curr Opin Cell Biol 25:420-7
Louie, Raymond J; Guo, Jingyu; Rodgers, John W et al. (2012) A yeast phenomic model for the gene interaction network modulating CFTR-ΔF508 protein biogenesis. Genome Med 4:103
Hou, Haitong; Zhou, Zhou; Wang, Yu et al. (2012) Csi1 links centromeres to the nuclear envelope for centromere clustering. J Cell Biol 199:735-44

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