Eukaryotic cells dedicate significant resources to the deployment of proteins to the membrane compartments that comprise the secretory pathway. Biogenesis of all membrane proteins starts with the folding and assembly of newly synthesized proteins within the endoplasmic reticulum (ER), often with the aid of cellular chaperones. Cells must strike a precise balance between ensuring that only fully folded proteins are allowed to leave the ER and avoiding the accumulation of misfolded proteins within the ER lumen. Uptake of newly synthesized cargo molecules into ER-derived transport vesicles only occurs once proteins are fully folded. These transport vesicles, known as COPII vesicles for the cytoplasmic coat proteins that drive membrane curvature and select cargo, thus play a critical role in regulating forward transport of new proteins. We study the close relationship between protein folding and packaging into ER- derived COPII vesicles in the model organism, Saccharomyces cerevisiae. Using a combination of genetics and biochemistry, we aim to define the cellular machinery that acts at the interface between protein folding and ER export. A model for examining this process is the yeast ABC transporter, Yor1, a plasma membrane protein that acts as a drug pump to clear toxic substances from the yeast cytoplasm. Yor1 is a homolog of the human cystic fibrosis transmembrane conductance regulator (CFTR), defects in which cause cystic fibrosis. Deletion of a Phe residue in Yor1, equivalent to the major disease-related mutation in human cystic fibrosis, causes Yor1, like mutant CFTR, to be ER-retained and degraded by the cytoplasmic ubiquitin/proteasome pathway. Thus Yor1 is a useful model that allows the direct comparison of the intracellular itineraries of native and aberrant forms of a single protein. This research proposal consists of four specific aims. (1) To define the molecular mechanisms that drive uptake of Yor1 into COPII vesicles and assess how protein folding influences this event. (2) To determine how cellular chaperones contribute to Yor1 biogenesis and assess how the kinetics of chaperone/client interactions may influence COPII binding and thereby regulate ER export. (3) To identify and characterize novel factors that may facilitate Yor1 biogenesis, including specific membrane chaperones and more general folding factors. (4) To determine the mechanisms by which the unfolded protein response improves the folding and/or transport of misfolded proteins. Ultimately, a better understanding of cellular machinery that acts to regulate protein folding and forward transport may lead to novel therapeutic approaches to treat the many diseases associated with aberrant protein folding within the secretory pathway. ? ? ?
| 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 |
| 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 |
| 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: |
| Louie, Raymond J; Guo, Jingyu; Rodgers, John W et al. (2012) A yeast phenomic model for the gene interaction network modulating CFTR-?F508 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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