Efficient and accurate protein secretion is a fundamental process that plays a pivotal role in the ability of al eukaryotic cells to function, grw 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). We study the biogenesis of ER-derived transport vesicles, aiming to uncover the rules that govern efficient capture of cargo proteins and the biophysical basis of membrane transformation events that give rise to spherical protein carriers. We use the model organism, Saccharomyces cerevisiae, to study this problem using a combination of genetic, biochemical and in vivo imaging approaches. Vesicle formation encompasses three fundamental processes: deformation of the donor membrane into a spherical transport carrier;selective recruitment of cargo proteins into the nascent bud;and scission of the membrane to release the vesicle. Traditionally, these processes were thought to be driven by discrete functions of the COPII coat, but our recent findings suggest a more intimate connection between the different events that conspire to yield a vesicle. For example, lumenally-oriented cargo proteins likely oppose the action of the COPII coat in deforming the lipid bilayer;the specific cargo composition at individual ER exit sites can thus directly impact the efficiency of vesicle formation by altering membrane properties. Furthermore, the GTP cycle of the coat, which controls coat assembly, vesicle scission and coat shedding, is regulated in part by a partnership between the cargo adaptor protein, Sec24, and the COPII accessory protein, Sec16, implicating cargo proteins in modulation of the GTP cycle. Taken together, these new findings place the cargo molecules as central players in the process of vesicle formation in vivo, which makes sense if we consider that vesicle formation must be an adaptable process that permits cells to respond to the changes in specific cargo burdens associated with changing environmental and developmental conditions. The current research proposal consists of two specific aims. (1) To unravel the complexity of the GTP cycle of the COPII coat, probing the impact of alterations to the GTPase activity of the coat and elucidating whether cargo plays a direct role in this process. (2) To characterize the influence of cargo proteins on membrane biophysical properties that impact the ability of the COPII coat to deform the ER membrane into transport vesicles. 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 defects in protein traffic within the secretory pathway.

Public Health Relevance

We study the process of vesicle formation from the eukaryotic endoplasmic reticulum to understand how cells govern the deployment of proteins to ensure accurate and efficient delivery to a variety of cellular compartments. This work has important implications for the growing number of diseases that are caused by defects in the trafficking and deployment of secretory proteins, including familial anemia, hemophilia and a variety of developmental disorders.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM085089-05
Application #
8438031
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Ainsztein, Alexandra M
Project Start
2008-09-01
Project End
2016-12-31
Budget Start
2013-01-01
Budget End
2013-12-31
Support Year
5
Fiscal Year
2013
Total Cost
$305,826
Indirect Cost
$110,726
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
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:
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
Miller, Elizabeth A (2013) The COPII cage sharpens its image. Nat Struct Mol Biol 20:139-40
Barlowe, Charles K; Miller, Elizabeth A (2013) Secretory protein biogenesis and traffic in the early secretory pathway. Genetics 193:383-410
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
Kung, Leslie F; Pagant, Silvere; Futai, Eugene et al. (2012) Sec24p and Sec16p cooperate to regulate the GTP cycle of the COPII coat. EMBO J 31:1014-27
Copic, Alenka; Latham, Catherine F; Horlbeck, Max A et al. (2012) ER cargo properties specify a requirement for COPII coat rigidity mediated by Sec13p. Science 335:1359-62
Pina, Francisco J; O'Donnell, Allyson F; Pagant, Silvere et al. (2011) Hph1 and Hph2 are novel components of the Sec63/Sec62 posttranslational translocation complex that aid in vacuolar proton ATPase biogenesis. Eukaryot Cell 10:63-71

Showing the most recent 10 out of 12 publications