In eukaryotic cells, intracellular transport between distinct membrane-bound compartments proceeds through dissociated carriers that bud from one membrane and then fuse selectively with another. This proposal will focus on the molecules and conserved mechanisms that catalyze vesicular transport between the endoplasmic reticulum (ER) and Golgi complex in Saccharomyces cerevisiae. Genetic approaches have identified essential components required for this transport pathway. However, many of the molecular mechanisms underlying this process remain obscure. Our studies combine molecular genetic approaches with in vitro assays that measure protein transport from the ER to early Golgi compartments. This transport reaction proceeds through the biochemically resolvable stages of COPII-dependent cargo selection and vesicle budding, Usolp-dependent vesicle tethering, and SNARE protein-dependent membrane fusion. We have reproduced these stages with isolated membranes and purified soluble molecules. The long-term goal of my research program is to elucidate catalytic mechanisms underlying these events though analysis of stage-specific assays and reconstitution experiments with defined protein and lipid fractions. The objectives of this proposal are to: dissect the mechanism by which the sorting adaptor Erv26p functions with the COPII coat to sort secretory proteins into ER-derived vesicles;determine the role of the integral membrane Yip1/Yif1/Yos1 complex in vesicle budding from the ER;investigate the role of specific lipid species in COPII vesicle formation;and analyze the fate of ER derived vesicles in homotypic and heterotypic membrane fusion reactions using newly established SNARE protein cysteine-disulfide cross-linking assays. It is estimated that 20-30% of cellular proteins enter the secretory pathway. Our experimental aims are designed to address fundamental questions on how secretory proteins are selectively exported from the ER and directed to the Golgi complex. This intracellular transport step is essential for virtually all cell growth and function. Therefore, the proposed studies are basic for understanding numerous health related issues and are specifically relevant to understanding cholesterol regulation, Alzheimer's disease and cystic fibrosis.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM052549-17
Application #
8067754
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Ainsztein, Alexandra M
Project Start
1995-05-01
Project End
2012-04-30
Budget Start
2011-05-01
Budget End
2012-04-30
Support Year
17
Fiscal Year
2011
Total Cost
$523,713
Indirect Cost
Name
Dartmouth College
Department
Biochemistry
Type
Schools of Medicine
DUNS #
041027822
City
Hanover
State
NH
Country
United States
Zip Code
03755
Anderson, Nadine S; Mukherjee, Indrani; Bentivoglio, Christine M et al. (2017) The Golgin protein Coy1 functions in intra-Golgi retrograde transport and interacts with the COG complex and Golgi SNAREs. Mol Biol Cell :
Mukherjee, Indrani; Barlowe, Charles (2016) Overexpression of Sly41 suppresses COPII vesicle-tethering deficiencies by elevating intracellular calcium levels. Mol Biol Cell 27:1635-49
Margulis, Neil G; Wilson, Joshua D; Bentivoglio, Christine M et al. (2016) Analysis of COPII Vesicles Indicates a Role for the Emp47-Ssp120 Complex in Transport of Cell Surface Glycoproteins. Traffic 17:191-210
Flanagan, John J; Mukherjee, Indrani; Barlowe, Charles (2015) Examination of Sec22 Homodimer Formation and Role in SNARE-dependent Membrane Fusion. J Biol Chem 290:10657-66
Barlowe, Charles (2015) Membrane trafficking: ER export encounters dualism. Curr Biol 25:R151-3
Shibuya, Aya; Margulis, Neil; Christiano, Romain et al. (2015) The Erv41-Erv46 complex serves as a retrograde receptor to retrieve escaped ER proteins. J Cell Biol 208:197-209
Brandizzi, Federica; Barlowe, Charles (2014) ER-Golgi transport: authors' response. Nat Rev Mol Cell Biol 15:1
Brandizzi, Federica; Barlowe, Charles (2013) Organization of the ER-Golgi interface for membrane traffic control. Nat Rev Mol Cell Biol 14:382-92
Brunet, Stephanie; Noueihed, Baraa; Shahrzad, Nassim et al. (2012) The SMS domain of Trs23p is responsible for the in vitro appearance of the TRAPP I complex in Saccharomyces cerevisiae. Cell Logist 2:28-42
Meiringer, Christoph T A; Rethmeier, Ralf; Auffarth, Kathrin et al. (2011) The Dsl1 protein tethering complex is a resident endoplasmic reticulum complex, which interacts with five soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptors (SNAREs): implications for fusion and fusion regulation. J Biol Chem 286:25039-46

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