Current estimates indicate that 25-30% of translated proteins enter the secretory pathway In eukaryotic cells. After folding and assembly of nascent secretory proteins in the endoplasmic reticulum (ER), the coat protein complex II (COPII) sorts folded cargo into transport vesicles that bud from the ER and are targeted to cis- Golgi compartments. Genetic and proteomic approaches have identified many of the components required for efficient transport between the ER and Golgi complex. However, the mechanisms by which diverse secretory cargo are sorted into COPII vesicles and how budded vesicles are then targeted to Golgi acceptor membranes remain obscure. Our research program applies a multidisciplinary approach to define molecular mechanisms that catalyze receptor-dependent sorting of secretory cargo into COPII vesicles and vesicular transport to the Golgi complex using yeast as a model organism. We exploit a cell-free transport assay that proceeds through the biochemically resolvable stages of COPIi-dependent cargo selection and vesicle budding, Uso1p-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 the catalytic mechanisms undertying these events though analysis of stage- specific assays and reconstitution experiments with defined protein and lipid fractions. Our recent progress has identified transmembrane cargo receptors (e.g. Erv26p and Erv29p) that select secretory proteins into COPII vesicles and function with the ER quality control pathway. In addition, we have made progress in characterizing specific lipid requirements, including PI(4)P, in distinct stages of ER to Golgi transport, in the next funding period we plan test molecular models to determine how cargo binding to export receptors is regulated;how cargo receptors function in ER quality control;the role of specific proteins and lipid species in organizing ER export sites and COPII budding;and the role of tethering factors and Pl(4) in transport to the Golgi complex.
; These 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 cell growth and regulation. Therefore these studies are basic for understanding numerous health related issues including cholesterol regulation, cystic fibrosis and diabetes.
|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|
|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|
|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|
|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|
|Wilson, Joshua D; Thompson, Sarah L; Barlowe, Charles (2011) Yet1p-Yet3p interacts with Scs2p-Opi1p to regulate ER localization of the Opi1p repressor. Mol Biol Cell 22:1430-9|
|Lorente-Rodriguez, Andres; Barlowe, Charles (2011) Requirement for Golgi-localized PI(4)P in fusion of COPII vesicles with Golgi compartments. Mol Biol Cell 22:216-29|
|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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