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 proteincomplex 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 requiredfor efficient transport between the ER and Golgi complex. However, the mechanisms by which diversesecretory cargo are sorted into COPII vesicles and how budded vesicles are then targeted to Golgi acceptormembranes remain obscure. Our research program applies a multidisciplinary approach to define molecularmechanisms that catalyze receptor-dependent sorting of secretory cargo into COPII vesicles and vesiculartransport to the Golgi complex using yeast as a model organism. We exploit a cell-free transport assay thatproceeds through the biochemically resolvable stages of COPIi-dependent cargo selection and vesiclebudding, Uso1p-dependent vesicle tethering, and SNARE protein-dependent membrane fusion. We havereproduced these stages with isolated membranes and purified soluble molecules. The long-term goal of myresearch 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 progresshas identified transmembrane cargo receptors (e.g. Erv26p and Erv29p) that select secretory proteins intoCOPII vesicles and function with the ER quality control pathway. In addition, we have made progress incharacterizing specific lipid requirements, including PI(4)P, in distinct stages of ER to Golgi transport, in thenext funding period we plan test molecular models to determine how cargo binding to export receptors isregulated; how cargo receptors function in ER quality control; the role of specific proteins and lipid species inorganizing ER export sites and COPII budding; and the role of tethering factors and Pl(4) in transport to theGolgi complex.
;These experimental aims are designed to address fundamental questions on how secretory proteins areselectively exported from the ER and directed to the Golgi complex. This intracellular transport step isessential for cell growth and regulation. Therefore these studies are basic for understanding numeroushealth related issues including cholesterol regulation, cystic fibrosis and diabetes.
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