title: The use of permeabilized cells to investigate secretory granule biogenesis.
abstract: To investigate the mechanism of secretory granule biogenesis in endocrine cells, our laboratory used rat anterior pituitary GH3 cells which secrete growth hormone and prolactin. Here we describe a simple and rapid procedure for generating permeabilized cells to dissect molecular mechanisms involved in nascent secretory vesicle budding from the trans-Golgi network (TGN). Using this system, we demonstrate that vesicle budding is temperature, energy, and cytosol dependent; in addition, cytosol from a variety of cells, including yeast (Saccharomyces cerevisiae), can support vesicle release. The budding of nascent secretory vesicles from the TGN is stimulated by a phospholipase D activity that is associated with Golgi membranes. Our results suggest that phospholipid metabolism plays an important role in the release of nascent secretory vesicles from the TGN.
authors.affiliation: Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York, 10461, USA.
authors.forename: W L
authors.initials: WL
authors.lastname: Ling
authors.forename: A
authors.initials: A
authors.lastname: Siddhanta
authors.forename: D
authors.initials: D
authors.lastname: Shields
mesh_terms: D020727:ADP-Ribosylation Factors; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D002463:Cell Membrane Permeability; D003584:Cytological Techniques; D003594:Cytoplasmic Granules / Q000378:metabolism*; D005089:Exocytosis; D019204:GTP-Binding Proteins / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism*; D013006:Growth Hormone / Q000378:metabolism; D008565:Membrane Proteins; D010739:Phospholipase D / Q000378:metabolism; D049294:Phospholipid Transfer Proteins; D010903:Pituitary Gland, Anterior / Q000166:cytology*; D051381:Rats
chemical_list: D002352:Carrier Proteins; D008565:Membrane Proteins; D049294:Phospholipid Transfer Proteins; D013006:Growth Hormone; D010739:Phospholipase D; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors