The use of permeabilized cells to investigate secretory granule biogenesis.
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.
Ling
W L
WL
Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York, 10461, USA.
Siddhanta
A
A
Shields
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D
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
D002352:Carrier Proteins; D008565:Membrane Proteins; D049294:Phospholipid Transfer Proteins; D013006:Growth Hormone; D010739:Phospholipase D; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors