Annual Meeting of the North American Society for Pediatric Gastroenterology and Nutrition; Orlando, October 22-24, 1998
Biogenesis In Vitro of Vesicles From Endosomes
The endocytosis and recycling of plasma membrane proteins allows the composition of the cell surface to be modulated. Transport of membrane proteins from endosomes to the surface or to other membranous compartments has been proposed to occur via vesicular intermediates. However, such intermediates have yet to be identified. We set out to determine whether endosomes can give rise to vesicular intermediates and if so, which cytosolic proteins are involved. We set up a system using CHO cells stably transfected with a myc-tagged version of the facilitative transporter, GLUT4. Endosomes are labeled by internalization of transferrin or anti-myc monoclonal antibodies. After disruption of the cells, labeled membranes are incubated with cytosol and an ATP regenerating system and warmed. We found that endosomal membranes incubated with cytosol from a variety of tissues as well as from yeast gave rise to small vesicles containing either transferrin or GLUT4. Vesicle formation was insensitive to the fungal metabolite brefeldin A but was completely inhibited by non-hydrolyzable forms of GTP (GTPγ-S). Cytosol from yeast strains carrying a disruption in clathrin heavy chain showed a reduced level of vesicle formation suggesting that clathrin is involved in vesicle biogenesis. When vesicles were incubated with cytosol and GTPγ-S, their density increased and clathrin was recruited. Our data suggests that endosomes can give to vesicular intermediates and that clathrin is one of the proteins involved in vesicle biogenesis from endosomes. We propose that such intermediates may represent those involved in transport of recycling membrane proteins from endosomes either directly to the plasma membrane or to other membranous compartments.
Transport/Cell Biology/Nutrition© 1998 Lippincott Williams & Wilkins, Inc.