Neuroendocrine synaptic vesicles are formed in vitro by both clathrin-dependent and clathrin-independent pathways

• Type of Material: Article
• Authors: Gongyi Shi, University of California, San Francisco; Victor Faundez, Emory University; Jack Roos, University of California, San Francisco; Esteban C. Dell'Angelica, University of California, San Francisco; Regis B. Kelly, University of California, San Francisco
• Language: English
• Date: 1998-11-16
• Publisher: Rockefeller University Press
• Publication Version: Final Published Version
• Copyright Statement: © The Rockefeller University Press,
• License: Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1083/jcb.143.4.947
• Title of Journal or Parent Work: Journal of Cell Biology
• ISSN: 0021-9525
• Volume: 143
• Issue: 4
• Start Page: 947
• End Page: 955
• Grant/Funding Information: J. Roos is supported by a postdoctoral fellowship from the American Cancer Society. V. Faundez was the recipient of an NIH Fogarty Fellowship.; This work was supported by grants to R.B. Kelly from Wheeler Center for the Neurobiology of Addiction, UCSF, and the National Institutes of Health (NIH; grant Nos. NS19878, NS15927, DA10154).
• Keywords: BREFELDIN-A; MEMBRANE; Cell Biology; COATED VESICLES; PC12 cells; TARGETING SIGNAL; Life Sciences & Biomedicine; Science & Technology; PROTEIN COMPLEX; DYNAMIN; VAMP; plasma membrane; MEDIATED ENDOCYTOSIS; PC12 CELLS; COMPARTMENT; synaptic vesicles; clathrin; RECEPTOR
• Research Categories: Chemistry, Biochemistry; Biology, Cell

Abstract

In the neuroendocrine cell line, PC12, synaptic vesicles can be generated from endosomes by a sorting and vesiculation process that requires the hetero-tetrameric adaptor protein AP3 and a small molecular weight GTPase of the ADP ribosylation factor (ARF) family. We have now discovered a second pathway that sorts the synaptic vesicle-associated membrane protein (VAMP) into similarly sized vesicles. For this pathway the plasma membrane is the precursor rather than endosomes. Both pathways require cytosol and ATP and are inhibited by GTPγS. The second pathway, however, uses AP2 instead of AP3 and is brefeldin A insensitive. The AP2-dependent pathway is inhibited by depletion of clathrin or by inhibitors of clathrin binding, whereas the AP3 pathway is not. The VAMP-containing, plasma membrane-derived vesicles can be readily separated on sucrose gradients from transferrin (Tf)-containing vesicles generated by incubating Tf-labeled plasma membrane preparations at 37°C. Dynamin-interacting proteins are required for the AP2-mediated vesiculation from the plasma membrane, but not from endosomes. Thus, VAMP is sorted into small vesicles by AP3 and ARF1 at endosomes and by AP2 and clathrin at the plasma membrane.