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J. Cell Biol., Volume 143, Number 4, November 16, 1998 947-955

Neuroendocrine Synaptic Vesicles Are Formed In Vitro by Both Clathrin-dependent and Clathrin-independent Pathways

Gongyi Shi,* Victor Faúndez,* Jack Roos,* Esteban C. Dell'Angelica,Dagger and Regis B. Kelly*

* Department of Biochemistry and Biophysics and the Hormone Research Institute, University of California, San Francisco, California 94143-0534; and Dagger  Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892 

In the neuroendocrine cell line, PC12, synaptic vesicles can be generated from endosomes by a sorting and vesiculation process that requires the heterotetrameric 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 GTPgamma S. 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.

Key words: synaptic vesiclesclathrinplasma membranePC12 cellsVAMP


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