NEUROENDOCRINE SYNAPTIC VESICLES ARE FORMED IN-VITRO BY BOTH CLATHRIN-DEPENDENT AND CLATHRIN-INDEPENDENT PATHWAYS

In the neuroendocrine cell line, PC12, synaptic vesicles can be genera ted 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 disco vered a second pathway that sorts the synaptic vesicle-associated memb rane protein (VAMP) into similarly sized vesicles. For this pathway th e plasma membrane is the precursor rather than endosomes. Both pathway s require cytosol and ATP and are inhibited by GTP gamma S. The second pathway, however, uses AP2 instead of AP3 and is brefeldin A insensit ive. The AP2-dependent pathway is inhibited by depletion of clathrin o r by inhibitors of clathrin binding, whereas the AP3 pathway is not. T he VAMP-containing, plasma membrane-derived vesicles can be readily se parated on sucrose gradients from transferrin (Tf)-containing vesicles generated by incubating Tf-labeled plasma membrane preparations at 37 degrees C. Dynamin-interacting proteins are required for the AP2-medi ated vesiculation from the plasma membrane, but not from endosomes. Th us, VAMP is sorted into small vesicles by AP3 and ARF1 at endosomes an d by AP2 and clathrin at the plasma membrane.