ATP-DEPENDENT FORMATION OF FREE SYNAPTIC VESICLES FROM PC12 MEMBRANESIN-VITRO

Synaptic vesicles are released from membranes during incubation at 37 degrees C in the presence of ATP (adenosine triphosphate). The donor m embranes are a rapidly sedimenting fraction derived from the neuroendo crine cell line PC12 (pheochromocytoma 12). These starting membranes c ontain the synaptic vesicle proteins, synaptophysin and SV2, and the e ndosomal markers transferrin receptor and cation-independent MPR (mann ose 6-phosphate receptor). Incubating the membranes in vitro increased the amount of organelles that migrate as synaptic vesicles in velocit y sedimentation gradients. The synaptic vesicle fractions that contain both synaptophysin and SV2 do not contain endosomal markers. A synapt ic vesicle increase in vitro is time-, cytosol-, ATP- and temperature- dependent and is inhibited by NEM (N-ethylmaleimide), BFA (brefeldin A ) and aluminum fluoride, but not GTP gamma S (guanosine-5'-O-C3-thiotr iphosphate). The production of synaptic vesicles under these condition s is unlike the de novo generation of vesicles from endosomes (1). Inc ubation in vitro under the conditions described here may allow the fin al stages of synaptic vesicle formation, uncoating or undocking, to oc cur but not the initiation of formation de novo.