RECONSTITUTION OF SYNAPTIC VESICLE BIOGENESIS FROM PC12 CELL-MEMBRANES

Neuroendocrine PC12 cells contain small microvesicles that closely res emble synaptic vesicles in their physical and chemical properties. Two defining characteristics of synaptic vesicles are their homogeneous s ize and their unique protein composition. Since synaptic vesicles aris e by endocytosis from the plasma membrane, nerve terminals and PC12 ce lls must contain the molecular machinery to sort synaptic vesicles fro m other membrane proteins and pinch off vesicles of the correct diamet er from a precursor compartment. A cell-free reconstitution system was developed that generates vesicles from PC12 membrane precursors in th e presence of ATP and brain cytosol and is temperature dependent. At 1 5 degrees C, surface-labeled synaptic vesicle proteins accumulate in a donor compartment, while labeled synaptic vesicles cannot be detected . The block of synaptic vesicle formation at 15 degrees C enables the use of the monoclonal antibody, KT3, a specific marker for the epitope -tagged synaptic vesicle protein, VAMP-TAg, to label precursors in the synaptic vesicle biogenesis pathway. From membranes labeled in vivo a t 15 degrees C, vesicles generated in vitro at 37 degrees C had the se dimentation characteristics of neuroendocrine synaptic vesicles on gly cerol velocity gradients, and excluded the transferrin receptor. There fore, vesiculation and sorting can be studied in this cell-free system . (C) 1998 Academic Press.