VAMP (synaptobrevin) is present in the plasma membrane of nerve terminals

Synaptic vesicle docking and exocytosis require the specific interaction of synaptic vesicle proteins (such as VAMP/synaptobrevin) with presynaptic pl asma membrane proteins (such as syntaxin and SNAP 25), These proteins form a stable, SDS-resistant, multimolecular complex, the SNARE complex. The sub cellular distribution of VAMP and syntaxin within Torpedo electric organ ne rve endings was studied by immunogoldlabeling of SDS-digested freeze-fractu re replicas (Fujimoto, 1995), This technique allowed us to visualize large surface areas of the presynaptic plasma membrane and numerous synaptic vesi cles from rapidly frozen nerve endings and synaptosomes. VAMP was found ass ociated with synaptic vesicles, as also shown by conventional electron micr oscopy immunolabeling, and to the presynaptic plasma membrane (P leaflet). Syntaxin was also detected in the nerve ending plasma membrane, without gol d labeling of synaptic vesicles. Comparison of gold particle densities sugg ests that the presynaptic plasma membrane contains 3 VAMP molecules per mol ecule of syntaxin. After biotinylation of intact synaptosomes, the synaptosomal plasma membran e was isolated on Streptavidin coated magnetic beads. Its antigenic content was compared to that of purified synaptic vesicles, VAMP was present in bo th membranes whereas syntaxin and SNAP 25 were highly enriched in the synap tosomal plasma membrane. This membrane has a low content of classical synap tic vesicle proteins (synaptophysin, SV2 and the vesicular acetylcholine tr ansporter). The VAMP to syntaxin stoechiometry in the isolated synaptosomal membrane was estimated by comparison with purified antigens and close to 2 , in accordance with morphological data. SDS-resistant SNARE complexes were detected in the isolated presynaptic membrane but absent in purified synap tic vesicles. Taken together, these results show that the presence of VAMP in the plasma membrane of nerve endings cannot result from exocytosis of sy naptic vesicles, a process which could, as far as SNAREs are concerned, ver y much ressemble homotypic fusion.