THE V-O SECTOR OF THE V-ATPASE, SYNAPTOBREVIN, AND SYNAPTOPHYSIN ARE ASSOCIATED ON SYNAPTIC VESICLES IN A TRITON X-100-RESISTANT, FREEZE-THAWING SENSITIVE, COMPLEX

Anti-synaptobrevin 2 immunoprecipitates obtained from freshly prepared Triton X-100 extracts of rat synaptosomes contained, in addition to s ynaptophysin, a 10-kDa band, which we identified by peptide sequencing and Western blotting as the c subunit of the vacuolar proton pump (V- ATPase) also called ductin or mediatophore, Ac39 and Ac116, two other transmembrane subunits of the V-0 sector of the V-ATPase, were also fo und by Western blotting to be enriched in the immunoprecipitates. None of these V-ATPase subunits, or synaptophysin, was present in anti-syn aptobrevin 2 immuno-precipitates obtained from frozen-thawed Triton X- 100 extracts, which were greatly enriched, instead, in SNAP-25 and syn taxin 1. Accordingly, V-ATPase subunit c was found in anti-synaptophys in immunoprecipitates generated from fresh, but not frozen-thawed extr acts, and was not found in anti syntaxin 1 immunoprecipitates. Thus, t he two complexes appear to be mutually exclusive. Subcellular fraction ation of rat brain demonstrated that V-ATPase subunit c is localized w ith synaptobrevin 2 and synaptophysin in synaptic vesicles. The coprec ipitation of V-ATPase subunit c with the synaptobrevin-synaptophysin c omplex suggests that this interaction may play a role in recruiting th e proton pump into synaptic vesicles. Freeze-thawing, which involves a mild denaturing step, may produce a conformational change which disso ciates the complex and mimics a change which occurs in, vivo as a prer equisite to SNARE complex formation.