The V0 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 synaptophysin, 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 V0 sector of the V-ATPase, were also found by Western blotting to be enriched in the immunoprecipitates. None of these V-ATPase subunits, or synaptophysin, was present in anti-synaptobrevin 2 immunoprecipitates obtained from frozen-thawed Triton X-100 extracts, which were greatly enriched, instead, in SNAP-25 and syntaxin 1. Accordingly, V-ATPase subunit c was found in anti-synaptophysin immunoprecipitates. Thus, the two complexes appear to be mutually exclusive. Subcellular fractionation of rat brain demonstrated that V-ATPase subunit c is localized with synaptobrevin 2 and synaptophysin in synaptic vesicles. The coprecipitation of V-ATPase subunit c with the synaptobrevin-synaptophysin complex suggests that this interaction may play a role in recruiting the proton pump into synaptic vesicles. Freeze-thawing, which involves a mild denaturing step, may produce a conformational change which dissociates the complex and mimics a change which occurs in vivo as a prerequisite to SNARE complex formation.
Galli
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T
Department of Cell Biology, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
McPherson
P S
PS
De Camilli
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P
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000648:ultrastructure; D019084:Fluorescent Antibody Technique, Indirect; D005615:Freezing; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D017830:Octoxynol / Q000737:chemistry; D006180:Proton-Translocating ATPases / Q000737:chemistry*; D050683:R-SNARE Proteins; D051381:Rats; D050600:SNARE Proteins; D013572:Synaptic Vesicles / Q000648:ultrastructure*; D016708:Synaptophysin / Q000737:chemistry*; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins
D046911:Macromolecular Substances; D008565:Membrane Proteins; D050683:R-SNARE Proteins; D050600:SNARE Proteins; C494213:Stx1a protein, rat; D016708:Synaptophysin; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins; D017830:Octoxynol; D006180:Proton-Translocating ATPases