The V
Sector of the V-ATPase, Synaptobrevin, and Synaptophysin Are Associated on Synaptic Vesicles in a Triton X-100-resistant,
Freeze-thawing Sensitive, Complex (*)
- From the Department of Cell Biology and Howard Hughes Medical Institute, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut 06510
- ¶ To whom correspondence should be addressed: Dept. of Cell Biology, Boyer Center for Molecular Medicine, 295 Congress Ave., New Haven, CT 06510. Tel.: 203-737-4465; Fax: 203-737-1762; pietro_decamilli{at}quickmail.yale.edu.
Abstract
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 V
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 generated from fresh, but not frozen-thawed extracts, and was not found in anti-syntaxin 1 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.
Footnotes
-
↵*This work was supported in part by National Institutes of Health Grant CA46128 and grants from the Juvenile Diabetes Foundation (to P. D. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵1 The abbreviations used are:
- SV
-
synaptic vesicle
- NSF
-
N-ethylmaleimide-sensitive fusion protein
- SNAP
-
soluble NSF attachment protein
- SNARE
-
SNAP receptor
- v-SNARE
-
vesicle SNARE
- t-SNARE
-
target SNARE
- PAGE
-
polyacrylamide gel electrophoresis.
-
- Received September 25, 1995.
- Revision received November 6, 1995.
- © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
