X-Message-Number: 8169
From: "Jan (John)" <>
Subject: Cryo Journal Club 10
Date: Sat, 3 May 1997 14:57:03 -0700

Cryo Journal Club 10



Volume 271, Number 4, Issue of January 26, 1996 pp. 2193-2198
©1996 by The American Society for Biochemistry and Molecular Biology, Inc. 
Thierry Galli ,  Peter S. McPherson,  Pietro De Camilli  

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<Picture: (0)> 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.

Response:

It is nice to know freeze-thawing is considered mild enough to mimic in
vivo conformational changes.

J.C.

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