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Journal of Neuroscience, Vol 13, 3895-3903, Copyright © 1993 by Society for Neuroscience
Synaptotagmin: a membrane constituent of neuropeptide-containing large dense-core vesicles
C Walch-Solimena, K Takei, KL Marek, K Midyett, TC Sudhof, P De Camilli and R Jahn
Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06510.
Synaptotagmin is known to be a major membrane protein of synaptic vesicles
(SVs) in neurons. We have now used an immunoisolation procedure to
demonstrate that synaptotagmin is also present in the membranes of peptide
containing large dense-core vesicles (LDCVs) of rat hypothalamus and bovine
posterior pituitary. Synaptotagmin bead- immunoisolated organelles from
these tissues primarily consisted of SVs but contained occasionally larger
structures reminiscent of LDCVs that were absent from vesicle populations
immunoisolated with a synaptophysin antibody. Furthermore, the vesicles
immunoisolated with synaptotagmin beads contained significant amounts of
neuropeptide Y (NPY). In contrast, vesicles immunoisolated with
synaptophysin beads did not contain detectable levels of NPY. Sucrose
density gradient fractionation of postnuclear supernatants obtained from
the bovine posterior pituitary resulted in a bimodal distribution of
synaptotagmin, corresponding to the positions of both SVs and
neurosecretory granules. A similar distribution was found for cytochrome
b561 and the 116 kDa subunit of the vacuolar proton pump. In contrast, the
SV proteins synaptophysin, SV2, and p29 were restricted to the
SV-containing fractions. Immunoisolation of small and large vesicles from
the sucrose gradient confirmed the differential distribution of
synaptotagmin and synaptophysin in the two types of secretory vesicles in
nerve endings of the posterior pituitary. We conclude that synaptotagmin is
a constituent of both SVs and peptide- containing secretory vesicles in the
nervous system. Since both types of organelles undergo Ca(2+)-dependent
exocytosis, these findings support a general role of synaptotagmin as an
exocytotic Ca2+ receptor.
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