Calcium-dependent Oligomerization of Synaptotagmins I and II
SYNAPTOTAGMINS I AND II ARE LOCALIZED ON THE SAME SYNAPTIC VESICLE AND HETERODIMERIZE IN THE PRESENCE OF CALCIUM*
- From the Molecular Neuropathobiology and ‡Cell Biophysics Laboratories, Imperial Cancer Research Fund, 44 Lincoln’s Inn Fields, London WC2A 3PX, United Kingdom
Abstract
Synaptotagmins constitute a large family of membrane proteins characterized by their distinct distributions and different biochemical features. Genetic evidence suggests that members of this protein family are likely to function as calcium sensors in calcium-regulated events in neurons, although the precise molecular mechanism remains ill defined. Here we demonstrate that different synaptotagmin isoforms (Syt I, II, and IV) are present in the same synaptic vesicle population from rat brain cortex. In addition, Syt I and II co-localize on the same small synaptic vesicle (SSV), and they heterodimerize in the presence of calcium with a concentration dependence resembling that of the starting phase of SSV exocytosis (EC50 = 6 ± 4 μm). The association between Syt I and Syt II was demonstrated by immunoprecipitation of the native proteins and the recombinant cytoplasmic domains and by using fluorescence resonance energy transfer (FRET). Although a subpopulation of SSV containing Syt I and IV can be isolated, these two isoforms do not show a calcium-dependent interaction. These results suggest that the self-association of synaptotagmins with different calcium binding features may create a variety of calcium sensors characterized by distinct calcium sensitivities. This combinatorial hypothesis predicts that the probability of a single SSV exocytic event is determined, in addition to the gating properties of the presynaptic calcium channels, by the repertoire and relative abundance of distinct synaptotagmin isoforms present on the SSV surface.
Footnotes
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↵* This work was supported in part by a fellowship of the Spanish Ministry of Culture and Education (to J. H.) and the Imperial Cancer Research Fund.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵§ To whom correspondence should be addressed. Tel.: 44-171-2693300; Fax: 44-171-2693417; E-mail: g.schiavo{at}icrf.icnet.uk.
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↵2 C. Thomas and G. Schiavo, unpublished results.
- Abbreviations:
- SSV
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small synaptic vesicle(s)
- Syt I
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II, and IV, synaptotagmin I, II, and IV, respectively
- FRET
-
fluorescence resonance energy transfer
- GST
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glutathione S-transferase
- OG
-
octyl-β-d-glucosopyranoside
- NSF
-
N-ethylmaleimide-sensitive factor
- SNAP
-
soluble NSF attachment protein
- SNARE
-
SNAP receptor
- VAMP
-
vesicle-associated membrane protein
- PAGE
-
polyacrylamide gel electrophoresis
- HA
-
hemagglutinin epitope.
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- Received March 25, 1998.
- Revision received October 21, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
