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Published online 14 July 2003. doi:10.1083/jcb.200302060
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© The Rockefeller University Press, 0021-9525/2003/7/199 $5.00
The Journal of Cell Biology, Volume 162, Number 2, 199-209

Article

Identification of synaptotagmin effectors via acute inhibition of secretion from cracked PC12 cells



Ward C. Tucker1, J. Michael Edwardson3, Jihong Bai1, Hyun-Jung Kim1, Thomas F.J. Martin2 and Edwin R. Chapman1

1 Department of Physiology, University of Wisconsin, Madison, WI 53706
2 Department of Biochemistry, University of Wisconsin, Madison, WI 53706
3 Department of Pharmacology, University of Cambridge, Cambridge CB2 1PD, UK

Address correspondence to Edwin R. Chapman, Dept. of Physiology, SMI 129, University of Wisconsin, 1300 University Ave., Madison, WI 53706. Tel.: (608) 263-1762. Fax: (608) 265-5512. E-mail: chapman{at}physiology.wisc.edu

T he synaptotagmins (syts) are a family of membrane proteins proposed to regulate membrane traffic in neuronal and nonneuronal cells. In neurons, the Ca2+-sensing ability of syt I is critical for fusion of docked synaptic vesicles with the plasma membrane in response to stimulation. Several putative Ca2+–syt effectors have been identified, but in most cases the functional significance of these interactions remains unknown. Here, we have used recombinant C2 domains derived from the cytoplasmic domains of syts I–XI to interfere with endogenous syt–effector interactions during Ca2+-triggered exocytosis from cracked PC12 cells. Inhibition was closely correlated with syntaxin–SNAP-25 and phosphatidylinositol 4,5-bisphosphate (PIP2)–binding activity. Moreover, we measured the expression levels of endogenous syts in PC12 cells; the major isoforms are I and IX, with trace levels of VII. As expected, if syts I and IX function as Ca2+ sensors, fragments from these isoforms blocked secretion. These data suggest that syts trigger fusion via their Ca2+-regulated interactions with t-SNAREs and PIP2, target molecules known to play critical roles in exocytosis.

Key Words: synaptotagmin; SNARE; membrane fusion; C2 domain; exocytosis

* Abbreviations used in this paper: LDCV, large dense core vesicle; PC, phosphatidylcholine; PIP2, phosphatidylinositol 4,5-bisphosphate; PS, phosphatidylserine; syt, synaptotagmin.


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