SNAP-25 Is Required for a Late Postdocking Step in Ca2+-dependent Exocytosis

doi:10.1074/jbc.271.34.20227

SNAP-25 Is Required for a Late Postdocking Step in Ca²⁺-dependent Exocytosis*

From the Departments of ^§ Biochemistry and
^‡ Food Microbiology and Toxicology, University of Wisconsin, Madison, Wisconsin 53706

^¶ To whom correspondence should be addressed:
Dept. of Biochemistry, University of Wisconsin, 420 Henry Mall, Madison, WI 53706.
Tel.: 608-263-2427; Fax: 608-262-3453; E-mail: tfmartin{at}macc.wisc.edu.

Abstract

The Ca²⁺-activated fusion of large dense core vesicles (LDCVs) with the plasma membrane is reconstituted in mechanically permeabilized PC12 cells by provision of millimolar MgATP and cytosolic proteins. Ca²⁺-activated LDCV exocytosis was inhibited completely by the type E but not the type A botulinum neurotoxin (BoNT) even though both BoNTs were equally effective in proteolytically cleaving the synaptosome-associated protein of 25 kDa (SNAP-25). The greater inhibition of exocytosis by BoNT E correlated with a greater destabilization of detergent-extracted complexes consisting of SNAP-25, synaptobrevin, and syntaxin. LDCVs in permeable PC12 cells can be poised at a late postdocking, prefusion state by MgATP-dependent priming processes catalyzed by N-ethylmaleimide sensitive factor and priming in exocytosis proteins. BoNT E completely blocked Ca²⁺-activated LDCV exocytosis in ATP-primed cells, whereas BoNT A was only slightly inhibitory, implying that the C-terminal region of SNAP-25 (Ile¹⁸¹-Gln¹⁹⁷) between the cleavage sites for BoNT E and BoNT A is essential for late postdocking steps. A required role for SNAP-25 at this stage was also indicated by inhibition of Ca²⁺-activated LDCV fusion in ATP-primed cells by a C-terminal peptide antibody. We conclude that plasma membrane SNAP-25, particularly residues 181-197, is required for Ca²⁺-regulated membrane fusion at a step beyond LDCV docking and ATP utilization.

Footnotes

↵* This work was supported by United States Public Health Service Grants DK25861, DK40428 (to T. F. J. M.), and NS17742 (to B. R. D.) and an award from the Northwestern Mutual Life Foundation, Wisconsin (to A. B.). 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
↵¹ The abbreviations used are:

SNAP-25

synaptosome-associated protein of 25 kDa

NE

norepinephrine

BoNT

botulinum neurotoxin

Tetx

tetanus toxin

LDCV

large dense core vesicle

NSF

N-ethylmaleimide-sensitive factor

SNAP

soluble NSF attachment protein

KGlu

potassium glutamate

BSA

bovine serum albumin

GMPPNP

guanylyl imidotriphosphate.
↵²A. Banerjee, V. A. Barry, B. R. DasGupta, and T. F. J. Martin (1996) J. Biol. Chem. 271, 20223-20230.
↵³T. F. J. Martin and J. A. Kowalchyk, unpublished results.
- Received June 6, 1996.