Identification of SNAREs Involved in Regulated Exocytosis in the Pancreatic Acinar Cell

doi:10.1074/jbc.274.32.22871

Identification of SNAREs Involved in Regulated Exocytosis in the Pancreatic Acinar Cell*

From the ^‡Department of Pharmacology, University of Cambridge, Cambridge, CB2 1QJ, United Kingdom and the ^¶Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Am Fassberg, D-37077 Göttingen, Germany

Abstract

The molecular basis of exocytotic membrane fusion in the pancreatic acinar cell was investigated using an in vitro assay that measures both zymogen granule-plasma membrane fusion and granule-granule fusion. These two fusion events were differentially sensitive to Ca²⁺, suggesting that they are controlled by different Ca²⁺-sensing mechanisms. Botulinum neurotoxin C (BoNT/C) treatment of the plasma membranes caused cleavage of syntaxin 2, the apical isoform of this Q-SNARE, but did not affect syntaxin 4, the basolateral isoform. BoNT/C also cleaved syntaxin 3, the zymogen granule isoform. BoNT/C treatment of plasma membranes abolished granule-plasma membrane fusion, whereas toxin treatment of the granules reduced granule-plasma membrane fusion and abolished granule-granule fusion. Tetanus toxin cleaved granule-associated synaptobrevin 2 but caused only a small reduction in both granule-plasma membrane fusion and granule-granule fusion. Our results indicate that syntaxin 2 is the isoform that mediates fusion between zymogen granules and the apical plasma membrane of the acinar cell. Syntaxin 3 mediates granule-granule fusion, which might be involved in compound exocytosis. In contrast, the major R-SNARE on the zymogen granule remains to be identified.

Footnotes

↵* This work was supported by Project Grant 053024 from the Wellcome Trust (to J. M. E.).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.
↵§ Recipient of a Medical Research Council studentship.
↵‖ To whom correspondence should be addressed: Dept. of Pharmacology, University of Cambridge, Tennis Court Rd., Cambridge, CB2 1QJ, United Kingdom. Tel.: 44-1223-334014; Fax: 44-1223-334040; E-mail: jme1000@cus.cam.ac.uk.
Abbreviations:

SNARE

solubleN-ethylmaleimide-sensitive fusion protein attachment protein receptor

SNAP-25

synaptosome-associated protein of molecular weight 25,000

SNAP-23

synaptosome-associated protein of molecular weight 23,000

TeNT

tetanus neurotoxin

BoNT/C

botulinum neurotoxin C

MBP

maltose-binding protein

PAGE

polyacrylamide gel electrophoresis

MES

2-morpholinoethanesulfonic acid

R18

octadecylrhodamine

PT

1,10-phenanthroline

VAMP

vesicle-associated membrane protein
- Received April 30, 1999.
- Revision received May 20, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.