The N-Ethylmaleimide-sensitive Fusion Protein and -SNAP Induce a Conformational Change in Syntaxin

doi:10.1074/jbc.270.28.16955

The N-Ethylmaleimide-sensitive Fusion Protein and -SNAP Induce a Conformational Change in Syntaxin (*)

From the (1)Department of Pharmacology and Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06510

^§ Supported by the Helen Hay Whitney Foundation. To whom correspondence should be addressed:
Howard Hughes Medical Institute, Boyer Center for Molecular Medicine, Rm. 247, Yale University Medical School, New Haven, CT 06510.
Tel.: 203-737-4455; Fax: 203-787-5334.

Abstract

The N-ethylmaleimide-sensitive fusion protein (NSF) plays an essential role in intracellular membrane fusion events and has been implicated in the exocytosis of synaptic vesicles. NSF binds through soluble NSF attachment proteins (SNAPs) to a complex of neuronal membrane proteins comprised of synaptobrevin, syntaxin, and SNAP-25. Disassembly of this complex by NSF is thought to be a critical step in the molecular events which lead to vesicle fusion with the plasma membrane. Here we have studied the interaction of α-SNAP and NSF with individual components of this complex and have identified syntaxin as a primary substrate for NSF/α-SNAP. We find that α-SNAP binds directly to syntaxin 1A as well as weakly to SNAP-25, while it does not bind to synaptobrevin II. NSF binds to syntaxin through α-SNAP and in the presence of ATP catalyzes a conformational rearrangement which abolishes binding of itself and α-SNAP. This reaction leads to the previously described disassembly of the fusion complex, since synaptobrevin binding to syntaxin is also reduced. α-SNAP binds to a carboxyl-terminal syntaxin fragment (residues 194-288) that also binds synaptobrevin and SNAP-25. However, NSF action on this syntaxin fragment has no effect on the binding of α-SNAP or synaptobrevin. This suggests that the conformational change normally induced by NSF in syntaxin depends on an interaction between carboxyl- and amino-terminal domains of syntaxin.

Footnotes

↵¶ Supported by the Deutsche Forschungsgemeinschaft.
↵* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

NSF

N-ethylmaleimide sensitive fusion protein

SNAP

soluble NSF attachment protein

SNARE

SNAP receptor

v-SNARE

vesicle SNARE

t-SNARE

target SNARE

SNAP-25

synaptosomal associated protein of 25 kDa

GST

glutathione S-transferase

HBS

HEPES-buffered saline

PAGE

polyacrylamide gel electrophoresis

ATPS

adenosine 5′-O-(thiotriphosphate).
↵² Otto, H., Hanson, P. I., Chapman, E. R., Blasi, J., and Jahn, R. (1995) Biochem. Biophys. Res. Commun., in press.
↵³ E. R. Chapman, P. I. Hanson, S. An, and R. Jahn (1995), manuscript submitted for publication.
↵⁴ P. Hanson and R. Jahn, unpublished observations.