Biochem. J. (2000) 345
(145–151) (Printed in Great Britain)
Palmitoylation of the 25-kDa synaptosomal protein (SNAP-25) in vitro occurs in the absence of an enzyme, but is stimulated by binding to syntaxin
Michael VEIT1
Department of Immunology and Molecular Biology, Vet.-Med. Faculty, Free University Berlin, Luisenstraße 56, 10117 Berlin, Germany
The neuronal N-ethylmaleimide-sensitive-factor-attachment-protein receptor (SNARE) proteins 25-kDa synaptosomal protein (SNAP-25), syntaxin 1 and synaptobrevin 2 interact to form the intermembrane SNARE complex, which mediates docking and fusion of synaptic vesicles with the plasma membrane. Assembly of the SNARE complex is accompanied by conformational changes, especially in SNAP-25. SNAP-25 is palmitoylated in vivo at cysteine residues located in the middle of the molecule. Acylation is required for membrane binding or membrane targeting of this intrinsically hydrophilic protein. Palmitoylation of recombinant SNAP-25 was studied in vitro in the absence of an enzyme source with [3H]palmitoyl-CoA as the lipid donor. [3H]Palmitate incorporation into unbound SNAP-25 was negligible, but was stimulated 100-fold when SNAP-25 was present in the SNARE complex. SNAP-25 in a binary complex with syntaxin 1 was palmitoylated with almost the same efficiency. A mutant of SNAP-25, which was not acylated in vivo, did not incorporate [3H]palmitate in this assay. [3H]Palmitate incorporation into wild-type SNAP-25 was blocked by chemical blocking of free SH groups, but slightly stimulated by reduction of disulfide-bonds. This shows that palmitoylation of SNAP-25 in vitro occurs at the same cysteine residues that are palmitoylated in vivo. This demonstrates that efficient palmitoylation of SNAP-25 depends on an interaction with a physiological binding partner. It suggests further that palmitoylation of SNAP-25 requires the a-helical conformation of the protein, which is induced by binding to syntaxin 1.
Abbreviations used: SNAP-25, 25-kDa synaptosomal protein; VAMP, vesicle-associated membrane protein; NEM,
N-ethylmaleimide; NSF,
N-ethylmaleimide-sensitive factor; SNAP, soluble NSF-attachment protein; SNARE, SNAP receptor; GST, glutathione S-transferase; DTT, dithiothreitol; TNE, Tris/NaCl/EDTA; TN, Tris/NaCl; Pal-CoA, palmitoyl-CoA.
1 e-mail mveit@city.vetmed.fu-berlin.de
Key words: membrane fusion, neurosecretion, palmitoyl-coenzyme A, SNARE complex, synaptobrevin.
Received 24 May 1999/17 September 1999; accepted 22 October 1999
The Biochemical Society, London © 2000
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