SNAP-25 is targeted to the plasma membrane through a novel membrane-binding domain

SNAP-25, syntaxin, and synaptobrevin are SNARE proteins that mediate fusion of synaptic vesicles with the plasma membrane. Membrane attachment of synt axin and synaptobrevin is achieved through a C-terminal hydrophobic tail, w hereas SNAP-25 association with membranes appears to depend upon palmitoyla tion of cysteine residues located in the center of the molecule. This proce ss requires an intact secretory pathway and is inhibited by brefeldin A. He re we show that the minimal plasma membrane-targeting domain of SNAP-25 map s to residues 85-120, This sequence is both necessary and sufficient to tar get a heterologous protein to the plasma membrane. Palmitoylation of this d omain is sensitive to brefeldin A, suggesting that it uses the same membran e-targeting mechanism as the full-length protein. As expected, the palmitoy lated cysteine cluster is present within this domain, but surprisingly memb rane anchoring requires an additional five-amino acid sequence that is high ly conserved among SNAP-25 family members. Significantly, the membrane-targ eting module coincides with the protease-sensitive stretch (residues 83-120 ) that connects the two alpha-helices that SNAP-25 contributes to the four- helix bundle of the synaptic SNARE complex. Our results demonstrate that re sidues 85-120 of SNAP-25 represent a protein module that is physically and functionally separable from the SNARE complex-forming domains.