Functional characterization of palmitoylated and nonacylated SNAP-25 purified from insect cells infected with recombinant baculovirus.

SNARE complex formation among syntaxin 1, VAMP 2, and SNAP-25 triggers fusion of synaptic vesicles with the presynaptic plasma membrane. After fusion the SNARE complex is disassembled by NSF and alpha-SNAP. These reactions have already been characterized with recombinant proteins lacking the authentic protein modifications. To study the role of palmitoylation of SNAP-25, we have purified 6xHis-SNAP-25, the wild-type protein, and mutants with deletions in the palmitoylation region from insect cells infected with recombinant baculovirus. Metabolic labeling with [(3)H]palmitate and Triton-X-114 phase distribution proved that SNAP-25 is palmitoylated in significant amounts. Palmitoylated and nonacylated SNAP-25 form SDS-resistant SNARE complexes which could then be disassembled by NSF and alpha-SNAP. Palmitoylated SNAP-25 attaches almost quantitatively to liposomes, whereas only small amounts of nonacylated SNAP-25 bind to these artificial membranes. Thus, palmitoylation of SNAP-25 is not required for assembly and disassembly of the SNARE complex, but for stable membrane anchoring of this intrinsically hydrophilic protein.