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.
Kammer
Bettina
B
Department of Immunology and Molecular Biology, Vet-Med Faculty, Free University, Berlin, Germany.
Schmidt
Michael F G
MF
Veit
Michael
M
D000215:Acylation; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002478:Cells, Cultured; D017353:Gene Deletion; D015967:Gene Expression Regulation, Viral; D007313:Insecta; D008081:Liposomes / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D019308:Palmitic Acid / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D014316:Tritium; D033921:Vesicular Transport Proteins
D008081:Liposomes; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D033921:Vesicular Transport Proteins; D014316:Tritium; D019308:Palmitic Acid