Cysteine residues of SNAP-25 are required for SNARE disassembly and exocytosis, but not for membrane targeting.
The release of neurotransmitter at a synapse occurs via the regulated fusion of synaptic vesicles with the plasma membrane. The fusion of the two lipid bilayers is mediated by a protein complex that includes the plasma membrane target soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein (SNAP) receptors (t-SNAREs), syntaxin 1A and synaptosome-associated protein of 25 kDa (SNAP-25), and the vesicle SNARE (v-SNARE), vesicle-associated membrane protein (VAMP). Whereas syntaxin 1A and VAMP are tethered to the membrane by a C-terminal transmembrane domain, SNAP-25 has been suggested to be anchored to the membrane via four palmitoylated cysteine residues. We demonstrate that the cysteine residues of SNAP-25 are not required for membrane localization when syntaxin 1A is present. Analysis of the 7 S and 20 S complexes formed by mutants that lack cysteine residues demonstrates that the cysteines are required for efficient SNARE complex dissociation. Furthermore, these mutants are unable to support exocytosis, as demonstrated by a PC12 cell secretion assay. We hypothesize that syntaxin 1A serves to direct newly synthesized SNAP-25 through the Golgi transport pathway to the axons and synapses, and that palmitoylation of cysteine residues is not required for targeting, but to optimize interactions required for SNARE complex dissociation.
Washbourne
P
P
Department of Neurosciences, University of New Mexico, 915 Camino de Salud, Albuquerque, NM 87131, USA.
Cansino
V
V
Mathews
J R
JR
Graham
M
M
Burgoyne
R D
RD
Wilson
M C
MC
D000818:Animals; D000954:Antigens, Surface / Q000378:metabolism; D002352:Carrier Proteins; D002478:Cells, Cultured; D006224:Cricetinae; D003545:Cysteine / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli; D005089:Exocytosis / Q000502:physiology; D008565:Membrane Proteins / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D016716:PC12 Cells; D017434:Protein Structure, Tertiary; D051381:Rats; D050600:SNARE Proteins; D013347:Subcellular Fractions; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D014162:Transfection; D033921:Vesicular Transport Proteins
D000954:Antigens, Surface; D002352:Carrier Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050600:SNARE Proteins; C494210:Snap25 protein, rat; C494213:Stx1a protein, rat; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; C090229:VAPP14 protein, rat; D033921:Vesicular Transport Proteins; D003545:Cysteine