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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.

ABSTRACT: 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 [(3)H]palmitoyl-CoA as the lipid donor. [(3)H]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 [(3)H]palmitate in this assay. [(3)H]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 alpha-helical conformation of the protein, which is induced by binding to syntaxin 1.

SUBMITTER: Veit M

PROVIDER: S-EPMC1220741 | biostudies-other | 2000 Jan

REPOSITORIES: biostudies-other

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Project description:SNAP-25 (synaptosomal-associated protein 25), syntaxin and synaptobrevin are the three SNARE [soluble NSF attachment protein receptor (where NSF = N-ethylmaleimide-sensitive fusion protein)] proteins that form the core complex involved in synaptic vesicle docking and subsequent fusion with the target membrane. The present study is aimed at understanding the mechanisms of fusion of vesicles carrying glucose transporter proteins with the plasma membrane in human insulin-responsive tissues. It describes the isolation and characterization of cDNA molecules encoding SNAP-25 A and B isoforms, syntaxin 4 and synaptobrevins (also known as vehicle-associated membrane proteins) from two major human insulin-responsive tissues, skeletal muscle and fat. The DNA and deduced amino acid sequences of SNAP-25 revealed perfect identity with the previously reported human neural SNAP-25 A and B isoforms. Our results indicate the presence of both isoforms both in insulin-responsive tissues and in in vitro cultured 3T3-L1 cells, but suggest a differential pattern of gene expression: isoform A is the major species in adipose tissue, and isoform B is the major species in skeletal muscle. The presence of SNAP-25 protein in 3T3-L1 cells was demonstrated by immunofluorescence microscopy using an anti-SNAP-25 monoclonal antibody. Immunoprecipitation experiments using the same monoclonal antibody also revealed the presence of SNAP-25 protein in plasma membrane fractions from rat epididymal fat pads. The syntaxin 4-encoding region from skeletal muscle contains five nucleotide differences from the previously reported placental cDNA sequence, two of which result in amino acid changes: Asp-174 to Glu and Val-269 to Ala. The synaptobrevin 1 cDNA from skeletal muscle contains two nucleotide differences when compared with the corresponding clone from neural tissues, one of which is silent and the other resulting in the amino acid change Thr-102 to Ala. The cDNA sequence of the protein from fat is identical with that of human synaptobrevin 1 from neural tissues. Furthermore, we have confirmed the presence of syntaxin 4 in fat and of synaptobrevin 2 in skeletal muscle by PCR amplification and Southern hybridization analysis. Using the yeast two-hybrid system, an interaction was observed between the full-length cytoplasmic domains of syntaxin 4 and synaptobrevin 2, a vesicle membrane SNARE previously shown by others to be associated with vesicles carrying the GLUT4 glucose transporter protein, but no interaction was seen with synaptobrevin 1. Flow cytometry of low-density microsomes isolated from fat cells was used to demonstrate the binding of syntaxin 4 to a subset of vesicles carrying GLUT4 protein; whereas SNAP-25 on its own bound poorly to these vesicles, the syntaxin 4-SNAP-25 complex gave a strong interaction.

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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.

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