Targeting of SNAP-25 to membranes is mediated by its association with the target SNARE syntaxin.
The docking and fusion of synaptic vesicles with the presynaptic plasma membrane require the interaction of the vesicle-associated membrane protein VAMP with the plasma membrane proteins syntaxin and SNAP-25. Both of these proteins behave as integral membrane proteins, although they are unusual in that they insert into membranes post-translationally. Whereas VAMP and syntaxin possess hydrophobic transmembrane domains, SNAP-25 does not, and it is widely believed that SNAP-25 traffics to and inserts into membranes by post-translational palmitoylation. In pulse-chase biosynthesis studies, we now show that SNAP-25 and syntaxin rapidly bind to each other while still in the cytosol of neuroendocrine and transfected heterologous cells. Cell fractionation studies revealed that cytosolic SNAP-25.syntaxin complexes then traffic to and insert into membranes. Furthermore, the association of SNAP-25 with membranes is dramatically enhanced by syntaxin, and the transmembrane domain of syntaxin is essential for this effect. Surprisingly, despite the importance of the SNAP-25 palmitoylation domain for membrane anchoring at steady state, removal of this domain did not inhibit the initial association of newly synthesized SNAP-25 with membranes in the presence of syntaxin. These data demonstrate that the initial attachment of newly synthesized SNAP-25 to membranes is a consequence of its association with syntaxin and that it is only after syntaxin-mediated membrane tethering that SNAP-25 is palmitoylated.
Vogel
K
K
Experimental Immunology Branch, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Cabaniols
J P
JP
Roche
P A
PA
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D019281:Dimerization; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D016716:PC12 Cells; D019308:Palmitic Acid / Q000378:metabolism; D011485:Protein Binding; D050765:Qa-SNARE Proteins; D051381:Rats; D050825:Synaptosomal-Associated Protein 25
D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050765:Qa-SNARE Proteins; C494208:SNAP25 protein, human; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; D019308:Palmitic Acid