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 mem brane require the interaction of the vesicle-associated membrane protein VA MP with the plasma membrane proteins syntaxin and SNAP-25, Both of these pr oteins behave as integral membrane proteins, although they are unusual in t hat they insert into membranes post-translationally. Whereas VAMP and synta xin 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 s how that SNAP-25 and syntaxin rapidly bind to each other while still in the cytosol of neuroendocrine and transfected heterologous cells. Cell fractio nation studies revealed that cytosolic SNAP-25 .syntaxin complexes then tra ffic 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 s teady state, removal of this domain did not inhibit the initial association of newly synthesized SNAP-25 with membranes in the presence of syntaxin. T hese 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 palm itoylated.