Targeting of SNAP-25 to Membranes Is Mediated by Its Association with the Target SNARE Syntaxin*
- From the Experimental Immunology Branch, NCI, National Institutes of Health, Bethesda, Maryland 20892
Abstract
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.
Footnotes
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↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ These authors contributed equally to this work.
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↵§ Present address: Inst. Pasteur, U277, Biologie Moleculaire du Gen, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France.
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↵¶ To whom correspondence should be addressed: NCI, NIH, Bldg. 10, Rm. 4B36, Bethesda, MD 20892. Tel.: 301-594-2595; Fax: 301-496-0887; E-mail: paul.roche@nih.gov.
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↵2 P. A. Roche, unpublished observations.
- Abbreviations:
- t-SNARE
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target SNARE
- PAGE
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polyacrylamide gel electrophoresis
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- Received September 16, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
