SNAP-25 traffics to the plasma membrane by a syntaxin-independent mechanism.
SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are essential for vesicle docking and fusion. SNAP-25, syntaxin 1A, and synaptobrevin/vesicle-associated membrane protein (VAMP) are SNARE proteins that mediate fusion of synaptic vesicles with the plasma membrane. It has been proposed that interactions of SNAP-25 with syntaxin 1A are required for initial membrane attachment of SNAP-25 (Vogel, K., Cabaniols, J.-P., and Roche, P. (2000) J. Biol. Chem. 275, 2959-2965). However, we have shown previously that residues 85-120 of the SNAP-25 interhelical domain, which do not interact with syntaxin, are necessary and sufficient for palmitoylation and plasma membrane localization of a green fluorescent protein reporter molecule (Gonzalo, S., Greentree, W. K., and Linder, M. E. (1999) J. Biol. Chem. 274, 21313-21318). To clarify the role of syntaxin in membrane targeting of SNAP-25, we studied a SNAP-25 point mutant (G43D) that does not interact with syntaxin. SNAP-25 G43D/green fluorescent protein was palmitoylated and localized at the plasma membrane. Newly synthesized SNAP-25 G43D had the same kinetics of membrane association as the wild-type protein. Furthermore, expression of a cytosolic mutant syntaxin 1A did not interfere with SNAP-25 membrane interactions or palmitoylation in the neuronal cell line NG108-15. Exogenously expressed SNAP-25 targets efficiently to the plasma membrane in cells of neuronal origin but only partially in HeLa cells, a neurosecretion-incompetent line. This phenotype was not rescued when syntaxin 1A was co-expressed with SNAP-25. Our data support a syntaxin-independent mechanism of membrane targeting for SNAP-25.
Loranger
Stephanie S
SS
Department of Cell Biology and Physiology, Washington University School of Medicine, 660 S. Euclid Avenue, St. Louis, MO 63110-1093, USA.
Linder
Maurine E
ME
D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology*; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D019308:Palmitic Acid / Q000378:metabolism; D017433:Protein Structure, Secondary; D050765:Qa-SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1
D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050765:Qa-SNARE Proteins; C494208:SNAP25 protein, human; C494212:STX1A protein, human; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D019308:Palmitic Acid