Syntaxin 1A drives fusion of large dense-core neurosecretory granules into a planar lipid bilayer.
The SNARE complex, involved in vesicular trafficking and exocytosis, is composed of proteins in the vesicular membrane (v-SNAREs) that intertwine with proteins of the target membrane (t-SNAREs). Our results show that modified large dense-core neurosecretory granules (NSGs), isolated from the bovine neurohypophysis, spontaneously fuse with a planar lipid membrane containing only the t-SNARE syntaxin 1A. This provides evidence that syntaxin alone is able to form a functional fusion complex with native v-SNAREs of the NSG. The fusion was similar to constitutive, not regulated, exocytosis because changes in free [Ca2+] had no effect on the syntaxin-mediated fusion. Several deletion mutants of syntaxin 1A were also tested. The removal of the regulatory domain did not significantly reduce spontaneous fusion. However, a syntaxin deletion mutant consisting of only the transmembrane domain was incapable of eliciting spontaneous fusion. Finally, a soluble form of syntaxin 1A (lacking its transmembrane domain) was used to saturate the free syntaxin-binding sites of modified NSGs. This treatment blocks spontaneous fusion of these granules to a bilayer containing full-length syntaxin 1A. This method provides an effective model system to study possible regulatory components affecting vesicle fusion.
McNally
James M
JM
Department of Physiology, University of Massachusetts Medical School, Worcester, MA, USA.
Woodbury
Dixon J
DJ
Lemos
José R
JR
D020816:Amino Acid Motifs; D000818:Animals; D001703:Biophysics / Q000379:methods; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D004492:Edetic Acid / Q000737:chemistry; D005089:Exocytosis; D017353:Gene Deletion; D006863:Hydrogen-Ion Concentration; D008051:Lipid Bilayers / Q000737:chemistry*; D009154:Mutation; D017434:Protein Structure, Tertiary; D022142:Secretory Vesicles / Q000378:metabolism*; D012995:Solubility; D050827:Syntaxin 1 / Q000378:metabolism*
D008051:Lipid Bilayers; D050827:Syntaxin 1; D004492:Edetic Acid; D002118:Calcium