SNAREs in native plasma membranes are active and readily form core complexes with endogenous and exogenous SNAREs.
During neuronal exocytosis, the vesicle-bound soluble NSF attachment protein (SNAP) receptor (SNARE) synaptobrevin 2 forms complexes with the plasma membrane-bound SNAREs syntaxin 1A and SNAP25 to initiate the fusion reaction. However, it is not known whether in the native membrane SNAREs are constitutively active or whether they are unable to enter SNARE complexes unless activated before membrane fusion. Here we used binding of labeled recombinant SNAREs to inside-out carrier supported plasma membrane sheets of PC12 cells to probe for the activity of endogenous SNAREs. Binding was specific, saturable, and depended on the presence of membrane-resident SNARE partners. Our data show that virtually all of the endogenous syntaxin 1 and SNAP-25 are highly reactive and readily form SNARE complexes with exogenously added SNAREs. Furthermore, complexes between endogenous SNAREs were not detectable when the membranes are freshly prepared, but they slowly form upon prolonged incubation in vitro. We conclude that the activity of membrane-resident SNAREs is not downregulated by control proteins but is constitutively active even if not engaged in fusion events.
Lang
Thorsten
T
Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Am Fassberg, D-37077 Göttingen, Germany.
Margittai
Martin
M
Hölzler
Helmut
H
Jahn
Reinhard
R
D000818:Animals; D000954:Antigens, Surface / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D016716:PC12 Cells / Q000378:metabolism; D011485:Protein Binding; D050683:R-SNARE Proteins; D051381:Rats; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D014407:Tumor Cells, Cultured; D033921:Vesicular Transport Proteins
D000954:Antigens, Surface; D046911:Macromolecular Substances; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050683:R-SNARE Proteins; D050600:SNARE Proteins; C494210:Snap25 protein, rat; C494213:Stx1a protein, rat; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins