title: N-Ethylmaleimide-sensitive factor acts at a prefusion ATP-dependent step in Ca2+-activated exocytosis.
abstract: An ATP-dependent activity of NSF (N-ethylmaleimide-sensitive factor) that rearranges soluble NSF attachment protein (SNAP) receptor (SNARE) protein complexes was proposed to be the driving force for membrane fusion. The Ca2+-activated fusion of secretory vesicles with the plasma membrane in permeable PC12 cells requires ATP; however, the ATP requirement is for a priming step that precedes the Ca2+-triggered fusion reaction. While phosphoinositide phosphorylation is a key reaction required for priming, additional ATP-dependent reactions are also necessary. Here we report that the NSF-catalyzed rearrangement of SNARE protein complexes occurs during ATP-dependent priming. NSF with alpha-SNAP (soluble NSF attachment protein) were required for ATP-dependent priming but not Ca2+-triggered fusion, indicating that NSF acts at an ATP-dependent prefusion step rather than at fusion itself. NSF-catalyzed activation of SNARE proteins may reorganize membranes to generate a vesicle-plasma membrane prefusion intermediate that is poised for conversion to full fusion by Ca2+-dependent mechanisms.
authors.affiliation: Department of Food Microbiology and Toxicology, University of Wisconsin, Madison, Wisconsin 53706, USA.
authors.forename: A
authors.initials: A
authors.lastname: Banerjee
authors.forename: V A
authors.initials: VA
authors.lastname: Barry
authors.forename: B R
authors.initials: BR
authors.lastname: DasGupta
authors.forename: T F
authors.initials: TF
authors.lastname: Martin
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000502:physiology*; D002135:Calcium-Binding Proteins; D002462:Cell Membrane / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D005089:Exocytosis; D008561:Membrane Fusion; D008562:Membrane Glycoproteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D016716:PC12 Cells; D011485:Protein Binding; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D051381:Rats; D050600:SNARE Proteins; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013572:Synaptic Vesicles / Q000378:metabolism; D050825:Synaptosomal-Associated Protein 25; D050857:Synaptotagmins; D033921:Vesicular Transport Proteins
chemical_list: D002135:Calcium-Binding Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D050600:SNARE Proteins; C494210:Snap25 protein, rat; D013439:Sulfhydryl Reagents; D050825:Synaptosomal-Associated Protein 25; D033921:Vesicular Transport Proteins; D050857:Synaptotagmins; D000255:Adenosine Triphosphate; D005033:Ethylmaleimide; D002118:Calcium