Capture and release of partially zipped trans-SNARE complexes on intact organelles.
Soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptors (SNAREs) are hypothesized to trigger membrane fusion by complexing in trans through their membrane-distal N termini and zippering toward their membrane-embedded C termini, which in turn drives the two membranes together. In this study, we use a set of truncated SNAREs to trap kinetically stable, partially zipped trans-SNARE complexes on intact organelles in the absence of hemifusion and content mixing. We show that the C-terminal zippering of SNARE cytoplasmic domains controls the onset of lipid mixing but not the subsequent transition from hemifusion to full fusion. Moreover, we find that a partially zipped nonfusogenic trans-complex is rescued by Sec17, a universal SNARE cochaperone. Rescue occurs independently of the Sec17-binding partner Sec18, and it exhibits steep cooperativity, indicating that Sec17 engages multiple stalled trans-complexes to drive fusion. These experiments delineate distinct functions within the trans-complex, provide a straightforward method to trap and study prefusion complexes on native membranes, and reveal that Sec17 can rescue a stalled, partially zipped trans-complex.
Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.
Matthew L
ML
Schwartz
Alexey J
AJ
Merz
D002118:Calcium / Q000494:pharmacology; D002459:Cell Fusion; D002462:Cell Membrane / Q000502:physiology*; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology; D007700:Kinetics; D008055:Lipids / Q000502:physiology; D008561:Membrane Fusion / Q000502:physiology; D008954:Models, Biological; D015388:Organelles / Q000502:physiology*; D011510:Proteolipids / Q000502:physiology; D050600:SNARE Proteins / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000502:physiology*; D014617:Vacuoles / Q000502:physiology
D008055:Lipids; D011510:Proteolipids; D050600:SNARE Proteins; C020485:proteoliposomes; D002118:Calcium