CAPS drives trans-SNARE complex formation and membrane fusion through syntaxin interactions.
Ca(2+)-dependent activator protein for secretion (CAPS) is an essential factor for regulated vesicle exocytosis that functions in priming reactions before Ca(2+)-triggered fusion of vesicles with the plasma membrane. However, the precise events that CAPS regulates to promote vesicle fusion are unclear. In the current work, we reconstituted CAPS function in a SNARE-dependent liposome fusion assay using VAMP2-containing donor and syntaxin-1/SNAP-25-containing acceptor liposomes. The CAPS stimulation of fusion required PI(4,5)P(2) in acceptor liposomes and was independent of Ca(2+), but Ca(2+) dependence was restored by inclusion of synaptotagmin. CAPS stimulated trans-SNARE complex formation concomitant with the stimulation of full membrane fusion at physiological SNARE densities. CAPS bound syntaxin-1, and CAPS truncations that competitively inhibited syntaxin-1 binding also inhibited CAPS-dependent fusion. The results revealed an unexpected activity of a priming protein to accelerate fusion by efficiently promoting trans-SNARE complex formation. CAPS may function in priming by organizing SNARE complexes on the plasma membrane.
James
Declan J
DJ
Department of Biochemistry, University of Wisconsin, Madison, WI 53706, USA.
Kowalchyk
Judith
J
Daily
Neil
N
Petrie
Matt
M
Martin
Thomas F J
TF
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism / Q000502:physiology; D005089:Exocytosis / Q000502:physiology; D006706:Homeostasis; D054709:Lecithins / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D008561:Membrane Fusion / Q000502:physiology; D016716:PC12 Cells / Q000502:physiology; D010718:Phosphatidylserines / Q000378:metabolism; D050765:Qa-SNARE Proteins / Q000378:metabolism*; D051381:Rats; D050600:SNARE Proteins / Q000378:metabolism*; D050825:Synaptosomal-Associated Protein 25 / Q000378:metabolism*; D050857:Synaptotagmins / Q000378:metabolism; D050827:Syntaxin 1 / Q000378:metabolism; D050988:Vesicle-Associated Membrane Protein 2 / Q000378:metabolism
D054709:Lecithins; D008081:Liposomes; D010718:Phosphatidylserines; D050765:Qa-SNARE Proteins; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D050988:Vesicle-Associated Membrane Protein 2; D050857:Synaptotagmins