title: Munc13-4 reconstitutes calcium-dependent SNARE-mediated membrane fusion.
abstract: Munc13-4 is a widely expressed member of the CAPS/Munc13 protein family proposed to function in priming secretory granules for exocytosis. Munc13-4 contains N- and C-terminal C2 domains (C2A and C2B) predicted to bind Ca(2+), but Ca(2+)-dependent regulation of Munc13-4 activity has not been described. The C2 domains bracket a predicted SNARE-binding domain, but whether Munc13-4 interacts with SNARE proteins is unknown. We report that Munc13-4 bound Ca(2+) and restored Ca(2+)-dependent granule exocytosis to permeable cells (platelets, mast, and neuroendocrine cells) dependent on putative Ca(2+)-binding residues in C2A and C2B. Munc13-4 exhibited Ca(2+)-stimulated SNARE interactions dependent on C2A and Ca(2+)-dependent membrane binding dependent on C2B. In an apparent coupling of membrane and SNARE binding, Munc13-4 stimulated SNARE-dependent liposome fusion dependent on putative Ca(2+)-binding residues in both C2A and C2B domains. Munc13-4 is the first priming factor shown to promote Ca(2+)-dependent SNARE complex formation and SNARE-mediated liposome fusion. These properties of Munc13-4 suggest its function as a Ca(2+) sensor at rate-limiting priming steps in granule exocytosis.
authors.affiliation: Department of Biochemistry, University of Wisconsin, Madison, WI 53706, USA.
authors.forename: Kristin L
authors.initials: KL
authors.lastname: Boswell
authors.forename: Declan J
authors.initials: DJ
authors.lastname: James
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Esquibel
authors.forename: Stephen
authors.initials: S
authors.lastname: Bruinsma
authors.forename: Ryutaro
authors.initials: R
authors.lastname: Shirakawa
authors.forename: Hisanori
authors.initials: H
authors.lastname: Horiuchi
authors.forename: Thomas F J
authors.initials: TF
authors.lastname: Martin
mesh_terms: D001792:Blood Platelets / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism; D005089:Exocytosis / Q000502:physiology; D006801:Humans; D008081:Liposomes / Q000378:metabolism; D008407:Mast Cells / Q000378:metabolism; D008561:Membrane Fusion / Q000502:physiology*; D008565:Membrane Proteins / Q000378:metabolism*; D055099:Neuroendocrine Cells / Q000378:metabolism; D050600:SNARE Proteins / Q000378:metabolism*; D050857:Synaptotagmins / Q000378:metabolism
chemical_list: D002135:Calcium-Binding Proteins; D008081:Liposomes; D008565:Membrane Proteins; D050600:SNARE Proteins; C507969:UNC13D protein, human; D050857:Synaptotagmins; D002118:Calcium