title: Synaptotagmin interaction with SNAP-25 governs vesicle docking, priming, and fusion triggering.
abstract: SNARE complex assembly constitutes a key step in exocytosis that is rendered Ca(2+)-dependent by interactions with synaptotagmin-1. Two putative sites for synaptotagmin binding have recently been identified in SNAP-25 using biochemical methods: one located around the center and another at the C-terminal end of the SNARE bundle. However, it is still unclear whether and how synaptotagmin-1 × SNARE interactions at these sites are involved in regulating fast neurotransmitter release. Here, we have used electrophysiological techniques with high time-resolution to directly investigate the mechanistic ramifications of proposed SNAP-25 × synaptotagmin-1 interaction in mouse chromaffin cells. We demonstrate that the postulated central binding domain surrounding layer zero covers both SNARE motifs of SNAP-25 and is essential for vesicle docking, priming, and fast fusion-triggering. Mutation of this site caused no further functional alterations in synaptotagmin-1-deficient cells, indicating that the central acidic patch indeed constitutes a mechanistically relevant synaptotagmin-1 interaction site. Moreover, our data show that the C-terminal binding interface only plays a subsidiary role in triggering but is required for the full size of the readily releasable pool. Intriguingly, we also found that mutation of synaptotagmin-1 interaction sites led to more pronounced phenotypes in the context of the adult neuronal isoform SNAP-25B than in the embryonic isoform SNAP-25A. Further experiments demonstrated that stronger synaptotagmin-1 × SNAP-25B interactions allow for the larger primed vesicle pool supported by SNAP-25 isoform B. Thus, synaptotagmin-1 × SNARE interactions are not only required for multiple mechanistic steps en route to fusion but also underlie the developmental control of the releasable vesicle pool.
authors.affiliation: Department of Physiology, University of Saarland, Homburg 66424, Germany. Ralf.Mohrmann@uks.eu
authors.forename: Ralf
authors.initials: R
authors.lastname: Mohrmann
authors.forename: Heidi
authors.initials: H
authors.lastname: de Wit
authors.forename: Emma
authors.initials: E
authors.lastname: Connell
authors.forename: Paulo S
authors.initials: PS
authors.lastname: Pinheiro
authors.forename: Charlotte
authors.initials: C
authors.lastname: Leese
authors.forename: Dieter
authors.initials: D
authors.lastname: Bruns
authors.forename: Bazbek
authors.initials: B
authors.lastname: Davletov
authors.forename: Matthijs
authors.initials: M
authors.lastname: Verhage
authors.forename: Jakob B
authors.initials: JB
authors.lastname: Sørensen
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D020033:Protein Isoforms; D021381:Protein Transport; D050825:Synaptosomal-Associated Protein 25 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D050863:Synaptotagmin I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D022161:Transport Vesicles / Q000378:metabolism*
chemical_list: D020033:Protein Isoforms; C494209:Snap25 protein, mouse; D050825:Synaptosomal-Associated Protein 25; D050863:Synaptotagmin I; C494311:Syt1 protein, mouse