title: The C-terminal transmembrane region of synaptobrevin binds synaptophysin from adult synaptic vesicles.
abstract: Synaptophysin and synaptobrevin are abundant membrane proteins of neuronal small synaptic vesicles. In mature, differentiated neurons they form the synaptophysin/synaptobrevin (Syp/Syb) complex. Synaptobrevin also interacts with the plasma membrane-associated proteins syntaxin and SNAP25, thereby forming the SNARE complex necessary for exocytotic membrane fusion. The two complexes are mutually exclusive. Synaptobrevin is a C-terminally membrane-anchored protein with one transmembrane domain. While its interaction with its SNARE partners is mediated exclusively by its N-terminal cytosolic region it has been unclear so far how binding to synaptophysin is accomplished. Here, we show that synaptobrevin can be cleaved in its synaptophysin-bound form by tetanus toxin and botulinum neurotoxin B, or by botulinum neurotoxin D, leaving shorter or longer C-terminal peptide chains bound to synaptophysin, respectively. A recombinant, C-terminally His-tagged synaptobrevin fragment bound to nickel beads specifically bound synaptophysin, syntaxin and SNAP25 from vesicular detergent extracts. After cleavage by tetanus toxin or botulinum toxin D light chain, the remaining C-terminal fragment no longer interacted with syntaxin or SNAP 25. In contrast, synaptophysin was still able to bind to the residual C-terminal synaptobrevin cleavage product. In addition, the His-tagged C-terminal synaptobrevin peptide 68-116 was also able to bind synaptophysin in detergent extracts from adult brain membranes. These data suggest that synaptophysin interacts with the C-terminal transmembrane part of synaptobrevin, thereby allowing the N-terminal cytosolic chain to interact freely with the plasma membrane-associated SNARE proteins. Thus, by binding synaptobrevin, synaptophysin may positively modulate neurotransmission.
authors.affiliation: AG Funktionelle Zellbiologie, Centrum für Anatomie, Charité-Hochschulmedizin Berlin, Philippstrasse 12, D-10115 Berlin, Germany.
authors.forename: Sowmya V
authors.initials: SV
authors.lastname: Yelamanchili
authors.forename: Clemens
authors.initials: C
authors.lastname: Reisinger
authors.forename: Anja
authors.initials: A
authors.lastname: Becher
authors.forename: Stefan
authors.initials: S
authors.lastname: Sikorra
authors.forename: Hans
authors.initials: H
authors.lastname: Bigalke
authors.forename: Thomas
authors.initials: T
authors.lastname: Binz
authors.forename: Gudrun
authors.initials: G
authors.lastname: Ahnert-Hilger
mesh_terms: D000818:Animals; D001905:Botulinum Toxins / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009411:Nerve Endings / Q000378:metabolism / Q000648:ultrastructure; D009419:Nerve Tissue Proteins / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D011485:Protein Binding; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D013572:Synaptic Vesicles / Q000378:metabolism*; D016708:Synaptophysin / Q000737:chemistry / Q000378:metabolism*; D050825:Synaptosomal-Associated Protein 25; D013744:Tetanus Toxin / Q000737:chemistry
chemical_list: D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins; C494210:Snap25 protein, rat; D016708:Synaptophysin; D050825:Synaptosomal-Associated Protein 25; D013744:Tetanus Toxin; D006639:Histidine; D001905:Botulinum Toxins