The C-terminal transmembrane region of synaptobrevin binds synaptophysin from adult synaptic vesicles.
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.
Yelamanchili
Sowmya V
SV
AG Funktionelle Zellbiologie, Centrum für Anatomie, Charité-Hochschulmedizin Berlin, Philippstrasse 12, D-10115 Berlin, Germany.
Reisinger
Clemens
C
Becher
Anja
A
Sikorra
Stefan
S
Bigalke
Hans
H
Binz
Thomas
T
Ahnert-Hilger
Gudrun
G
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
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