Ca2+-independent syntaxin binding to the C2B effector region of synaptotagmin

Abstract

Although synaptotagmin I, which is a calcium (Ca(2+))-binding synaptic vesicle protein, may trigger soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated synaptic vesicle exocytosis, the mechanisms underlying the interaction between these proteins remain controversial, especially with respect to the identity of the protein(s) in the SNARE complex that bind(s) to synaptotagmin and whether Ca(2+) is required for their highly effective binding. To address these questions, native proteins were solubilized, immunoprecipitated from rat brain extracts, and analyzed by immunoblotting. SNARE complexes comprising syntaxin 1, 25-kDa synaptosomal-associated protein (SNAP-25), and synaptobrevin 2 were coprecipitated with synaptotagmin I in the presence of ethylene glycol tetraacetic acid. The amount of coprecipitated proteins was significantly unaltered by the addition of Ca(2+) to the brain extract. To identify the component of the SNARE complex that bound to synaptotagmin, SNARE was coexpressed with synaptotagmin in HEK293 cells and immunoprecipitated. Syntaxin, but not SNAP-25 and synaptobrevin, bound to synaptotagmin in a Ca(2+)-independent manner, and the binding was abolished in the presence of 1M NaCl. Synaptotagmin contains 2 Ca(2+)-binding domains (C(2)A, C(2)B). Mutating the positively charged lysine residues in the putative effector-binding region of the C(2)B domain, which are critical for transmitter release, markedly inhibited synaptotagmin-syntaxin binding, while similar mutations in the C(2)A domain had no effect on binding. Synaptotagmin-syntaxin binding was reduced by mutating multiple negatively charged glutamate residues in the amino-terminal half of the syntaxin SNARE motif. These results indicate that synaptotagmin I binds to syntaxin 1 electrostatically through its C(2)B domain effector region in a Ca(2+)-independent fashion, providing biochemical evidence that synaptotagmin I binds SNARE complexes before Ca(2+) influx into presynaptic nerve terminals.

Architecture of the synaptotagmin–SNARE machinery for neuronal exocytosis

“…The effect of the Syt1 R322E, K325E mutant ( Fig. 4 ) as well as reported observations 29 , 30 , 35 , 44 , 45 suggests a functional role for the polybasic region.…”

PRIP (Phospholipase C-related but Catalytically Inactive Protein) Inhibits Exocytosis by Direct Interactions with Syntaxin 1 and SNAP-25 through Its C2 Domain

“…There are several C2 domains that directly interact with SNARE proteins (32-36, 46, 47) with differing specificity. The C2 domains of rabphilin and Rab3-interacting molecule bind only to SNAP-25 (34,35,46), and the C2 domains of synaptotagmin, DOC2, and otoferlin bind to both SNAP-25 and syntaxins, although the Ca 2ϩ dependence and preference for distinct syntaxin isoforms vary (32,33,36,47). PRIP-C2 interacted with both syntaxin 1 and SNAP-25, but the C2 domain of PLC-␦1, despite its structural similarity, did not bind to either syntaxin 1 or SNAP-25.…”

Models of synaptotagmin‐1 to trigger Ca2+‐dependent vesicle fusion

“…In addition to these Ca 2+ -binding loops, the C2B domain contains a polybasic region (KKKK, 324-327; Fig. 1) that is close to Ca 2+ -binding loops and is enriched with lysine residues [23] that interact with anionic phospholipids [26][27][28][29][30][31][32] or the SNARE complex [33][34][35][36][37][38][39][40][41][42][43][44]. Evidence in 1992 showed that Syt1 interacts with anionic membrane [19] or with SNARE proteins [39] by electrostatic interaction.…”

“…Many groups have demonstrated Syt1 interaction with SNARE proteins [33][34][35][36][37][38][39][40][41][42][43][44]. The polybasic region in the C2B domain of Syt1 (KKKK, 324-327; Fig.…”

“…The polybasic region in the C2B domain of Syt1 (KKKK, 324–327; Fig. ) is associated with SNAREs . NMR structure suggested that the polybasic region of the C2B domain (KKKK, 324–327; Fig.…”