Department of Physiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama-shi, Okayama 700-8558, Japan.
Department of Physiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama-shi, Okayama 700-8558, Japan.
Mol Cell Neurosci. 2012 Jan;49(1):1-8. doi: 10.1016/j.mcn.2011.09.007. Epub 2011 Oct 8.
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.
虽然突触结合蛋白 I（synaptotagmin I）是一种钙（Ca(2+)）结合的突触囊泡蛋白，可能触发可溶性 N-乙基马来酰亚胺敏感因子附着蛋白受体（SNARE）介导的突触囊泡胞吐，但这些蛋白质之间相互作用的机制仍存在争议，特别是关于 SNARE 复合物中与突触结合蛋白 I 结合的蛋白质（s）的身份，以及 Ca(2+) 是否是其高效结合所必需的。为了解决这些问题，从大鼠脑提取物中溶解、免疫沉淀天然蛋白，并通过免疫印迹进行分析。在乙二胺四乙酸存在的情况下，包含突触融合蛋白 1、25kDa 突触相关蛋白（SNAP-25）和突触融合蛋白 2 的 SNARE 复合物与突触结合蛋白 I 共沉淀。向脑提取物中添加 Ca(2+)并不会显著改变共沉淀蛋白的数量。为了确定与突触结合蛋白结合的 SNARE 复合物的成分，将 SNARE 与突触结合蛋白 I 在 HEK293 细胞中共表达并进行免疫沉淀。结果发现，以 Ca(2+)非依赖的方式，只有突触融合蛋白 1，而不是 SNAP-25 和突触融合蛋白 2，与突触结合蛋白 I 结合，而在 1M NaCl 存在的情况下，结合被废除。突触结合蛋白 I 包含 2 个 Ca(2+)结合结构域（C(2)A、C(2)B）。突变 C(2)B 结构域中假定的效应物结合区域的带正电荷的赖氨酸残基，这些残基对于递质释放至关重要，显著抑制了突触结合蛋白 I-突触融合蛋白 1 的结合，而 C(2)A 结构域中的类似突变对结合没有影响。通过突变突触融合蛋白 1 SNARE 基序氨基端一半的多个负电荷谷氨酸残基，降低了突触结合蛋白 I-突触融合蛋白 1 的结合。这些结果表明，突触结合蛋白 I 通过其 C(2)B 结构域效应区域以 Ca(2+)非依赖的方式静电结合突触融合蛋白 1，提供了生化证据表明，在 Ca(2+)流入突触前神经末梢之前，突触结合蛋白 I 就与 SNARE 复合物结合。
