Kee Y, Scheller R H
Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Stanford University Medical Center, California 94305, USA.
J Neurosci. 1996 Mar 15;16(6):1975-81. doi: 10.1523/JNEUROSCI.16-06-01975.1996.
Synaptotagmin, an abundant calcium- and phospholipid-binding protein of synaptic vesicles, has been proposed to regulate neurotransmitter release at the nerve terminal. To understand better the biochemical mechanism of neurotransmitter release, we have investigated the calcium-dependent and -independent protein-protein interactions between synaptotagmin I and syntaxin 1a, a subunit of the receptor for synaptic vesicles on the presynaptic plasma membrane. Soluble syntaxin 1a binds to synaptotagmin glutathione S-transferase (GST) fusion protein, and the binding was decreased in the presence of calcium. A synaptotagmin fragment containing the second C2 repeat (Syt3-5) had the same binding profile as the whole cytoplasmic domain; however, fragments containing the first C2 repeat (Syt1-3 and Syt2-3) showed calcium-dependent binding to syntaxin. In addition, the soluble full-length cytoplasmic domain of synaptotagmin binds to a syntaxin GST fusion protein in a calcium-dependent manner. Syntaxin domains required for calcium-dependent and -independent synaptotagmin-binding were localized using syntaxin deletion mutants. Amino acids 241-266 of the syntaxin C terminus were required for calcium-independent binding of synaptotagmin. The minimal domain required for calcium-dependent binding of synaptotagmin to syntaxin was localized to amino acids 220-266. The syntaxin domains required for synaptotagmin binding overlap with the domains for vesicle-associated membrane protein (or VAMP) and alpha-soluble N-ethyl-maleimide-sensitive fusion protein attachment protein (or alphaSNAP) interactions. The data suggest both calcium-dependent and -independent roles of synaptotagmin in regulating synaptic vesicle release and/or recycling.
突触结合蛋白是突触小泡中一种丰富的钙和磷脂结合蛋白，被认为可调节神经末梢的神经递质释放。为了更好地理解神经递质释放的生化机制，我们研究了突触结合蛋白I与 syntaxin 1a（突触前质膜上突触小泡受体的一个亚基）之间的钙依赖性和非钙依赖性蛋白质-蛋白质相互作用。可溶性syntaxin 1a与突触结合蛋白谷胱甘肽S-转移酶（GST）融合蛋白结合，并且在有钙的情况下结合减少。包含第二个C2重复序列的突触结合蛋白片段（Syt3-5）具有与整个细胞质结构域相同的结合模式；然而，包含第一个C2重复序列的片段（Syt1-3和Syt2-3）显示出与syntaxin的钙依赖性结合。此外, 突触结合蛋白的可溶性全长细胞质结构域以钙依赖性方式与syntaxin GST融合蛋白结合。使用syntaxin缺失突变体定位了与突触结合蛋白的钙依赖性和非钙依赖性结合所需的syntaxin结构域。突触结合蛋白的非钙依赖性结合需要syntaxin C末端的氨基酸241-266。突触结合蛋白与syntaxin的钙依赖性结合所需的最小结构域定位于氨基酸220-266。突触结合蛋白结合所需的syntaxin结构域与囊泡相关膜蛋白（或VAMP）和α-可溶性N-乙基马来酰亚胺敏感融合蛋白附着蛋白（或αSNAP）相互作用的结构域重叠。数据表明突触结合蛋白在调节突触小泡释放和/或再循环中具有钙依赖性和非钙依赖性作用。
