Fukuda M, Kanno E, Ogata Y, Mikoshiba K
Laboratory for Developmental Neurobiology, Brain Science Institute, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
J Biol Chem. 2001 Oct 26;276(43):40319-25. doi: 10.1074/jbc.M105356200. Epub 2001 Aug 20.
Synaptotagmin I (Syt I), a proposed major Ca(2+) sensor in the central nervous system, has been hypothesized as functioning in an oligomerized state during neurotransmitter release. We previously showed that Syts I, II, VII, and VIII form a stable SDS-resistant, beta-mercaptoethanol-insensitive, and Ca(2+)-independent oligomer surrounding the transmembrane domain (Fukuda, M., and Mikoshiba, K. (2000) J. Biol. Chem. 275, 28180-28185), but little is known about the molecular mechanism of the Ca(2+)-independent oligomerization by the synaptotagmin family. In this study, we analyzed the Ca(2+)-independent oligomerization properties of Syt I and found that it shows two distinct forms of self-oligomerization activity: stable SDS-resistant self-oligomerization activity and relatively unstable SDS-sensitive self-oligomerization activity. The former was found to be mediated by a post-translationally modified (i.e. fatty-acylated) cysteine (Cys) cluster (Cys-74, Cys-75, Cys-77, Cys-79, and Cys-82) at the interface between the transmembrane and spacer domains of Syt I. We also show that the number of Cys residues at the interface between the transmembrane and spacer domains determines the SDS- resistant oligomerizing capacity of each synaptotagmin isoform: Syt II, which contains seven Cys residues, showed the strongest SDS-resistant oligomerizing activity in the synaptotagmin family, whereas Syt XII, which has no Cys residues, did not form any SDS-resistant oligomers. The latter SDS-sensitive self-oligomerization of Syt I is mediated by the spacer domain, because deletion of the whole spacer domain, including the Cys cluster, abolished it, whereas a Syt I(CA) mutant carrying Cys to Ala substitutions still exhibited self-oligomerization. Based on these results, we propose that the oligomerization of the synaptotagmin family is regulated by two distinct mechanisms: the stable SDS-resistant oligomerization is mediated by the modified Cys cluster, whereas the relatively unstable (SDS-sensitive) oligomerization is mediated by the environment of the spacer domain.
突触结合蛋白I（Syt I）是中枢神经系统中一种被认为主要的Ca(2+)传感器，据推测在神经递质释放过程中以寡聚化状态发挥作用。我们之前表明，Syt I、II、VII和VIII在跨膜结构域周围形成一种稳定的、抗十二烷基硫酸钠（SDS）、对β-巯基乙醇不敏感且不依赖Ca(2+)的寡聚体（福田，M.，和三木茂，K.（2000年）《生物化学杂志》275，28180 - 28185），但对于突触结合蛋白家族不依赖Ca(2+)的寡聚化分子机制知之甚少。在本研究中，我们分析了Syt I不依赖Ca(2+)的寡聚化特性，发现它表现出两种不同形式的自寡聚化活性：稳定的抗SDS自寡聚化活性和相对不稳定的对SDS敏感的自寡聚化活性。前者被发现是由Syt I跨膜结构域和间隔结构域之间界面处的一个翻译后修饰（即脂肪酰化）的半胱氨酸（Cys）簇（Cys - 74、Cys - 75、Cys - 77、Cys - 79和Cys - 82）介导的。我们还表明，跨膜结构域和间隔结构域之间界面处的半胱氨酸残基数量决定了每个突触结合蛋白异构体的抗SDS寡聚化能力：含有7个半胱氨酸残基的Syt II在突触结合蛋白家族中表现出最强的抗SDS寡聚化活性，而没有半胱氨酸残基的Syt XII则不形成任何抗SDS寡聚体。Syt I后者对SDS敏感的自寡聚化是由间隔结构域介导的，因为删除包括Cys簇在内的整个间隔结构域会消除它，而携带Cys到Ala替换的Syt I(CA)突变体仍表现出自寡聚化。基于这些结果，我们提出突触结合蛋白家族的寡聚化受两种不同机制调控：稳定的抗SDS寡聚化由修饰的Cys簇介导，而相对不稳定的（对SDS敏感的）寡聚化由间隔结构域的环境介导。
