Conserved N-terminal cysteine motif is essential for homo- and heterodimer formation of synaptotagmins III, V, VI, and X
Conserved N-terminal cysteine motif is essential for homo- and heterodimer formation of synaptotagmins III, V, VI, and X
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DOI：
10.1074/jbc.274.44.31421
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发表时间：
1999-10-29
影响因子：
4.8
通讯作者：
Mikoshiba, K
中科院分区：
文献类型：
--
作者：
Fukuda, M;Kanno, E;Mikoshiba, K
突触素现在构成了一个膜蛋白大家族，其特征是一个跨膜区和两个C2结构域。突触聚集素(SYT)I是一种低亲和力的神经递质释放感受器，它的二聚化被认为在突触小泡的胞吐过程中对功能的表达具有重要作用。然而，人们对其他异构体的自二聚特性知之甚少。在本研究中，我们证明了Synaptopagmins(III，V，VI和X)的一个子类(Ibata，K.，Fukuda，Ri.和Mikoshiba，K.(1998)J.Biol.化学。12267-12273)形成对β-硫醇敏感的同源二聚体，并在N端鉴定了三个进化上保守的半胱氨酸残基(N-末端半胱氨酸基序，位于小鼠SYT III的第10、21和33位氨基酸)，它们在其他异构体中不保守。这些半胱氨酸残基的定点突变和免疫共沉淀实验清楚地表明，第一个半胱氨酸残基对于SYT III、V或VI稳定的同源二聚体形成以及Syts II、V、VI和X之间的异源二聚体形成是必不可少的。我们还表明，来自小鼠大脑的天然SYT III形成了对β-硫醇敏感的同源二聚体。我们的结果表明，SYT III和SYT V、VI或X之间基于半胱氨酸的异二聚化具有不同的生化性质，这可能调节了SYT III作为高亲和力钙感受器释放神经递质的功能。
The synaptotagmins now constitute a large family of membrane proteins characterized by one transmembrane region and two C2 domains. Dimerization of synaptotagmin (Syt) I, a putative low affinity Ca2+ sensor for neurotransmitter release, is thought to be important for expression of function during exocytosis of synaptic vesicles. However, little is known about the self-dimerization properties of other isoforms. In this study, we demonstrate that a subclass of synaptotagmins (III, V, VI, and X) (Ibata, K., Fukuda, Ri., and Mikoshiba, K. (1998) J. Biol. Chem. 273, 12267-12273) forms beta-mercaptoethanol-sensitive homodimers and identify three evolutionarily conserved cysteine residues at the N terminus (N-terminal cysteine motif, at amino acids 10, 21, and 33 of mouse Syt III) that are not conserved in other isoforms. Site-directed mutagenesis of these cysteine residues and co-immunoprecipitation experiments clearly indicate that the first cysteine residue is essential for the stable homodimer formation of Syt III, V, or VI, and heterodimer formation between Syts II, V, VI, and X. We also show that native Syt III from mouse brain forms a beta-mercaptoethanol-sensitive homodimer. Our results suggest that the cysteine-based heterodimerization between Syt III and Syt V, VI, or X, which have different biochemical properties, may modulate the proposed function of Syt III as a putative high affinity Ca2+ sensor for neurotransmitter release.