The calcium-binding loops of the tandem C2 domains of synaptotagmin VII cooperatively mediate calcium-dependent oligomerization

被引:29
作者
Fukuda, M
Katayama, E
Mikoshiba, K
机构
[1] RIKEN, Fukuda Initiat Res Unit, Inst Phys & Chem Res, Wako, Saitama 3510198, Japan
[2] RIKEN, Brain Sci Inst, Dev Neurobiol Lab, Wako, Saitama 3510198, Japan
[3] Univ Tokyo, Inst Med Sci, Dept Basic Med Sci, Div Biomol Imaging,Minato Ku, Tokyo 1088639, Japan
[4] JST, PRESTO, Kawaguchi, Saitama 3320012, Japan
[5] Univ Tokyo, Inst Med Sci, Dept Basic Med Sci, Div Mol Neurobiol,Minato Ku, Tokyo 1088639, Japan
关键词
D O I
10.1074/jbc.M201697200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Synaptotagmin VII (Syt VII), a proposed regulator for Ca2+-dependent exocytosis, showed a robust Ca2+-dependent oligomerization property via its two C2 domains (Fukuda, M., and Mikoshiba, K. (2001) J. Biol. Chem. 276, 27670-27676), but little is known about its structure or the critical residues directly involved in the oligomerization interface. In this study, site-directed mutagenesis and chimeric analysis between Syt I and Syt VII showed that three Asp residues in Ca2+-binding loop 1 or 3 (Asp-172, Asp-303, and Asp-357) are crucial to robust Ca2+-dependent oligomerization. Unlike Syt 1, however, the polybasic sequence in the 134 strands of the C2 structures (so-called "C2 effector domain") is not involved in the Ca2+-dependent oligomerization of Syt VII. The results also showed that the Ca2+-binding loops of the two C2 domains cooperatively mediate Syt VII oligomerization (i.e. the presence of redundant Ca2+-binding site(s)) as well as the importance of Ca2+-dependent oligomerization of Syt VII in Ca2+-regulated secretion. Expression of wild-type tandem C2 domains of Syt VII in PC12 cells inhibited Ca2+-dependent neuropeptide Y release, whereas mutant fragments lacking Ca2+-dependent oligomerization activity had no effect. Finally, rotary-shadowing electron microscopy showed that the Ca2+-dependent oligomer of Syt VII is "a large linear structure," not an irregular aggregate. By contrast, in the absence of Ca2+ Syt VII molecules were observed to form a globular structure. Based on these results, we suggest that the linear Ca2+-dependent oligomer may be aligned at the fusion site between vesicles and plasma membrane and modulate Ca2+-regulated exocytosis by opening or dilating fusion pores.
引用
收藏
页码:29315 / 29320
页数:6
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