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标题：Transmembrane tethering of synaptotagmin to synaptic vesicles controls multiple modes of neurotransmitter release
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作者：Jihye Lee ; J. Troy Littleton
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：12
页码：3793-3798
DOI：10.1073/pnas.1420312112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceSynaptotagmin 1 (Syt1) is widely considered to act as the fast Ca2+ sensor for synchronous synaptic vesicle fusion through its tandem Ca2+-binding C2 domains. Here we demonstrate that Syt1's C2 domains activate rapid synchronous fusion only if they are in the proper orientation and specifically tethered to the synaptic vesicle with an appropriate linker distance. Although expression of the cytoplasmic C2 domains of Syt1 alone did not support fast synchronous release, it did enhance the asynchronous component of exocytosis. These findings demonstrate that synaptic vesicle tethering of Syt1 positions the protein to allow its C2 domains to regulate the kinetics of vesicle fusion. Synaptotagmin 1 (Syt1) is a synaptic vesicle integral membrane protein that regulates neurotransmitter release by activating fast synchronous fusion and suppressing slower asynchronous release. The cytoplasmic C2 domains of Syt1 interact with SNAREs and plasma membrane phospholipids in a Ca2+-dependent manner and can substitute for full-length Syt1 in in vitro membrane fusion assays. To determine whether synaptic vesicle tethering of Syt1 is required for normal fusion in vivo, we performed a structure-function study with tethering mutants at the Drosophila larval neuromuscular junction. Transgenic animals expressing only the cytoplasmic C2 domains or full-length Syt1 tethered to the plasma membrane failed to restore synchronous synaptic vesicle fusion, and also failed to clamp spontaneous vesicle release. In addition, transgenic animals with shorter, but not those with longer, linker regions separating the C2 domains from the transmembrane segment abolished Syt1's ability to activate synchronous vesicle fusion. Similar defects were observed when C2 domain alignment was altered to C2B-C2A from the normal C2A-C2B orientation, leaving the tether itself intact. Although cytoplasmic and plasma membrane-tethered Syt1 variants could not restore synchronous release in syt1 null mutants, they were very effective in promoting fusion through the slower asynchronous pathway. As such, the subcellular localization of Syt1 within synaptic terminals is important for the temporal dynamics that underlie synchronous and asynchronous neurotransmitter release.
关键词：synaptotagmin ; Drosophila ; synaptic vesicle ; neurotransmitter release ; exocytosis