Structure-function study of mammalian Munc18-1 and C. elegans UNC-18 implicates domain 3b in the regulation of exocytosis.
Structure-function study of mammalian Munc18-1 and C. elegans UNC-18 implicates domain 3b in the regulation of exocytosis.
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DOI：
10.1371/journal.pone.0017999
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发表时间：
2011-03-21
期刊：
影响因子：
3.7
通讯作者：
Barclay JW
中科院分区：
文献类型：
--
作者：
Graham ME;Prescott GR;Johnson JR;Jones M;Walmesley A;Haynes LP;Morgan A;Burgoyne RD;Barclay JW
Munc 18 -1是一种重要的突触蛋白，在胞吐过程的多个阶段发挥作用，包括囊泡募集、对接和融合。这些功能需要许多不同的突触融合蛋白依赖的相互作用，然而，Munc 18 -1也调节囊泡融合通过突触融合蛋白独立的相互作用与其他胞吐蛋白。虽然Munc 18 -1蛋白参与闭合构象突触融合蛋白结合的结构区域已被彻底研究，但参与其他相互作用的蛋白质区域的特征很差。为了研究这一点，我们进行了随机转座子诱变，确定Munc 18 -1的结构域3b作为蛋白质的功能重要区域。转座子插入在该结构域内的暴露环中特异性地破坏Mint 1结合，尽管留下对闭合构象突触融合蛋白的亲和力和与SNARE复合物的结合不受影响。插入突变显着减少了胞吐的总量，在嗜铬细胞中通过碳纤维安培法测量。通过涕灭威敏感性评估，在秀丽隐杆线虫中引入E118的等效突变也减少了神经递质的释放。使用先前确定的Munc 18 -1突变E466 K功能增益（嗜铬细胞胞吐增加和C. elegans）。这些数据暗示了Munc 18 -1结构域3b中暴露的环在独立于闭合构象突触融合蛋白结合的囊泡融合的转导调节中的新作用。
Munc18-1 is an essential synaptic protein functioning during multiple stages of the exocytotic process including vesicle recruitment, docking and fusion. These functions require a number of distinct syntaxin-dependent interactions; however, Munc18-1 also regulates vesicle fusion via syntaxin-independent interactions with other exocytotic proteins. Although the structural regions of the Munc18-1 protein involved in closed-conformation syntaxin binding have been thoroughly examined, regions of the protein involved in other interactions are poorly characterised. To investigate this we performed a random transposon mutagenesis, identifying domain 3b of Munc18-1 as a functionally important region of the protein. Transposon insertion in an exposed loop within this domain specifically disrupted Mint1 binding despite leaving affinity for closed conformation syntaxin and binding to the SNARE complex unaffected. The insertion mutation significantly reduced total amounts of exocytosis as measured by carbon fiber amperometry in chromaffin cells. Introduction of the equivalent mutation in UNC-18 in Caenorhabditis elegans also reduced neurotransmitter release as assessed by aldicarb sensitivity. Correlation between the two experimental methods for recording changes in the number of exocytotic events was verified using a previously identified gain of function Munc18-1 mutation E466K (increased exocytosis in chromaffin cells and aldicarb hypersensitivity of C. elegans). These data implicate a novel role for an exposed loop in domain 3b of Munc18-1 in transducing regulation of vesicle fusion independent of closed-conformation syntaxin binding.
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影响因子：
5.3
作者：
Gulyas-Kovacs, Attila;de Wit, Heidi;Sorensen, Jakob B.
通讯作者：
Sorensen, Jakob B.
影响因子：
3.9
作者：
Graham, ME;Fisher, RJ;Burgoyne, RD
通讯作者：
Burgoyne, RD
影响因子：
3.3
作者：
Coppola, T;Frantz, C;Regazzi, R
通讯作者：
Regazzi, R
影响因子：
11.4
作者：
Dulubova, I;Sugita, S;Rizo, J
通讯作者：
Rizo, J
DOI：
10.1073/pnas.0611318104
发表时间：
2007-02-20
影响因子：
11.1
作者：
Dulubova, Irina;Khvotchev, Mikhail;Rizo, Josep
通讯作者：
Rizo, Josep