Bin Na-Ryum, Jung Chang Hun, Kim Byungjin, Chandrasegram Prashanth, Turlova Ekaterina, Zhu Dan, Gaisano Herbert Y, Sun Hong-Shuo, Sugita Shuzo
Division of Fundamental Neurobiology, Toronto Western Research Institute, University Health Network, Krembil Discovery Tower, Toronto, ON M5T 2S8, Canada Department of Physiology, University of Toronto, Toronto, ON M5S 1A8, Canada.
Division of Fundamental Neurobiology, Toronto Western Research Institute, University Health Network, Krembil Discovery Tower, Toronto, ON M5T 2S8, Canada.
J Cell Sci. 2015 May 15;128(10):1946-60. doi: 10.1242/jcs.165662. Epub 2015 Mar 20.
Understanding how Munc18 proteins govern exocytosis is crucial because mutations of this protein cause severe secretion deficits in neuronal and immune cells. Munc18-2 has indispensable roles in the degranulation of mast cell, partly by binding and chaperoning a subset of syntaxin isoforms. However, the key syntaxin that, crucially, participates in the degranulation – whose levels and intracellular localization are regulated by Munc18-2 – remains unknown. Here, we demonstrate that double knockdown of Munc18-1 and Munc-2 in mast cells results in greatly reduced degranulation accompanied with strikingly compromised expression levels and localization of syntaxin-3. This phenotype is fully rescued by wild-type Munc18 proteins but not by the K46E, E59K and K46E/E59K mutants of Munc-18 domain 1, each of which exhibits completely abolished binding to 'closed' syntaxin-3. Furthermore, knockdown of syntaxin-3 strongly impairs degranulation. Collectively, our data argue that residues Lys46 and Glu59 of Munc18 proteins are indispensable for mediating the interaction between Munc18 and closed syntaxin-3, which is essential for degranulation by chaperoning syntaxin-3. Our results also indicate that the functional contribution of these residues differs between immune cell degranulation and neuronal secretion.
了解Munc18蛋白如何调控胞吐作用至关重要，因为该蛋白的突变会导致神经元和免疫细胞出现严重的分泌缺陷。Munc18-2在肥大细胞脱颗粒过程中发挥着不可或缺的作用，部分原因是它能结合并陪伴一部分 syntaxin 异构体。然而，关键参与脱颗粒过程的syntaxin（其水平和细胞内定位受Munc18-2调控）仍然未知。在此，我们证明在肥大细胞中同时敲低Munc18-1和Munc-2会导致脱颗粒显著减少，同时 syntaxin-3的表达水平和定位也受到明显损害。野生型Munc18蛋白能完全挽救这一表型，但Munc-18结构域1的K46E、E59K和K46E/E59K突变体则不能，这些突变体与“封闭”的syntaxin-3的结合均完全丧失。此外，敲低syntaxin-3会严重损害脱颗粒。总的来说，我们的数据表明，Munc18蛋白的赖氨酸46和谷氨酸59残基对于介导Munc18与封闭的syntaxin-3之间的相互作用是不可或缺的，而这种相互作用对于通过陪伴syntaxin-3进行脱颗粒至关重要。我们的结果还表明，这些残基在免疫细胞脱颗粒和神经元分泌中的功能贡献有所不同。
