The calcium-binding protein ALG-2 regulates protein secretion and trafficking via interactions with MISSL and MAP1B proteins
The calcium-binding protein ALG-2 regulates protein secretion and trafficking via interactions with MISSL and MAP1B proteins
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DOI：
10.1074/jbc.m117.800201
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发表时间：
2017-09
期刊：
影响因子：
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通讯作者：
Terunao Takahara;Kuniko Inoue;Yumika Arai;K. Kuwata;H. Shibata;M. Maki
中科院分区：
文献类型：
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作者：
Terunao Takahara;Kuniko Inoue;Yumika Arai;K. Kuwata;H. Shibata;M. Maki
细胞内钙离子的动员对于广泛的细胞过程是必不可少的，包括信号转导、凋亡和囊泡运输。一些证据表明，骨化相关基因2（ALG-2，也称为PDCD 6），钙结合蛋白，作为钙传感器连接钙水平与有效的囊泡运输，特别是在内质网（ER）到高尔基体的运输步骤。然而，ALG-2如何调节这些过程在很大程度上仍不清楚。在这里，我们报告说，MAPK 1相互作用和纺锤体稳定（MISS）样（MISSL），以前未知的蛋白质，与ALG-2的钙依赖性的方式相互作用。活细胞成像显示，在细胞内钙离子水平上升时，GFP标记的MISSL（GFP-MISSL）以点状模式动态重新定位并与ALG-2共定位。MISSL敲低导致内质网出口部位、内质网-高尔基体中间区室和高尔基体的组成部分解体。重要的是，MISSL或ALG-2的敲低减弱了分泌型碱性磷酸酶（SEAP）（一种模型分泌型货物蛋白）的分泌，通过单蛋白和双蛋白敲低具有类似的分泌减少，表明MISSL和ALG-2以相同的途径起作用以调节分泌过程。此外，ALG-2或MISSL敲低延迟了I型前胶原的ER到高尔基体的转运。我们还发现，ALG-2和MISSL与微管相关蛋白1B（MAP 1B）相互作用，MAP 1B敲低可逆转由MISSL或ALG-2耗竭引起的SEAP分泌减少。这些结果表明，细胞内钙水平的变化通过ALG-2与MISSL和MAP 1B的相互作用在分泌途径的调节中起作用。
Mobilization of intracellular calcium is essential for a wide range of cellular processes, including signal transduction, apoptosis, and vesicular trafficking. Several lines of evidence have suggested that apoptosis-linked gene 2 (ALG-2, also known as PDCD6), a calcium-binding protein, acts as a calcium sensor linking calcium levels with efficient vesicular trafficking, especially at the endoplasmic reticulum (ER)-to-Golgi transport step. However, how ALG-2 regulates these processes remains largely unclear. Here, we report that MAPK1-interacting and spindle-stabilizing (MISS)-like (MISSL), a previously uncharacterized protein, interacts with ALG-2 in a calcium-dependent manner. Live-cell imaging revealed that upon a rise in intracellular calcium levels, GFP-tagged MISSL (GFP-MISSL) dynamically relocalizes in a punctate pattern and colocalizes with ALG-2. MISSL knockdown caused disorganization of the components of the ER exit site, the ER-Golgi intermediate compartment, and Golgi. Importantly, knockdown of either MISSL or ALG-2 attenuated the secretion of secreted alkaline phosphatase (SEAP), a model secreted cargo protein, with similar reductions in secretion by single- and double-protein knockdowns, suggesting that MISSL and ALG-2 act in the same pathway to regulate the secretion process. Furthermore, ALG-2 or MISSL knockdown delayed ER-to-Golgi transport of procollagen type I. We also found that ALG-2 and MISSL interact with microtubule-associated protein 1B (MAP1B) and that MAP1B knockdown reverts the reduced secretion of SEAP caused by MISSL or ALG-2 depletion. These results suggest that a change in the intracellular calcium level plays a role in regulation of the secretory pathway via interaction of ALG-2 with MISSL and MAP1B.