Takahara Terunao, Inoue Kuniko, Arai Yumika, Kuwata Keiko, Shibata Hideki, Maki Masatoshi
From the Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, and
From the Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, and.
J Biol Chem. 2017 Oct 13;292(41):17057-17072. doi: 10.1074/jbc.M117.800201. Epub 2017 Sep 1.
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 ), 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 APK1-nteracting and pindle-tabilizing (MISS)-ike (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 creted lkaline hosphatase (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.
细胞内钙的动员对于广泛的细胞过程至关重要，包括信号转导、细胞凋亡和囊泡运输。多条证据表明，凋亡相关基因2（ALG-2，也称为 ），一种钙结合蛋白，作为一种钙传感器，将钙水平与高效的囊泡运输联系起来，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，我们报告了一种以前未被表征的蛋白质——APK1相互作用和平行稳定（MISS）样（MISSL），它以钙依赖的方式与ALG-2相互作用。活细胞成像显示，当细胞内钙水平升高时，绿色荧光蛋白标记的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位点、内质网-高尔基体中间腔室和高尔基体的成分紊乱。重要的是，MISSL或ALG-2的敲低减弱了分泌性碱性磷酸酶（SEAP）的分泌，SEAP是一种模型分泌货物蛋白，单蛋白和双蛋白敲低后的分泌减少相似，这表明MISSL和ALG-2在同一途径中起作用来调节分泌过程。此外，ALG-2或MISSL敲低延迟了I型前胶原从内质网到高尔基体的运输。我们还发现ALG-2和MISSL与微管相关蛋白1B（MAP1B）相互作用，并且MAP1B敲低可逆转由MISSL或ALG-2缺失引起的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MAP1B的相互作用在分泌途径的调节中起作用。
