Pull down assay for GTP-bound form of Sar1a reveals its activation during morphological differentiation
Pull down assay for GTP-bound form of Sar1a reveals its activation during morphological differentiation
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DOI：
10.1016/j.bbrc.2018.07.157
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发表时间：
2018-09-10
影响因子：
3.1
通讯作者：
Yamauchi, Junji
中科院分区：
文献类型：
--
作者：
Urai, Yuri;Yamawaki, Minami;Yamauchi, Junji
细胞内分子转运系统是一种基本且通用的细胞机制，受一系列信号分子的调节。 Sar1 小 GTP 酶是在内质网 (ER) 和高尔基体之间控制载体运输中发挥关键作用的分子。与其他小 GTP 酶一样，Sarla 的活性取决于其鸟嘌呤核苷酸结合状态，而鸟嘌呤核苷酸交换因子 (GEF) 和 GTP 酶激活蛋白 (GAP) 调节该状态。尽管哺乳动物 Sarl 在细胞内转运系统中的功能众所周知，但人们对 Sarl 在细胞形态变化过程中何时以及如何被激活知之甚少。在这里，我们表明，Sarla 效应蛋白 Sec23A 和 Sec23B 的 C 端区域（而非 N 端）与 Sarla 的活性 GTP 结合形式特异性结合。使用 Sec23B 重组 C 末端区域的亲和沉淀（下拉）测定表明，Sarla 在神经元细胞系分化后被激活。在神经元 N1E-115 细胞中，当细胞延长神经元突起时，GTP 结合的 Sarla 会增加。在少突胶质细胞 FBD-102b 细胞的形态分化中观察到类似的结果。此外，催乳素调节元件结合 (PREB)，即 Sar1（Sar1 激活剂）的 GEF，在发生形态分化时增加了与无核苷酸形式 Sarla 的结合能力。无核苷酸的小 GTP 酶优先与同源的活性 GEF 相互作用。这些结果提供了证据，表明使用先前未报告的 Pull Down 测定表明，Sar1 和 PREB 在诱导形态分化后上调，表明在形态分化过程中通过 Sar1a 进行信号传导的潜在作用。 (C) 2018 Elsevier Inc. 保留所有权利。
The intracellular molecular transport system is a basic and general cellular mechanism that is regulated by an array of signaling molecules. Sar1 small GTPases are molecules that play a key role in controlling vehicle transport between the endoplasmic reticulum (ER) and Golgi bodies. Like other small GTPases, the activities of Sarla depend on their guanine-nucleotide-binding states, which are regulated by guanine-nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Despite the well-known function of mammalian Sarl in the intracellular transport system, little is known about when and how Sarl is activated during cell morphological changes. Here we show that the C-terminal, but not the N-terminal, regions of Sec23A and Sec23B, the effector proteins of Sarla, specifically bind to the active, GTP-bound form of Sarla. An affinity precipitation (pull-down) assay using a recombinant C-terminal region of Sec23B reveals that Sarla is activated following differentiation in neuronal cell lines. In neuronal N1E-115 cells, GTP-bound Sarla is increased when cells elongate neuronal processes. Similar results are observed in morphological differentiation in oligodendroglial FBD-102b cells. Additionally, prolactin regulatory element binding (PREB), the GEF for Sar1 (Sar1 activator), increases the binding ability to the nucleotide-free form of Sarla when morphological differentiation occurs. Nucleotide-free small GTPases preferentially interact with the cognate, active GEFs. These results provide evidence that using previously unreported pull down assays reveals that Sar1 and PREB are upregulated following the induction of morphological differentiation, suggesting the potential role of signaling through Sar1a during morphological differentiation. (C) 2018 Elsevier Inc. All rights reserved.