ALG-2 directly binds Sec31A and localizes at endoplasmic reticulum exit sites in a Ca2+-dependent manner
ALG-2 directly binds Sec31A and localizes at endoplasmic reticulum exit sites in a Ca2+-dependent manner
复制标题
DOI：
10.1016/j.bbrc.2006.12.101
复制
发表时间：
2007-02-16
影响因子：
3.1
通讯作者：
Maki, Masatoshi
中科院分区：
文献类型：
--
作者：
Shibata, Hideki;Suzuki, Hironori;Maki, Masatoshi
采用免疫荧光共聚焦显微镜技术，利用多克隆抗体，研究了penta-EF-hand钙离子结合蛋白ALG-2在HeLa细胞中的细胞内定位。除了其存在于细胞核中之外，发现ALG-2在细胞质中以点状模式分布，其中它与内质网（ER）出口位点标记物p125部分共染色。体外GST下拉分析表明，ALG-2及其可变剪接异构体与COPII组分Sec 31 A以Ca 2+依赖性方式相互作用，生物素标记的ALG-2覆盖试验显示ALG-2与Sec 31 A直接结合。生物化学和免疫荧光显微镜分析表明，ALG-2富集在Sec 31 A定位的膜隔室刺激后，与Ca 2+离子载体A23187。相反，用膜渗透性Ca 2+螯合剂BAPTA-AM处理细胞导致ALG-2在整个细胞中分散，并导致核周区域中Sec 31 A的显著损失。这些发现确立了Sec 31 A作为ALG-2的新靶点，并为研究ALG-2在ER-高尔基体转运中的作用提供了框架。(c)2006爱思唯尔公司All rights reserved.
Intracellular localization of the penta-EF-hand Ca2+-binding protein ALG-2 in HeLa cells was investigated by immunofluorescent confocal microscopy using a polyclonal antibody. In addition to its presence in the nucleus, ALG-2 was found to be distributed in a punctate pattern in the cytoplasm, where it was partly co-stained with an endoplasmic reticulum (ER) exit site marker p125. In vitro GST pull down analysis demonstrated that ALG-2 and its alternatively spliced isoform interact with the COPII component Sec31A in a Ca2+-dependent manner, and a biotin-labeled ALG-2 overlay assay revealed direct binding of ALG-2 to Sec31A. Biochemical and immunofluorescent microscopic analyses showed that ALG-2 was enriched at the Sec31A-localizing membrane compartments upon stimulation with the Ca2+ ionophore A23187. In contrast, treatment of cells with the membrane-permeant Ca2+ chelator BAPTA-AM led to a dispersion of ALG-2 throughout the cells and to a significant loss of Sec31A in the perinuclear region. These findings establish Sec31A as a novel target for ALG-2 and provide a framework for studies on the roles of ALG-2 in ER-Golgi transport. (c) 2006 Elsevier Inc. All rights reserved.