Calreticulin-2 is localized in the lumen of the endoplasmic reticulum but is not a Ca2+-binding protein
Calreticulin-2 is localized in the lumen of the endoplasmic reticulum but is not a Ca2+-binding protein
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DOI：
10.1007/s00418-011-0817-z
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发表时间：
2011-06-01
影响因子：
2.3
通讯作者：
Senda, Takao
中科院分区：
文献类型：
--
作者：
Nomura, Ryuji;Orii, Minami;Senda, Takao
钙网蛋白（CRT）-1是内质网腔中的主要钙缓冲蛋白。人类和小鼠CRT-2在2002年被分离，但亚细胞定位和功能仍不清楚。在这里，我们研究了细胞内的定位和功能的CRT-2与血凝素标记（HA-）的人CRT-2。Western印迹显示HA-CRT-2在50 kDa处为单一条带。使用免疫荧光显微镜培养的成纤维细胞和上皮细胞转染HA-CRT-2的cDNA，标记HA-CRT-2被视为一个网状网络的核膜模式，共定位与钙连接蛋白和蛋白质二硫键异构酶。免疫电镜证实HA-CRT-2定位于内质网腔内。尽管HA-CRT-1免疫沉淀物被染成蓝色，但检测Ca 2+结合蛋白的方法--全染色不能染色HA-CRT-2的免疫沉淀物。这些结果表明，在SDS-PAGE上未标记的CRT-2的分子量为49 kDa，CRT-2以及CRT-1定位于内质网的内腔中，但CRT-2结合Ca 2+的能力可能不存在或远低于CRT-1。
Calreticulin (CRT)-1 is a major Ca2+-buffering protein in the lumen of the endoplasmic reticulum. Human and murine CRT-2 was isolated in 2002, but the subcellular localization and function is still unclear. Here, we studied the intracellular localization and function of CRT-2 with hemagglutinin-tagged (HA-) human CRT-2. Western blotting revealed HA-CRT-2 as a single band at 50 kDa. Using immunofluorescence microscopy of cultured fibroblasts and epithelial cells transfected with HA-CRT-2 cDNA, labeling for HA-CRT-2 was seen as a reticular network with a nuclear envelope pattern that colocalized with calnexin and protein disulfide isomerase. Immunoelectron microscopy confirmed that HA-CRT-2 was localized in the lumen of the endoplasmic reticulum. Stains-all staining, a method to detect Ca2+-binding proteins, could not stain the immunoprecipitate of HA-CRT-2, although HA-CRT-1 immunoprecipitate was stained blue. These results indicate that the molecular weight of the non-tagged CRT-2 on SDS-PAGE is 49 kDa, and that CRT-2, as well as CRT-1, is localized in the lumen of the endoplasmic reticulum, but that CRT-2 capacity for Ca2+-binding may be absent or much lower than that of CRT-1.