Anti-calreticulin Antibody Binds to a Membrane Protein in Caveolae
Anti-calreticulin Antibody Binds to a Membrane Protein in Caveolae
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DOI：
10.1267/ahc.38.43
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发表时间：
2005
影响因子：
2.4
通讯作者：
R. Nomura;T. Aoki;H. Hagiwara;T. Senda;T. Fujimoto
中科院分区：
文献类型：
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作者：
R. Nomura;T. Aoki;H. Hagiwara;T. Senda;T. Fujimoto
细胞膜小窝中含有大量与钙动员有关的蛋白质，包括1，4，5-三磷酸肌醇受体样蛋白和质膜钙泵。在寻找其他钙相关蛋白的过程中，我们发现钙网蛋白（CRT）（一种内质网（ER）钙结合蛋白）的多克隆抗体结合到小窝的胞质表面。通过培养的成纤维细胞的免疫荧光显微镜，发现由抗CRT抗体标记与小窝蛋白-1共定位。冰冻切片的免疫金电子显微镜显示抗体装饰小窝的胞质表面。小窝标记可能是由于CRT泄漏出ER，但即使用10 mM EDTA、1 M NaCl或0.1 M Na 2CO 3（pH 11）处理分离的质膜制剂，标记仍持续存在。此外，在SDS消化的冷冻断裂复制品，标记与质膜的P面的小窝。然而，在转染血凝素（HA）标记的cDNA的细胞中，通过抗HA的标记只观察到在ER中，并没有发现在小窝。两者合计，结果表明，结构上类似于CRT的蛋白质定位在小窝作为一个完整的膜蛋白，并且该蛋白质可能在功能上与细胞内钙稳态。
Caveolae have been reported to contain a number of proteins related to Ca mobilization, including an inositol 1,4,5trisphosphate receptor-like protein, and a plasmalemmal calcium pump. In search of other Ca -related proteins, we found that a polyclonal antibody to calreticulin (CRT), an endoplasmic reticulum (ER) Ca -binding protein, binds to the cytoplasmic surface of caveolae. By immunofluorescence microscopy of cultured fibroblasts, labeling by the anti-CRT antibody was found colocalized with caveolin-1. Immunogold electron microscopy of ultrathin frozen sections showed that the antibody decorates the cytoplasmic surface of caveolae. The caveolar labeling could be due to CRT that leaked out of the ER, but the labeling persisted even when isolated plasmalemmal preparations were treated with 10 mM EDTA, 1 M NaCl, or 0.1 M Na2CO3 (pH 11). Furthermore, in SDS-digested freeze-fracture replicas, labeling was associated with caveolae in the P face of the plasma membrane. However, in cells transfected with hemagglutinin (HA)-tagged cDNA, the labeling by anti-HA was observed only in the ER and was not found in caveolae. Taken together, the results suggest that a protein structurally similar to CRT is localized in caveolae as an integral membrane protein, and that the protein could be functionally related to the intracellular Ca homeostasis.