Calreticulin-2 is localized in the lumen of the endoplasmic reticulum but is not a Ca2+-binding protein
- First Online:
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.
KeywordsCalreticulin Endoplasmic reticulum Immunofluorescence Immunoelectron microscopy Stains-all
- Bastianutto C, Clementi E, Codazzi F, Podini P, De Giorgi F, Rizzuto R, Meldolesi J, Pozzan T (1995) Overexpression of calreticulin increases the Ca2+ capacity of rapidly exchanging Ca2+ stores and reveals aspects of their lumenal microenvironment and function. J Cell Biol 130:847–855PubMedCrossRefGoogle Scholar
- Hayashi E, Matsuzaki Y, Hasegawa G, Yaguchi T, Kurihara S, Fujita T, Kageshita T, Sano M, Kawakami Y (2007) Identification of a novel cancer-testis antigen CRT2 frequently expressed in various cancers using representational differential analysis. Clin Cancer Res 13:6267–6274PubMedCrossRefGoogle Scholar