Histochemistry and Cell Biology

, Volume 135, Issue 6, pp 531–538

Calreticulin-2 is localized in the lumen of the endoplasmic reticulum but is not a Ca2+-binding protein

Original Paper


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.


Calreticulin Endoplasmic reticulum Immunofluorescence Immunoelectron microscopy Stains-all 


  1. Baksh S, Michalak M (1991) Expression of calreticulin in Escherichia coli and identification of its Ca2+ binding domains. J Biol Chem 266:21458–21465PubMedGoogle Scholar
  2. 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
  3. Bedard K, Szabo E, Michalak M, Opas M (2005) Cellular functions of endoplasmic reticulum chaperones calreticulin, calnexin, and ERp57. Int Rev Cytol 245:91–121PubMedCrossRefGoogle Scholar
  4. Campbell KP, MacLennan DH, Jorgensen AO (1983) Staining of the Ca2+-binding proteins, calsequestrin, calmodulin, troponin C, and S-100, with the cationic carbocyanine dye “Stains-all”. J Biol Chem 258:11267–11273PubMedGoogle Scholar
  5. Edman JC, Ellis L, Blacher RW, Roth RA, Rutter WJ (1985) Sequence of protein disulphide isomerase and implications of its relationship to thioredoxin. Nature 317:267–270PubMedCrossRefGoogle Scholar
  6. Fujita A, Cheng J, Hirakawa M, Furukawa K, Kusunoki S, Fujimoto T (2007) Gangliosides GM1 and GM3 in the living cell membrane form clusters susceptible to cholesterol depletion and chilling. Mol Biol Cell 18:2112–2122PubMedCrossRefGoogle Scholar
  7. Gelebart P, Opas M, Michalak M (2005) Calreticulin, a Ca2+-binding chaperone of the endoplasmic reticulum. Int J Biochem Cell Biol 37:260–266PubMedCrossRefGoogle Scholar
  8. 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
  9. Louvard D, Reggio H, Warren G (1982) Antibodies to the Golgi complex and the rough endoplasmic reticulum. J Cell Biol 92:92–107PubMedCrossRefGoogle Scholar
  10. Mesaeli N, Nakamura K, Zvaritch E, Dickie P, Dziak E, Krause KH, Opas M, MacLennan DH, Michalak M (1999) Calreticulin is essential for cardiac development. J Cell Biol 144:857–868PubMedCrossRefGoogle Scholar
  11. Michalak M, Milner RE, Burns K, Opas M (1992) Calreticulin. Biochem J 285(Pt 3):681–692PubMedGoogle Scholar
  12. Michalak M, Corbett EF, Mesaeli N, Nakamura K, Opas M (1999) Calreticulin: one protein, one gene, many functions. Biochem J 344(Pt2):281–292PubMedCrossRefGoogle Scholar
  13. Michalak M, Groenendyk J, Szabo E, Gold LI, Opas M (2009) Calreticulin, a multi-process calcium-buffering chaperone of the endoplasmic reticulum. Biochem J 417:651–666PubMedCrossRefGoogle Scholar
  14. Nakamura K, Zuppini A, Arnaudeau S, Lynch J, Ahsan I, Krause R, Papp S, De Smedt H, Parys JB, Muller-Esterl W, Lew DP, Krause KH, Demaurex N, Opas M, Michalak M (2001) Functional specialization of calreticulin domains. J Cell Biol 154:961–972PubMedCrossRefGoogle Scholar
  15. Nomura R, Aoki T, Hagiwara H, Senda T, Fujimoto T (2005) Anti-calreticulin antibody binds to a membrane protein in caveolae. Acta Histocem Cytochem 38:43–52CrossRefGoogle Scholar
  16. Persson S, Rosenquist M, Sommarin M (2002) Identification of a novel calreticulin isoform (Crt2) in human and mouse. Gene 297:151–158PubMedCrossRefGoogle Scholar
  17. Terasaki M, Song J, Wong JR, Weiss MJ, Chen LB (1984) Localization of endoplasmic reticulum in living and glutaraldehyde-fixed cells with fluorescent dyes. Cell 38:101–108PubMedCrossRefGoogle Scholar
  18. Tokuyasu KT (1986) Application of cryoultramicrotomy to immunocytochemistry. J Microsc 143:139–149PubMedGoogle Scholar
  19. Wada I, Rindress D, Cameron PH, Ou WJ, Doherty JJ 2nd, Louvard D, Bell AW, Dignard D, Thomas DY, Bergeron JJ (1991) SSR alpha and associated calnexin are major calcium binding proteins of the endoplasmic reticulum membrane. J Biol Chem 266:19599–19610PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  1. 1.Department of Anatomy IFujita Health University School of MedicineToyoakeJapan
  2. 2.Department of Legal MedicineFujita Health University School of MedicineToyoakeJapan

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