Original Paper

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

  • Ryuji NomuraAffiliated withDepartment of Anatomy I, Fujita Health University School of Medicine Email author 
  • , Minami OriiAffiliated withDepartment of Anatomy I, Fujita Health University School of MedicineDepartment of Legal Medicine, Fujita Health University School of Medicine
  • , Takao SendaAffiliated withDepartment of Anatomy I, Fujita Health University School of Medicine

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Abstract

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.

Keywords

Calreticulin Endoplasmic reticulum Immunofluorescence Immunoelectron microscopy Stains-all