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

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.