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.
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Acknowledgments
We are grateful to Drs. Akiko Iizuka-Kogo (Fujita-Health University), Akikazu Fujita, and Toyoshi Fujimoto (Nagoya University) for helpful discussions and technical advice. We also wish to thank Kazuhiro Yanagisawa (Fujita-Health University) for excellent technical assistance. This work was supported by grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology of the Japanese Government to R.N.
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Nomura, R., Orii, M. & Senda, T. Calreticulin-2 is localized in the lumen of the endoplasmic reticulum but is not a Ca2+-binding protein. Histochem Cell Biol 135, 531–538 (2011). https://doi.org/10.1007/s00418-011-0817-z
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DOI: https://doi.org/10.1007/s00418-011-0817-z