Identification of the Ca2+-binding domains in reticulocalbin, an endoplasmic reticulum resident Ca2+-binding protein with multiple EF-hand motifs

Reticulocalbin (RCN) is a member of the EF-hand Ca2+-binding protein family and is a luminal protein of the endoplasmic reticulum (ER) with a molecular weight of 44,000 [Ozawa, M. and Muramatsu, T. (1993) J. Biol. Chem. 268, 699-705]. Although RCN has six repeats of a domain containing an EF-hand motif, the varying degrees of divergence of the amino acid sequences of these domains from the EF-hand consensus sequences suggested that some domains might have lost their Ca2+-binding capability and adopted new functions, To identify the domains involved in Ca2+-binding, discrete domains of RCN were expressed in Escherichia coli, using the glutathione S-transferase fusion protein system, Ca-45(2+) blot analysis of the resultant fusion proteins revealed that the first, fourth, fifth, and sixth domains bind Ca2+, however, the second and third ones do not. The fusion proteins containing all six domains, and the first and second domains, respectively, showed Ca2+-dependent increases in their electrophoretic mobilities, suggesting that Ca2+ induces a conformational change in reticulocalbin.