Identification of the Ca2+-Binding Domains in Reticulocalbin, an Endoplasmic Reticulum Resident Ca2+-Binding Protein with Multiple EF-Hand Motifs

1 January 1997

journal article
research article
Published by Oxford University Press (OUP) in The Journal of Biochemistry

Vol. 121 (1) , 145-149
https://doi.org/10.1093/oxfordjournals.jbchem.a021557

Abstract

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. ⁴⁵Ca²⁺ 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.

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