cDNA cloning and molecular characterization of a glycoprotein with Ca^<2+> -binding EF-hand motifs.

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A novel Ca^<2+>-binding protein, designated reticulocalbin, has been identified and characterized. Reticulocalbin is a luminal protein of the endoplasmic reticulum with an Mr of 44,000 as revealed by biochemical analysis such as cell fractionation and immunoflurescence staining.The cDNA of reticulocalbin encodes a protein of 325 amino acids with an amino-terminal signal sequence of 20 amino acids. The protein has six repeats of a domain containing the high affinity Ca^<2+> -binding motif, the EF-hand. Although oxygencontaining amino acids important for the positioning of Ca^<2+> are conserved in all six domains, the conserved glycine reidues in the central portion of the EF-hand motif are absent in three (in sites II, III, and VI) of them. Calcium blots showed that recombinant reticulocalbin expressed in bacterial cells and purified from them binds Ca^<2+>.The protein has the sequence His-Asp-Glu-Leu (HDEL) at its carboxyl terminus. This sequence is similar to the Lys-Asp-Glu-Leu(KDEL) sequence, which serves as a signal to retain the resident proteins in the endoplasmic reticulum of animal cells. A mutant protein lacking the HDEL sequence produced by in vitro mutagenesis has been shown to be secreted into the medium in transient expression assays.