RETICULOCALBIN, A NOVEL ENDOPLASMIC-RETICULUM RESIDENT CA2+-BINDING PROTEIN WITH MULTIPLE EF-HAND MOTIFS AND A CARBOXYL-TERMINAL HDEL SEQUENCE

A novel Ca2+-binding protein, tentatively designated reticulocalbin, has been identified and characterized. Reticulocalbin is a luminal protein of the endoplasmic reticulum with an M(r) of 44,000 as revealed by biochemical analysis and immunofluorescence 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 Ca2+-binding motif, the EF-hand. Although oxygen-containing amino acids important for the positioning of Ca2+ are conserved in all six domains, the conserved glycine residues in the central portion of the EF-hand motif are absent in three of them. Calcium blots showed that recombinant reticulocalbin expressed in bacterial cells binds Ca2+. The protein has the sequence His-Asp-Glu-Leu (HDEL) at its carboxyl terminus. This is similar to the Lys-Asp-Glu-Leu 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 medium in transient expression assays.