Abstract
We have identified and characterized a cDNA encoding a novel Ca2+-binding protein named calumenin from mouse heart by the signal sequence trap method. The deduced amino acid sequence (315 residues) of calumenin contains an amino-terminal signal sequence and six Ca2+-binding (EF-hand) motifs and shows homology with reticulocalbin, Erc-55, and Cab45. These proteins seem to form a new subset of the EF-hand protein family expressed in the lumen of the endoplasmic reticulum (ER) and Golgi apparatus. Purified calumenin had Ca2+-binding ability. The carboxyl-terminal tetrapeptide His-Asp-Glu-Phe was shown to be responsible for retention of calumenin in ER by the retention assay, immunostaining with a confocal laser microscope, and the deglycosylation assay. This is the first report indicating that the Phe residue is included in the ER retention signal. Calumenin is expressed most strongly in heart of adult and 18.5-day embryos. The calumenin gene (Calu) was mapped at the proximal portion of mouse chromosome 7.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) U81829
Cloning and Sequencing of a Novel cDNA C39 —4.4 3 105 yeast transformants from 9.5 dpc embryonic heart cDNA library were screened, and 386 positive clones were obtained by the signal sequence trap method described previously [30, 31]
Calumenin is translocated and glycosylated in the endoplasmic reticulum (ER) and not modified in the Golgi apparatus, because Endo H completely cleaved oligosaccharides on FLAG-calumenin and FLAG-calumenin-rHDEL. These results indicate that calumenin resides in the ER and that the C-terminal tetrapeptide HDEF works as the ER retention signal like HDEL [16, 17]
Summary
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) U81829 Cardiac myocytes develop their specialized ER, called the sarcoplasmic reticulum, as the Ca21 storage compartment and produce the rhythmical Ca21 oscillation between the sarcoplasmic reticulum and the cytosol [27] Molecules involved in this Ca21 oscillation are reported to be present very early in mouse cardiogenesis [28, 29]. We here report cloning and characterization of cDNA encoding calumenin that binds Ca21 and carries a new ER retention signal, HDEF, at the carboxyl terminus. Calumenin is most strongly expressed in the heart of adult and 18.5-day embryos
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