|Budget Amount *help
¥5,400,000 (Direct Cost : ¥5,400,000)
Fiscal Year 1994 : ¥2,100,000 (Direct Cost : ¥2,100,000)
Fiscal Year 1993 : ¥3,300,000 (Direct Cost : ¥3,300,000)
Reticulocalbin, an endoplasmic reticulum (ER) resident Ca^<2+>-binding protein, is a member of the EF-hand Ca^<2+>-binding protein superfamily [Ozawa, M.and Muramatsu, T.(1993) J.Biol.Chem.268,699-705]. The genomic structure of this unique Ca^<2+>-binding protein was analyzed. Southern blot analysis of mouse genomic DNA revealed that there is a single copy of the reticulocalbin gene per hapliod genome. The gene spans over 13 kilobase pairs and encodes six separate exons. Thus, reticulocalbin differs from the cytosolic Ca^<2+>-binding protein calbindin D28 which also has six EF-hand motif domains but the gene for which is divided into 11 exons. Comparison of the gene organization of reticulocalbin with that of other EF-hand proteins revealed that reticulocalbin diverged very early from other members of the EF-hand protein superfamily. Consistent with the observation that reticulocalbin is expressed by different types of cells, the 5'-flanking region of the gene contains a GC-rich (more
than 80%) area including two Spl binding motifs and a CCAAT box, but no apparent TATA box was present between the CCAAT box and the transcription start site, a feature being found in many housekeeping genes.
We have cloned the human homologue of reticulocalbin. The sequence of this clone revealed a novel protein with 95 percent identity in ammino acid sequence to the mouse reticulocalbin, indicating that this molecule has been evolutionarily conserved in mammals. As was found for the mouse reticulocalbin, the human homologue showed the six repeats of a domain containing EF-hand motifs. Interestingly, the conservation of amino acid sequence was not restricted to the Ca^<2+>-binding motifs, consistent with the possibility that reticulocalbin plays some role (s) besides Ca^<2+>-binding. As was found for the mouse homologue, the protein has the HDEL sequence at its carboxy terminus instead of the KDEL sequence, which is more common as a signal to retain resident proteins in the ER of animal cells. The conservation of the HDEL sequence in reticulocalbin in both species raises the possibility that this sequence has some roles in the function (s) of this protein family. Less