Molecular cloning of the high affinity calcium-binding protein (calreticulin) of skeletal muscle sarcoplasmic reticulum.
A cDNA clone encoding the high affinity Ca2+-binding protein (HACBP) of rabbit skeletal muscle sarcoplasmic reticulum was isolated and sequenced. The cDNA encoded a protein of 418 amino acids, but a comparison of the deduced amino acid sequence with the NH2-terminal amino acid sequence of the purified protein indicates that a 17-residue NH2-terminal signal sequence was removed during synthesis. This was confirmed by studies of in vitro translation of mRNA encoding the protein. Structural predictions did not reveal any potential transmembrane segments in the protein. The COOH-terminal sequence of the high affinity Ca2+-binding protein, Lys-Asp-Glu-Leu, is the same as that proposed to be an endoplasmic reticulum retention signal (Munro, S., and Pelham, H. R. B. (1987) Cell 48, 899-907). All of these characteristics suggest that the protein is localized in the lumen of the sarcoplasmic reticulum. The mature protein of Mr 46,567 contains 109 acidic and 52 basic amino acids. Structural predictions suggest that the first half of the molecule forms a globular domain of 8 anti-parallel beta-strands with a helix-turn-helix motif at the extreme NH2 terminus. The next one-third of the sequence is proline-rich. This segment can be subdivided into a charged region which contains a 17-amino acid repeat, followed by a proline, serine, and threonine-rich segment extending from Pro-246 to Thr-316. Thirty-seven acidic residues are clustered within 56 amino acids at the COOH terminus of the protein. Although the protein binds 1 mol of Ca2+/mol with high affinity, no "EF-hand" consensus sequence was observed in the protein. The acidic COOH terminus, however, could account for the low affinity, high capacity Ca2+ binding observed in the protein. In agreement with other involved laboratories, we have chosen the name calreticulin for the protein.
Fliegel
L
L
Department of Pediatrics, University of Alberta, Edmonton, Canada.
Burns
K
K
MacLennan
D H
DH
Reithmeier
R A
RA
Michalak
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002135:Calcium-Binding Proteins / Q000235:genetics*; D037282:Calreticulin; D002474:Cell-Free System; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D008969:Molecular Sequence Data; D009132:Muscles / Q000378:metabolism*; D009928:Organ Specificity; D014176:Protein Biosynthesis; D011487:Protein Conformation; D012333:RNA, Messenger / Q000235:genetics; D011817:Rabbits; D015183:Restriction Mapping; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D037282:Calreticulin; D012333:RNA, Messenger; D004247:DNA