Abstract
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.
Highlights
A cDNA clone encoding the high affinity Ca2+-bind- The sarcoplasmic reticulum is an intracellular membrane ing protein (HACBP) of rabbit skeletal muscle sarcoplasmicreticulum was isolated and sequenced
During differentiation of myoblasts be subdivided into a charged region which contains a to myotubes, the synthesis of calsequestrin and the high affinity Ca2+-binding protein (HACBP)
We show that theCOOH-terminal sequence from various rabbit tissues [38]and 10-pg samplesof each RNAwere of the protein terminates with the residues Lys-Asp-Glu-LeuCOOH (KDEL-COOH), which are believed to be an important signal in the retention of peripheral endoplasmic reticulum membrane proteins [30,31].Comparison of our sequence with that of CRP 5Ei3 established the identity of the separated by electrophoresis in a denaturing formaldehyde, 0.8% agarose gel [39], stained with ethidium bromide, and blotted onto nitrocellulose or nylon membranes [40]
Summary
A cDNA clone encoding the high affinity Ca2+-bind- The sarcoplasmic reticulum is an intracellular membrane ing protein (HACBP) of rabbit skeletal muscle sarcoplasmicreticulum was isolated and sequenced. (Department of Pharmacology, University of Milano) has This clone contained nucleotides -5 to 1754, encoding all of shown that rat andrabbit liver and brain microsomes contain the mature protein and five-sixths of 2amino acids in the signal a soluble, luminal protein of identical size to HACBP that binds large quantities of Ca2+with low affinity.2 Since calsequestrin is absent from these endoplasmic reticulum preparations, it is conceivable that the HACBP may, represent the major Ca2+sequestering protein inthe endoplasmic sequence. Bp encoding the complete amino acid sequence of HACBP, including
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