Abstract
A cDNA clone encoding 55-kDa multifunctional, thyroid hormone binding protein of rabbit skeletal muscle sarcoplasmic reticulum was isolated and sequenced. The cDNA encoded a protein of 509 amino acids, and a comparison of the deduced amino acid sequence with the NH2-terminal amino acid sequence of the purified protein indicates that an 18-residue NH2-terminal signal sequence was removed during synthesis. The deduced amino acid sequence of the rabbit muscle clone suggested that this protein is related to human liver thyroid hormone binding protein, rat liver protein disulfide isomerase, human hepatoma beta-subunit of prolyl 4-hydroxylase and hen oviduct glycosylation site binding protein. The protein contains two repeated sequences Trp-Cys-Gly-His-Cys-Lys proposed to be in the active sites of protein disulfide isomerase. Northern blot analysis showed that the mRNA encoding rabbit skeletal muscle form of the protein is present in liver, kidney, brain, fast- and slow-twitch skeletal muscle, and in the myocardium. In all tissues the cDNA reacts with mRNA of 2.7 kilobases in length. The 55-kDa multifunctional thyroid hormone binding protein was identified in isolated sarcoplasmic reticulum vesicles using a monoclonal antibody specific to the 55-kDa thyroid hormone binding protein from rat liver endoplasmic reticulum. The mature protein of Mr 56,681 contains 95 acidic and 61 basic amino acids. The COOH-terminal amino acid sequence of the protein is highly enriched in acidic residues with 17 of the last 29 amino acids being negatively charged. Analysis of hydropathy of the mature protein suggests that there are no potential transmembrane segments. The COOH-terminal sequence of the protein, Arg-Asp-Glu-Leu (RDEL), is similar to but different from that proposed to be an endoplasmic reticulum retention signal; Lys-Asp-Glu-Leu (KDEL) (Munro, S., and Pelham, H.R.B. (1987) Cell 48, 899-907). This variant of the retention signal may function in a similar manner to the KDEL sequence, to localize the protein to the sarcoplasmic or endoplasmic reticulum. The positively charged amino acids Lys and Arg may thus interchange in this retention signal.
Highlights
Molecular Cloning of cDNA Encoding a 55-kDa Multifunctional Thyroid Hormone Binding Protein of Skeletal Muscle Sarcoplasmic Reticulum*
The deduced amino acid sequence of the rabbit muscle clone suggested that this protein is related to human liver thyroid hormone binding protein, rat liver protein disulfide isomerase, human hepatoma
It has recently been shown that a single 55kDa polypeptide may be a multifunctional protein with the activities of protein disulfide isomerase (PDI) (XI), glycosylation site binding protein of oligosaccharyl transferase
Summary
T,BP, thyroid hormone binding protein; PDI, protein disulfide isomerase; Tar 3,5,3’-triiodo-L-thyronine; SDS, sodium dodecyl sulfate; bp, base pairs; kb, kilobases. It has recently been shown that a single 55kDa polypeptide may be a multifunctional protein with the activities of protein disulfide isomerase (PDI) (XI), glycosylation site binding protein of oligosaccharyl transferase [16], the P-subunit of prolyl. All of these proteins have shown homology in their amino acid sequences and predicted protein structures [16,20,21,22,23,24] They are localized to the lumen of the endoplasmic reticulum [16, 19,20,21, 25, 26]. We report the deduced amino acid sequence of the clone and show that it is homologous to PDI, the p subunit of prolyl 4-hydroxylase, glycosylation site binding protein and T7BP. For the sake of clarity, throughout this paper, we refer to this protein as the 55-kDa multifunctional TZIBP
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