Purification, properties, and molecular cloning of a novel Ca2+ -binding protein in radish vacuoles
2000
Yuasa, K. | Maeshima, M.
To understand the roles of plant vacuoles, we have purified and characterized a major soluble protein from vacuoles of radish (Raphanus sativus cv Tokinashi-daikon) taproots. The results showed that it is a novel radish vacuole Ca2+ -binding protein (RVCaB). RVCaB was released from the vacuolar membrane fraction by sonication, and purified by ion exchange and gel filtration column chromatography. RVCaB is an acidic protein and migrated on sodium dodecyl sulfate-polyacrylamide gel with an apparent molecular mass of 43 kD. The Ca2+ -binding activity was confirmed by the 45Ca2+ -overlay assay. RVCaB was localized in the lumen, as the protein was recovered in intact vacuoles prepared from protoplasts and was resistant to trypsin digestion. Plant vacuoles store Ca2+ using two active Ca2+ uptake systems, namely Ca2+ -ATPase and Ca2+/H+ antiporter. Vacuolar membrane vesicles containing RVCaB accumulated more Ca2+ than sonicated vesicles depleted of the protein at a wide range of Ca2+ concentrations. A cDNA (RVCaB) encoding a 248-amino acid polypeptide was cloned. Its deduced sequence was identical to amino acid sequences obtained from several peptide fragments of the purified RVCaB. The deduced sequence is not homologous to that of other Ca2+ -binding proteins such ascalreticulin. RVCaB has a repetitive unique acidic motif, but not the EF-hand motif. The recombinant RVCaB expressed in Escherichia coli-bound Ca2+ as evidenced by staining with Stains-all and migrated with an apparent molecular mass of 44 kD. These results suggest that RVCaB is a new type Ca2+ -binding protein with high capacity and low affinity for Ca2+ and that the protein could function as a Ca2+ -buffer and/or Ca2+ -sequestering protein in the vacuole.
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