Purification, properties, and molecular cloning of a novel Ca2+-binding protein in radish vacuoles

To understand the roles of plant vacuoles, we have purified and characteriz ed a major soluble protein from vacuoles of radish (Raphanus sativus cv Tok inashi-daikon) taproots. The results showed that it is a novel radish vacuo le Ca2+-binding protein (RVCaB). RVCaB was released from the vacuolar membr ane fraction by sonication, and purified by ion exchange and gel filtration column chromatography. RVCaB is an acidic protein and migrated on sodium d odecyl sulfate-polyacrylamide gel with an apparent molecular mass of 43 kD. The Ca2+-binding activity was confirmed by the Ca-45(2+)-overlay assay. RV CaB was localized in the lumen, as the protein was recovered in intact vacu oles prepared from protoplasts and was resistant to trypsin digestion. Plan t vacuoles store Ca2+ using two active Ca2+ uptake systems, namely Ca2+-ATP ase and Ca2+/H+ antiporter. Vacuolar membrane vesicles containing RVCaB acc umulated more Ca2+ than sonicated vesicles depleted of the protein at a wid e range of Ca2+ concentrations. A cDNA (RVCaB) encoding a 248-amino acid po lypeptide was cloned. Its deduced sequence was identical to amino acid sequ ences obtained from several peptide fragments of the purified RVCaB. The de duced sequence is not homologous to that of other Ca2+-binding proteins suc h as calreticulin. RVCaB has a repetitive unique acidic motif, but not the EF-hand motif. The recombinant RVCaB expressed in Escherichia coli-bound Ca 2+ as evidenced by staining with Stains-all and migrated with an apparent m olecular 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.