Purification, properties, and molecular cloning of a novel Ca(2+)-binding protein in radish vacuoles.
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 Ca(2+)-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 Ca(2+)-binding activity was confirmed by the (45)Ca(2+)-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 Ca(2+) using two active Ca(2+) uptake systems, namely Ca(2+)-ATPase and Ca(2+)/H(+) antiporter. Vacuolar membrane vesicles containing RVCaB accumulated more Ca(2+) than sonicated vesicles depleted of the protein at a wide range of Ca(2+) 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 Ca(2+)-binding proteins such 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 molecular mass of 44 kD. These results suggest that RVCaB is a new type Ca(2+)-binding protein with high capacity and low affinity for Ca(2+) and that the protein could function as a Ca(2+)-buffer and/or Ca(2+)-sequestering protein in the vacuole.
Yuasa
K
K
Laboratory of Biochemistry, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
Maeshima
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D019607:Brassicaceae / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D018517:Plant Roots / Q000166:cytology / Q000378:metabolism; D020539:Sequence Analysis, Protein; D014617:Vacuoles / Q000737:chemistry / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D010940:Plant Proteins; C423442:RVCaB protein, Raphanus sativus; D002118:Calcium