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Plant Physiol, November 2000, Vol. 124, pp. 1069-1078
Purification, Properties, and Molecular Cloning of a Novel
Ca2+-Binding Protein in Radish
Vacuoles1
Koji
Yuasa and
Masayoshi
Maeshima*
Laboratory of Biochemistry, Graduate School of Bioagricultural
Sciences, Nagoya University, Nagoya 464-8601, Japan
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 as calreticulin. 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.
1
This work was supported in part by Grants-in-Aid
for Scientific Research (nos. 11163212 and 10219203 to M.M.) from the
Ministry of Education, Science, Sports and Culture of Japan.
*
Corresponding author; e-mail maeshima{at}agr.nagoya-u.ac.jp; fax
81-52-789-4094.
© 2000 American Society of Plant Physiologists
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