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First published online July 15, 2005; 10.1104/pp.105.064352 Plant Physiology 138:1939-1946 (2005) © 2005 American Society of Plant Biologists Characterization of Vacuolar Transport of the Endogenous Alkaloid Berberine in Coptis japonica1Division of Integrated Life Science, Graduate School of Biostudies (M.O., F.S.), and Division of Applied Life Sciences, Graduate School of Agriculture (K.S., F.S.), Kyoto University, Kyoto 606–8502, Japan; Laboratory of Plant Gene Expression, Research Institute for Sustainable Humanosphere, Kyoto University, Gokasho, Uji 611–0011, Japan (N.S., K.Y.); and Zurich Basel Plant Science Center, University of Zurich, Plant Biology, CH–8008 Zurich, Switzerland (E.M.)
Alkaloids comprise one of the largest groups of plant secondary metabolites. Many of them exhibit strong biological activities, and, in most cases, they are accumulated in the central vacuole of alkaloid-producing plants after synthesis. However, the mechanisms involved in alkaloid transport across the tonoplast are only poorly understood. In this study, we analyzed the vacuolar transport mechanism of an isoquinoline alkaloid, berberine, which is produced and accumulated in the vacuole of cultured cells of Coptis japonica. The characterization of berberine transport using intact vacuoles and a tonoplast vesicle system showed that berberine uptake was stimulated by Mg/ATP, as well as GTP, CTP, UTP, and Mg/pyrophosphate. Berberine uptake was strongly inhibited by NH4+ and bafilomycin A1, while vanadate, which is commonly used to inhibit ATP-binding cassette transporters, had only a slight effect, which suggests the presence of a typical secondary transport mechanism. This is contrary to the situation in the plasma membrane of this plant cell, where the ATP-binding cassette transporter is involved in berberine transport. Model experiments with liposomes demonstrated that an ion-trap mechanism was hardly implicated in berberine transport. Further studies suggested that berberine was transported across the tonoplast via an H+/berberine antiporter, which has a Km value of 43.7 µM for berberine. Competition experiments using various berberine analogs, as well as other classes of alkaloids, revealed that this transporter is fairly specific, but not exclusive, for berberine.
1 This work was supported in part by a grant from the Research for the Future program "Molecular mechanisms on regulation of morphogenesis and metabolism leading to increased plant productivity" (no. 00L01605 to K.Y.), by a Grant-in-Aid for Scientific Research (no. 15031217 and no. 17051018 to K.Y.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by an additional grant from the Uehara Memorial Foundation. 2 These authors contributed equally to the paper. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.064352. * Corresponding author; e-mail yazaki{at}rish.kyoto-u.ac.jp; fax 81–774–38–3623. Received April 19, 2005; returned for revision May 19, 2005; accepted May 19, 2005. This article has been cited by other articles:
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