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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

Characterization of Vacuolar Transport of the Endogenous Alkaloid Berberine in Coptis japonica¹

Division 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.)

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
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 K_m 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.

On the other hand, alkaloid-producing plant cells seem to be insensitive to their own metabolites, probably because they have a detoxification mechanism to prevent the cytotoxicity of alkaloids. However, such plant detoxification of secondary metabolites is not well understood. One possible explanation is the compartmentation of alkaloids into the plant vacuole. Many alkaloids are presumed to be synthesized in the cytosol and on the endoplasmic reticulum (ER) and then transported through the tonoplast to be sequestrated in the vacuolar matrix. A simple model for alkaloid accumulation in plant vacuoles is the ion-trap mechanism (Matile, 1976). According to this model, alkaloids, which are in a lipophilic state in the neutral pH of cytosol, can freely pass through the tonoplast by simple diffusion. However, under the acidic conditions inside the vacuole, they are protonated to form hydrophilic cations, and therefore become unable to permeate through the membrane and are trapped inside. Contrary to this hypothesis, several studies have demonstrated that the vacuolar transport of alkaloids was managed by specific carriers in an energy-requiring manner, which may involve a proton-antiport carrier system (Deus-Neumann and Zenk, 1984, 1986; Mende and Wink, 1987; Wink and Mende, 1987). A third possible mechanism was recently proposed; i.e. a directly energized transporter, the ATP-binding cassette (ABC) transporter, is present on the tonoplast and might be responsible for the transport of secondary metabolites (Klein et al., 2000). However, there have been few detailed biochemical analyses of the vacuolar membrane transport of endogenous alkaloids.

To study the mechanism of alkaloid transport in plant vacuoles, we have been using a yellow isoquinoline alkaloid, berberine, which was the first alkaloid to have its biosynthesis fully described at the enzyme level (Zenk, 1995). Berberine is stable and widely used as an antibacterial and antimalaria drug in many countries (Yamamoto et al., 1993; Iwasa et al., 1998). While it is one of the most widely occurring alkaloids in many plant families, we used cell cultures of Coptis japonica (Ranunculaceae) as a model system. C. japonica is a perennial medicinal plant grown in Asian countries, and berberine is highly accumulated in its rhizome as its main alkaloid. Cultured C. japonica cells produce berberine and accumulate it exclusively in the vacuole (Sato et al., 1990, 1994). Moreover, exogenous berberine added to the culture medium is actively taken up by C. japonica cells (Sato et al., 1990, 1994), and is also transported into the vacuole and stably accumulated in the lumen (Sato et al., 1993; Sakai et al., 2002). Our previous studies to identify the berberine transporter in C. japonica cells showed that CjMDR1, a multidrug-resistance protein-type ABC transporter, was involved in the transport of berberine at the plasma membrane (Shitan et al., 2003). However, it is still unclear how berberine is transported across the tonoplast.

In this study, we analyzed the vacuolar transport mechanism of berberine by using intact vacuoles and tonoplast vesicles of C. japonica cells. Berberine transport into tonoplast vesicles was stimulated by Mg/ATP and was inhibited by NH₄⁺ and bafilomycin A1, but not by vanadate, which suggested that uptake involved an H⁺/berberine-antiporter.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Uptake of [³H]Berberine by Isolated Vacuoles

To clarify the mechanism of the vacuolar uptake of berberine, we first isolated intact vacuoles from 2-week-old C. japonica suspension cultures. Vacuoles of C. japonica cells appear to be bright yellow because of the high concentration of endogenous berberine, which reaches more than 72 mM. Their diameter was 11 to 28 µm and the average volume was calculated to be 4.9 pL. The vacuolar pH of C. japonica cells was approximately 5.5. These isolated vacuoles were incubated with radiolabeled berberine in an assay buffer of pH 7.8, which mimicked the endogenous state of vacuoles in terms of the pH across the tonoplast. Incubated vacuoles were passed through a silicon oil layer by centrifugal filtration and [³H]berberine in the vacuoles recovered from the bottom layer was estimated by scintillation counting. Burst vacuoles, which were prepared by sonication in the presence of Triton, were used as a negative control. As a result, the clear uptake of berberine by intact vacuoles was observed in the absence of Mg/ATP, and berberine uptake was somewhat stimulated by Mg/ATP, but not by Mg/ADP and Mg/ATP--S, a nonhydrolyzable analog of ATP (Fig. 1). This suggests that berberine is apparently taken up by intact vacuoles, probably due to the preexisting pH in the assay condition (incubation buffer, pH 7.8), and that ATP enhances berberine transport into the vacuole, whereas the effect is seemingly indirect. However, further detailed analyses of berberine uptake using various inhibitors were hampered because of the instability of the vacuoles, i.e. it was very difficult to perform the assays using solvents such as dimethyl sulfoxide (DMSO). For this reason, we decided to further analyze berberine transport using tonoplast vesicles.

View larger version (67K): [in this window] [in a new window]   Figure 1. Effects of Mg/ATP and different compounds on the uptake of berberine into isolated C. japonica vacuoles. A, Isolated protoplasts of C. japonica cells. B, Isolated vacuoles of C. japonica cells. Bars = 50 µm. C, Vacuoles were incubated in the presence of 1 µM [3H]berberine and 5 mM of the compounds listed. The pH of the incubation medium was adjusted to 7.8 and berberine uptake was measured after 20 min. Double bars indicate the data of two independent experiments. In the negative control, burst vacuoles were used for berberine uptake.

We purified tonoplast vesicles by fractionating microsomes of C. japonica cells on a discontinuous Suc density gradient. Vanadate-sensitive ATPase and KNO₃-sensitive ATPase activities were measured as marker enzymes for plasma membrane and tonoplasts, respectively. Tonoplast-rich membrane vesicles were recovered at 0/20% Suc fraction (Fig. 2A). The enrichment of the tonoplast in this fraction was also confirmed by immunodetection of vacuolar pyrophosphatase (V-PPase), a tonoplast marker, whereas plasma membrane H⁺-ATPase and luminal binding protein (BiP), an ER marker, sedimented at positions different from V-PPase (Fig. 2B). The tonoplast vesicle fraction showed clear [³H]berberine uptake, while other fractions, which contain a large portion of plasma membrane and ER, did not show the uptake activity even in the presence of ATP (Fig. 2C). These results suggested that the tonoplast vesicles had the berberine transport activity, which was not observed in plasma or ER membranes. Using the tonoplast vesicles, we measured the time course of [³H]berberine uptake (Fig. 2D). Rapid uptake was observed in the presence of Mg/ATP, whereas no uptake was seen in the absence of Mg/ATP, which indicates that Mg/ATP is required for berberine uptake in this assay system. This berberine transport activity was reproducibly observed among each preparation of the tonoplast vesicles, i.e. the uptake within the range between 12 and 18 pmol/µg protein was observed for 5 min in the presence of Mg/ATP.

View larger version (13K): [in this window] [in a new window]   Figure 2. Time-dependent uptake of berberine into tonoplast vesicles. A, Tonoplast vesicles were prepared by Suc gradient fractionation. Vanadate-sensitive ATPase () and KNO3-sensitive ATPase () activities were measured as marker enzymes for plasma membrane and tonoplast content, respectively. B, Plasma membrane H+-ATPase, V-PPase, and BiP were immunodetected to confirm the purity of tonoplast vesicles. C, Each membrane fraction was incubated with 50 µM of [3H]berberine in the presence () or absence () of 5 mM Mg/ATP. D, Tonoplast vesicles (0/20% Suc fraction) were incubated with 50 µM of [3H]berberine in the presence () or absence () of 5 mM Mg/ATP. Berberine uptake was monitored at 25°C, as described in "Materials and Methods." Data are an average of three replicates and are presented as pmol/µg protein.

View larger version (17K): [in this window] [in a new window]   Figure 3. Effect of pH on ATP-dependent berberine uptake. Reaction mixtures were as described in "Materials and Methods." The pH was adjusted with HCl. Berberine uptake was determined after 5 min of incubation. Results are mean ± SD of three replicates.

View larger version (15K): [in this window] [in a new window]   Figure 4. Effects of Mg/ATP, different nucleotides, and pyrophosphate on berberine uptake. Tonoplast vesicles were incubated in the presence 50 µM [3H]berberine and the compounds listed. Results are mean ± SD of three replicates. Asterisk indicates statistically significant difference compared to –ATP (P < 0.01).

View larger version (15K): [in this window] [in a new window]   Figure 5. Berberine uptake into liposomes in the presence and absence of a pH gradient. A, [14C]Methylamine uptake into liposomes. B, [3H]Berberine uptake into liposomes. A pH gradient was generated as described in "Materials and Methods." Liposomes were incubated at 25°C for 10 min with 20 µM [14C]methylamine (approximately 1 µCi/mL) or 50 µM [3H]berberine, and uptake was determined as described for the measurement of berberine transport. To destroy the pH gradient, NH4Cl (10 mM) was added to the incubation mixture. Results are mean ± SD of three replicates. Asterisk indicates statistically significant difference (*, P < 0.05; **, P < 0.01).

The uptake of berberine by tonoplast vesicles exhibited K_m-type saturation kinetics, as shown in Figure 6. The K_m and V_max values were calculated to be 43.7 µM and 13.5 pmol/µg protein per min, respectively.

View larger version (12K): [in this window] [in a new window]   Figure 6. Uptake of [3H]berberine into tonoplast vesicles shows Km-type saturation kinetics. A, Tonoplast vesicles were incubated in the presence of [3H]berberine. B, Representative saturation experiments are illustrated as a Hanes-Woolf plot. Km value measured in three independent experiments ranged between 10 and 50 µM.

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
Biosynthetic reactions of alkaloids occur in various organelles in plant cells, which may vary according to the type of alkaloid involved, e.g. quinolizidine alkaloids of legumes appear to be biosynthesized in mesophyll chloroplasts of green leaves (De Luca and St. Pierre, 2000). However, the final intracellular destination of most biosynthesized alkaloid molecules is vacuoles. In plants, four major uptake mechanisms have been proposed for secondary metabolite transportation across the tonoplast, i.e. H⁺ antiporter, ion trap, conformational trap (Rataboul et al., 1985), and directly energized transport by ABC transporters (Martinoia et al., 2000). The first three use the electrochemical gradient of H⁺ across the tonoplast, whereas ABC transporters directly use the energy of Mg/ATP hydrolysis to pump their substrates into the vacuolar lumen, i.e. independent of electrochemical force. This study demonstrated that an H⁺ electrochemical gradient was necessary for berberine transport across C. japonica vacuoles. The liposome experiment showed that the ion-trap mechanism was not applicable to berberine accumulation in this plant, but rather a certain carrier-mediated transport mechanism is involved. Based on these results, we conclude that berberine was transported across the tonoplast via an H⁺/berberine antiporter, which uses the proton gradient formed by the two vacuolar proton pumps, vacuolar H⁺-ATPase and PPase, localized at the tonoplast (Fig. 7).

View larger version (35K): [in this window] [in a new window]   Figure 7. Proposed model for berberine transport and accumulation. Exogenous berberine is taken up relatively slowly by CjMDR1, an ABC transporter, while the H+/berberine antiporter effluxes berberine very rapidly from the cytosol to the vacuole via a proton gradient formed by V-ATPase and V-PPase, resulting in a low berberine concentration in the cytosol.

It is not yet clear why C. japonica cells have two different types of transporters for berberine; i.e. ABC transporter (CjMDR1) at the plasma membrane (Shitan et al., 2003) and H⁺/berberine antiporter at the tonoplast, but the difference in their V_max might offer one possible explanation. Compared to the antiporter at the tonoplast, CjMDR1 appears to pump berberine relatively slowly (the V_max of CjMDR1 for berberine is 0.08 nmol/mg protein) to the cytosol from the apoplast (Shitan et al., 2003), whereas the transport across the tonoplast by the H⁺/berberine antiporter seems to be more rapid (the V_max of the antiporter is 13.5 nmol/mg protein). If this difference in transport efficiency is valid for C. japonica cells in vivo, the rapid transport by the antiporter would largely contribute to the detoxification of this cytotoxic alkaloid; i.e. the cytosolic concentration of berberine in C. japonica cells can be kept at a low level by the scheme shown in Figure 7. Therefore C. japonica cells are capable of safely accumulating berberine in vacuoles even if they are cultured in the presence of 750 µM berberine in the medium, which is highly toxic to tobacco (Nicotiana tabacum) and other plant cells, but not to C. japonica cells (Sakai et al., 2002).

In bacteria, several antiporters that accept berberine as their substrate have been reported, although Na⁺ is the counterpart instead of H⁺, and their roles as drug efflux pumps for the purpose of detoxification have been demonstrated. For example, NorM from Vibrio parahaemolyticus and its homolog in Escherichia coli, YdhE, which are both multidrug and toxic compound extrusion (MATE)-type transporters, efflux several toxic compounds, including berberine, and confer multidrug resistance to these microorganisms (Xu et al., 2003). In plants, a plasma membrane-localized MATE-type transporter of Arabidopsis (Arabidopsis thaliana), AtDTX1, which also recognizes berberine as an exogenous substrate (Li et al., 2002), has been reported so far as an H⁺ antiporter that acts as a detoxifier. Sequence data of these known antiporters can be used to clone the H⁺/berberine antiporter of C. japonica, although the MATE family in Coptis may be different from these known MATE members since the former may only be responsible for the endogenous alkaloid, not other exogenous compounds.

Plant alkaloids are often translocated from the source organ to a sink organ (Hashimoto and Yamada, 1994; Hartmann, 1999). Berberine in C. japonica is also translocated from root tissues to rhizome, which involves transport across several membranes, i.e. at the plasma membrane in root and rhizome and at the tonoplast in cortex cells of rhizome. Together with our previous work on CjMDR1 at the plasma membrane, this study on the tonoplast should help to clarify the entire molecular mechanism of the translocation of alkaloids in plants.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Chemicals

Chemicals used in this study were purchased from Wako Pure Chemicals (Osaka) or Nakalai Tesque (Kyoto).

Cultured Cells

High berberine-producing cultures of Coptis japonica, which were originally induced from the rootlets of C. japonica Makino var. dissecta (Yamabe), were maintained as described (Sato and Yamada, 1984). Two-week-old cells were used for the experiments.

Isolation of Vacuoles

Protoplasts were isolated from cultured cells by the procedure of Sato et al. (1990). Isolation of vacuoles from protoplasts was carried out by the procedure of Sato et al. (1992). Isolated purified vacuoles were used immediately for the uptake experiment.

Preparation of Tonoplast Vesicles

The tonoplast vesicles used in this study were prepared from C. japonica cells by the procedure of Rocha Facanha and de Meis (1998) with some modifications. Cells were collected and homogenized in 2 mL/g of ice-cold homogenizing buffer (10% [v/v] glycerol, 0.5% [w/v] polyvinylpolypyrrolidone, 5 mM EDTA, and 0.1 M Tris-HCl adjusted to pH 8.0 and autoclaved). Prior to use, 150 mM KCl, 3.3 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride were added to the buffer and the mixture was stirred for 1 min. The following modifications were also applied: Homogenate was strained through Miracloth (Merck, Rahway, NJ) instead of cheesecloth; 20%, 30%, 40% (w/v) discontinuous Suc gradient was used to fractionate the microsomes; and each fraction recovered from interfaces was resuspended with resuspension buffer (10% [v/v] glycerol, 1 mM EDTA, and 10 mM Tris-HCl adjusted to pH 7.6 and autoclaved) instead of water. Isolated membrane vesicles were stored at –80°C in resuspension buffer containing 10 µg/mL leupeptin, 2 µg/mL aprotinin, 2 µg/mL pepstatin, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride. Vanadate-sensitive ATPase and KNO₃-sensitive ATPase activities were measured as marker enzymes for plasma membrane and tonoplast contents, respectively. For the berberine uptake, the membrane vesicles obtained from 0/20% Suc fraction were used as the tonoplast vesicles. The purity of tonoplast vesicles was checked by immunodetection. The antibodies used for immunodetection were against plasma membrane H⁺-ATPase, vacuolar H⁺-pyrophosphatase from Arabidopsis (Arabidopsis thaliana), and ER luminal BiP (see "Acknowledgments" for the sources of antibodies).

Preparation of [³H]Berberine

[³H]Berberine (specific activity, 7.4 mCi/mol) was prepared by labeling berberine with NaB[³H]₄. First, 25 mg of cold NaBH₄ were added to 10 mCi of NaB[³H]₄ (Amersham Biosciences, Buckinghamshire, UK) for dilution, which was dissolved in 100 µL of 0.2% NaOH. To this solution was slowly added berberine hydrochloride (50 mg) dissolved in 10 mL MeOH, and the mixture was stirred for 8 h. Next, 100 mg I₂ were added to oxidize the tetrahydroberberine and the mixture was stirred for 1 h. Excess I₂ was decomposed with 120 mg NaHSO₃ by stirring for 30 min. The berberine iodide that precipitated in the solution was recovered by filtration, and berberine chloride was formed by treatment with 200 mg AgCl. The yielded berberine chloride was further purified by preparative thin-layer chromatography, using the solvent system by the procedure of Ikuta and Itokawa (1988). Berberine was recovered by elution with methanol, which was evaporated to dryness and dissolved in water for transport assay.

Measurement of Berberine Transport

Uptake of [³H]berberine by intact vacuoles was measured by the centrifuged filtration technique through silicone oil as described in the literature (Heldt and Sauer, 1971) with some modifications. Isolated vacuoles were incubated with 1 µM [³H]berberine at 25°C in vacuole resuspension buffer (0.7 M Glycinebetaine, 1 mM NaH₂PO₄, 2 mM HEPES-NaOH, 1 mg/mL bovine serum albumin, pH 7.8) for 20 min. As the negative control, burst vacuoles prepared by sonication in the presence of 2% Triton-X were used. Filtering centrifugation was carried out at room temperature using 400-µL plastic tubes. The tube contained (from the bottom) 50 µL bottom buffer (25% Percoll, 0.7 M sorbitol, 2% Triton-X), 150 µL silicone oil (SH550:SH556 = 77:23; Toray Dow Corning Co. Ltd, Tokyo), and 70 µL vacuoles in incubation buffer, and was centrifuged at 6,000g for 1 min. The contents of the plastic tube were then frozen in liquid N₂, the bottom layer was recovered by cutting off the tube with a knife, and radioactivity was estimated by a scintillation counter (Beckman Instruments, Fullerton, CA).

Uptake of [³H]berberine by tonoplast vesicles was measured at 25°C for 5 min in 200 µL of reaction mixture containing 50 mM Tris-MES buffer (pH 7.5), 0.1 M KCl, 5 mM Mg/ATP, 50 µM [³H]berberine, and tonoplast vesicles for 40 µg protein, unless otherwise stated. Inhibitors and competitors were added to the above mixture to give a final volume of 200 µL. After incubation, 130 µL of the reaction mixture were loaded on a Sephadex (G-50 fine) spin column and centrifuged at 2,000 rpm for 2 min. The radioactivity of 100 µL of filtrate was determined by a liquid scintillation counter. The inhibitors used in this study (ammonium chloride and vanadate) were dissolved in water, whereas bafilomycin A1 was dissolved in DMSO. In the control, DMSO was added to the reaction mixture at a final concentration of 0.1%. DMSO did not affect berberine uptake at this concentration. Statistical analysis was performed using Student's unpaired t test.

Transport Assay with Liposomes

Liposomes having transmembrane pH gradients were prepared by the procedure of Mayer et al. (1990) with some modifications. Briefly, asolectin (Wako Pure Chemicals) was dissolved in 300 mM citrate buffer (pH 5.5) at a concentration of 5 mg/mL. Multilamellar vesicles were subjected to five freeze-thaw cycles (freezing in liquid N₂ and thawing at 40°C). Transmembrane pH gradients were established by passing the vesicles through a Sephadex G-25 column (PD-10) equilibrated in 20 mM HEPES containing 150 mM NaCl (pH 7.5). Liposomes (1.25 mg/mL) were incubated at 25°C for 10 min with 20 µM [¹⁴C]methylamine (approximately 1 µCi/mL) or 50 µM [³H]berberine. The uptake of methylamine or berberine by liposomes was measured by the spin-column assay, as described above, for the measurement of berberine transport.

	ACKNOWLEDGMENTS

 
We thank Dr. Yoshinori Moriyama of Okayama University and Dr. Eduardo Blumwald of the University of California for their helpful technical advice regarding the transport assay. We are also grateful to Dr. M. Boutry, Université Catholique de Louvain, for providing anti-H⁺-ATPase antibodies, Dr. M. H. Sato, Kyoto University, for anti-V-PPase antibodies, and Dr. N. Koizumi, Nara Institute of Science and Technology, for anti-BiP antibodies.

Received April 19, 2005; returned for revision May 19, 2005; accepted May 19, 2005.

	FOOTNOTES

 
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.

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