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Tonoplast-localized Abc2 Transporter Mediates Phytochelatin Accumulation in Vacuoles and Confers Cadmium Tolerance*

Open AccessPublished:October 11, 2010DOI:https://doi.org/10.1074/jbc.M110.155408
      Phytochelatins mediate tolerance to heavy metals in plants and some fungi by sequestering phytochelatin-metal complexes into vacuoles. To date, only Schizosaccharomyces pombe Hmt1 has been described as a phytochelatin transporter and attempts to identify orthologous phytochelatin transporters in plants and other organisms have failed. Furthermore, recent data indicate that the hmt1 mutant accumulates significant phytochelatin levels in vacuoles, suggesting that unidentified phytochelatin transporters exist in fungi. Here, we show that deletion of all vacuolar ABC transporters abolishes phytochelatin accumulation in S. pombe vacuoles and abrogates 35S-PC2 uptake into S. pombe microsomal vesicles. Systematic analysis of the entire S. pombe ABC transporter family identified Abc2 as a full-size ABC transporter (ABCC-type) that mediates phytochelatin transport into vacuoles. The S. pombe abc1 abc2 abc3 abc4 hmt1 quintuple and abc2 hmt1 double mutant show no detectable phytochelatins in vacuoles. Abc2 expression restores phytochelatin accumulation into vacuoles and suppresses the cadmium sensitivity of the abc quintuple mutant. A novel, unexpected, function of Hmt1 in GS-conjugate transport is also shown. In contrast to Hmt1, Abc2 orthologs are widely distributed among kingdoms and are proposed as the long-sought vacuolar phytochelatin transporters in plants and other organisms.

      Introduction

      Heavy metal contamination is a serious worldwide environmental problem caused by almost two centuries of intense industrial and mining activities, combined with an inappropriate disposal of residual waste (
      • Ogunseitan O.A.
      • Schoenung J.M.
      • Saphores J.D.
      • Shapiro A.A.
      ,
      • Satarug S.
      • Garrett S.H.
      • Sens M.A.
      • Sens D.A.
      ). Detrimental effects of heavy metals on human health may arise from occupational exposure, food intake and long-term exposure to metals in the environment, and have been linked to diabetes, hypertension, myocardial infarction, diminished lung function and certain types of cancer (
      • Ryan J.A.
      • Scheckel K.G.
      • Berti W.R.
      • Brown S.L.
      • Casteel S.W.
      • Chaney R.L.
      • Hallfrisch J.
      • Doolan M.
      • Grevatt P.
      • Maddaloni M.
      • Mosby D.
      ,
      • Guo Y.
      • Huo X.
      • Li Y.
      • Wu K.
      • Liu J.
      • Huang J.
      • Zheng G.
      • Xiao Q.
      • Yang H.
      • Wang Y.
      • Chen A.
      • Xu X.
      ). Non-essential heavy metals such as cadmium (Cd), lead (Pb), and mercury (Hg) interfere with the function of essential metals, including copper, zinc and manganese, by displacing them from their functional binding sites in proteins, thus interfering with biochemical and physiological functions (for reviews see Refs.
      • Mendoza-Cózatl D.
      • Loza-Tavera H.
      • Hernández-Navarro A.
      • Moreno-Sánchez R.
      ,
      • Clemens S.
      ,
      • Verbruggen N.
      • Hermans C.
      • Schat H.
      ). Because of the intrinsically high reactivity of heavy metals, exposure to elevated concentrations of both essential and non-essential metals impairs metabolism. Therefore, organisms have developed mechanisms to sense, transport, and mobilize essential metals, and other mechanisms to detoxify non-essential heavy metals (
      • Mendoza-Cózatl D.
      • Loza-Tavera H.
      • Hernández-Navarro A.
      • Moreno-Sánchez R.
      ,
      • Clemens S.
      ,
      • Verbruggen N.
      • Hermans C.
      • Schat H.
      ,
      • Zenk M.H.
      ). One of the most studied mechanisms mediating heavy metal detoxification involves phytochelatins, which are present in plants, algae, Schizosaccharomyces pombe and, surprisingly, Caenorhabditis elegans (
      • Mendoza-Cózatl D.
      • Loza-Tavera H.
      • Hernández-Navarro A.
      • Moreno-Sánchez R.
      ,
      • Clemens S.
      ,
      • Grill E.
      • Winnacker E.L.
      • Zenk M.H.
      ,
      • Mutoh N.
      • Hayashi Y.
      ,
      • Hayashi Y.
      • Isobe M.
      • Mutoh N.
      • Nakagawa C.W.
      • Kawabata M.
      ,
      • Clemens S.
      • Schroeder J.
      • Degenkolb T.
      ,
      • Vatamaniuk O.K.
      • Bucher E.A.
      • Ward J.T.
      • Rea P.A.
      ).
      Phytochelatins (PCs)
      The abbreviations used are: PC
      phytochelatin
      abc1–4
      S. pombe abc1 abc2 abc3 abc4 quadruple mutant
      abc1–4hmt1
      S. pombe abc1 abc2 abc3 abc4 hmt1 quintuple mutant
      ABC transporters
      ATP-binding cassette transporters
      ATM
      ABC transporters of the mitochondrion
      Cd
      cadmium
      CdS
      cadmium-sulfur clusters
      FeS
      iron-sulfur clusters
      GS-bimane
      bimane-S-glutathione
      Hmt1
      heavy metal tolerance factor 1
      HMWC
      high molecular weight complexes
      HPLC-MS
      reversed phase high performance liquid chromatography coupled to mass spectrometry
      MCB
      monochlorobimane
      MRP
      multidrug resistance protein
      YFP
      yellow fluorescent protein.
      are glutathione-derived peptides synthesized in the cytosol by the enzyme phytochelatin synthase (
      • Ha S.B.
      • Smith A.P.
      • Howden R.
      • Dietrich W.M.
      • Bugg S.
      • O'Connell M.J.
      • Goldsbrough P.B.
      • Cobbett C.
      ,
      • Vatamaniuk O.K.
      • Mari S.
      • Lu Y.P.
      • Rea P.A.
      ,
      • Clemens S.
      • Kim E.J.
      • Neumann D.
      • Schroeder J.I.
      ). In plants and S. pombe, cytosolic phytochelatin-metal complexes are transported into vacuoles (
      • Vögeli-Lange R.
      • Wagner G.J.
      ,
      • Salt D.E.
      • Rauser W.E.
      ,
      • Ortiz D.F.
      • Ruscitti T.
      • McCue K.F.
      • Ow D.W.
      ,
      • Van Belleghem F.
      • Cuypers A.
      • Semane B.
      • Smeets K.
      • Vangronsveld J.
      • d'Haen J.
      • Valcke R.
      ). In plants, PCs can also undergo long distance transport between shoots and roots (
      • Gong J.M.
      • Lee D.
      • Schroeder J.I.
      ,
      • Chen A.
      • Komives E.A.
      • Schroeder J.I.
      ,
      • Mendoza-Cózatl D.G.
      • Butko E.
      • Springer F.
      • Torpey J.W.
      • Komives E.A.
      • Kehr J.
      • Schroeder J.I.
      ) and this transport affects the shoot-to-root distribution of Cd. In the case of plants and S. pombe, PC-Cd complexes inside the vacuole bind sulfide and free Cd2+, forming high molecular weight complexes (HMWCs) that surround cadmium-sulfur clusters, providing enhanced stability and vacuolar Cd accumulation capacity (
      • Reese R.N.
      • Winge D.R.
      ,
      • Wu J.S.
      • Sung H.Y.
      • Juang R.H.
      ,
      • Ortiz D.F.
      • Kreppel L.
      • Speiser D.M.
      • Scheel G.
      • McDonald G.
      • Ow D.W.
      ,
      • Speiser D.M.
      • Ortiz D.F.
      • Kreppel L.
      • Scheel G.
      • McDonald G.
      • Ow D.W.
      ). Cd2+, as a free ion, can also be transported into vacuoles by vacuolar Ca2+/H+ antiporters (
      • Ortiz D.F.
      • Ruscitti T.
      • McCue K.F.
      • Ow D.W.
      ,
      • Salt D.E.
      • Wagner G.J.
      ,
      • Shigaki T.
      • Barkla B.J.
      • Miranda-Vergara M.C.
      • Zhao J.
      • Pantoja O.
      • Hirschi K.D.
      ). As for phytochelatin transporters, early studies in isolated vacuoles indicated that PC transport was mediated by ABC-type transporters (
      • Salt D.E.
      • Rauser W.E.
      ,
      • Ortiz D.F.
      • Ruscitti T.
      • McCue K.F.
      • Ow D.W.
      ). Furthermore, a vacuolar half-size ABC transporter required for Cd tolerance, Hmt1 (heavy metal tolerance 1), was proposed to function as the S. pombe vacuolar PC transporter (
      • Ortiz D.F.
      • Ruscitti T.
      • McCue K.F.
      • Ow D.W.
      ,
      • Ortiz D.F.
      • Kreppel L.
      • Speiser D.M.
      • Scheel G.
      • McDonald G.
      • Ow D.W.
      ).
      For more than 10 years the role of Hmt1 in Cd tolerance was undisputed, but attempts to identify Hmt1 orthologs in plants were unsuccessful (
      • Sánchez-Fernández R.
      • Davies T.G.
      • Coleman J.O.
      • Rea P.A.
      ,
      • Rea P.A.
      ). This was particularly intriguing after the completion of the Arabidopsis genome project, where it was clear that this reference plant lacks an Hmt1 ortholog, but retains the capacity to sequester PCs and Cd in vacuoles to form HMWCs (
      • Sánchez-Fernández R.
      • Davies T.G.
      • Coleman J.O.
      • Rea P.A.
      ,
      • Howden R.
      • Goldsbrough P.B.
      • Andersen C.R.
      • Cobbett C.S.
      ). Hmt1-like proteins are not abundant in nature, being restricted to a limited number of organisms, and their function is not completely understood (
      • Sooksa-Nguan T.
      • Yakubov B.
      • Kozlovskyy V.I.
      • Barkume C.M.
      • Howe K.J.
      • Thannhauser T.W.
      • Rutzke M.A.
      • Hart J.J.
      • Kochian L.V.
      • Rea P.A.
      • Vatamaniuk O.K.
      ). The closest Hmt1 homologues in Arabidopsis are the ATM transporters localized in the mitochondrion (
      • Sánchez-Fernández R.
      • Davies T.G.
      • Coleman J.O.
      • Rea P.A.
      ,
      • Rea P.A.
      ,
      • Sooksa-Nguan T.
      • Yakubov B.
      • Kozlovskyy V.I.
      • Barkume C.M.
      • Howe K.J.
      • Thannhauser T.W.
      • Rutzke M.A.
      • Hart J.J.
      • Kochian L.V.
      • Rea P.A.
      • Vatamaniuk O.K.
      ,
      • Chen S.
      • Sánchez-Fernández R.
      • Lyver E.R.
      • Dancis A.
      • Rea P.A.
      ). ATM3 mediates the maturation of iron-sulfur clusters (
      • Kushnir S.
      • Babiychuk E.
      • Storozhenko S.
      • Davey M.W.
      • Papenbrock J.
      • De Rycke R.
      • Engler G.
      • Stephan U.W.
      • Lange H.
      • Kispal G.
      • Lill R.
      • Van Montagu M.
      ) and functions in the synthesis of molybdenum cofactor (
      • Teschner J.
      • Lachmann N.
      • Schulze J.
      • Geisler M.
      • Selbach K.
      • Santamaria-Araujo J.
      • Balk J.
      • Mendel R.R.
      • Bittner F.
      ). Interestingly, overexpression of ATM3 in Arabidopsis enhances cadmium resistance (
      • Kim D.Y.
      • Bovet L.
      • Kushnir S.
      • Noh E.W.
      • Martinoia E.
      • Lee Y.
      ).
      The first evidence indicating that Hmt1 may have a role in Cd tolerance other than PC transport came from work in C. elegans, where PC synthase and Hmt1 homologues were identified (
      • Clemens S.
      • Schroeder J.
      • Degenkolb T.
      ,
      • Vatamaniuk O.K.
      • Bucher E.A.
      • Ward J.T.
      • Rea P.A.
      ,
      • Vatamaniuk O.K.
      • Bucher E.A.
      • Sundaram M.V.
      • Rea P.A.
      ). RNAi studies, and more recently deletion analyses, revealed that the C. elegans Hmt1 and PC synthase have an additive effect on Cd tolerance, suggesting that Hmt1 and PC synthase do not function in a simple linear pathway (
      • Vatamaniuk O.K.
      • Bucher E.A.
      • Sundaram M.V.
      • Rea P.A.
      ,
      • Schwartz M.S.
      • Benci J.L.
      • Selote D.S.
      • Sharma A.K.
      • Chen A.G.
      • Dang H.
      • Fares H.
      • Vatamaniuk O.K.
      ). In addition, an HMT1 protein from the non-PC producing Drosophila melanogaster was able to rescue the Cd sensitivity of the S. pombe hmt1 mutant (
      • Sooksa-Nguan T.
      • Yakubov B.
      • Kozlovskyy V.I.
      • Barkume C.M.
      • Howe K.J.
      • Thannhauser T.W.
      • Rutzke M.A.
      • Hart J.J.
      • Kochian L.V.
      • Rea P.A.
      • Vatamaniuk O.K.
      ). Conversely, S. pombe Hmt1 conferred tolerance to Cd in Escherichia coli and Saccharomyces cerevisiae, organisms devoid of phytochelatins (
      • Prévéral S.
      • Gayet L.
      • Moldes C.
      • Hoffmann J.
      • Mounicou S.
      • Gruet A.
      • Reynaud F.
      • Lobinski R.
      • Verbavatz J.M.
      • Vavasseur A.
      • Forestier C.
      ). Furthermore, recent data showed that the S. pombe hmt1 mutant accumulates significant levels of PCs in vacuoles (
      • Sooksa-Nguan T.
      • Yakubov B.
      • Kozlovskyy V.I.
      • Barkume C.M.
      • Howe K.J.
      • Thannhauser T.W.
      • Rutzke M.A.
      • Hart J.J.
      • Kochian L.V.
      • Rea P.A.
      • Vatamaniuk O.K.
      ), suggesting that other, yet unidentified proteins mediate PC transport into vacuoles. Altogether these results might explain why the search for a Hmt1-related PC transporters in other organisms has been futile and re-opens the original question regarding the identity of vacuolar PC uptake transporters.
      Here we report that Abc2, a full-size ABC transporter of the MRP/ABCC subfamily, mediates accumulation of PCs in S. pombe vacuoles. Systematic analyses of ABC transporter deletion mutants, Cd sensitivity assays, complementation experiments, PC content, and HMWCs in purified vacuoles demonstrate that Abc2 mediates PC accumulation into vacuoles. We further show a new role for Hmt1 in GS-conjugate transport. The identification of Abc2 as a PC transporter expands the current model of Cd tolerance mediated by PCs and opens the possibility to identify the long-sought PC transporters in plants and other organisms. These transporters, in combination with other genes, hold the potential to enhance the heavy metal tolerance and accumulation capacity of organisms for their use in the bioremediation of soils and waters contaminated with heavy metals, or to circumvent the accumulation of non-essential metals in the edible parts of crop plants.

      DISCUSSION

      Since its discovery 15 years ago, S. pombe Hmt1 has stood alone as the only known PC transporter (
      • Ortiz D.F.
      • Ruscitti T.
      • McCue K.F.
      • Ow D.W.
      ), despite numerous genetic screens to identify PC transporters in plants and other organisms. Here, we demonstrate through systematic analyses of deletion mutants, Cd tolerance assays, PC content in purified vacuoles and complementation experiments, that S. pombe Abc2, a full-size ABC transporter, also mediates PC accumulation into vacuoles.

      PC Synthase and Hmt1 Mediate Cd Tolerance through Independent Mechanisms

      The capacity of Hmt1 to confer Cd tolerance in a PC-independent manner suggests that Hmt1 may have other functions in addition to PC transport (
      • Vatamaniuk O.K.
      • Bucher E.A.
      • Sundaram M.V.
      • Rea P.A.
      ,
      • Schwartz M.S.
      • Benci J.L.
      • Selote D.S.
      • Sharma A.K.
      • Chen A.G.
      • Dang H.
      • Fares H.
      • Vatamaniuk O.K.
      ,
      • Prévéral S.
      • Gayet L.
      • Moldes C.
      • Hoffmann J.
      • Mounicou S.
      • Gruet A.
      • Reynaud F.
      • Lobinski R.
      • Verbavatz J.M.
      • Vavasseur A.
      • Forestier C.
      ) (Fig. 1). This is also evident in the S. pombe hmt1 mutant, which is Cd sensitive despite the fact that it retains the ability to synthesize PCs and transport them into vacuoles to form HMWCs (Figs. 1, 2, 3E, and 4E). These results have encouraged several groups to re-assess the function of Hmt1 (
      • Sooksa-Nguan T.
      • Yakubov B.
      • Kozlovskyy V.I.
      • Barkume C.M.
      • Howe K.J.
      • Thannhauser T.W.
      • Rutzke M.A.
      • Hart J.J.
      • Kochian L.V.
      • Rea P.A.
      • Vatamaniuk O.K.
      ,
      • Prévéral S.
      • Gayet L.
      • Moldes C.
      • Hoffmann J.
      • Mounicou S.
      • Gruet A.
      • Reynaud F.
      • Lobinski R.
      • Verbavatz J.M.
      • Vavasseur A.
      • Forestier C.
      ) (Fig. 1). The first suggestion that Hmt1 may have a function other than PC transport came from work in C. elegans, where it was shown that hmt1–1 and pcs-1 have an additive effect on Cd tolerance compared with single mutants (
      • Vatamaniuk O.K.
      • Bucher E.A.
      • Sundaram M.V.
      • Rea P.A.
      ). An additive effect of Hmt1 is not expected in a linear model when PC synthase is deleted. We have found that Hmt1 and PC synthase also have additive effects on Cd tolerance in both S. cerevisiae and S. pombe (Fig. 1), suggesting that they do not function in a strictly linear pathway (
      • Ortiz D.F.
      • Ruscitti T.
      • McCue K.F.
      • Ow D.W.
      ). Moreover, in C. elegans, Hmt1 and PC synthase localize to different tissues, supporting the hypothesis that they cannot operate in a simple linear metal detoxification pathway (
      • Schwartz M.S.
      • Benci J.L.
      • Selote D.S.
      • Sharma A.K.
      • Chen A.G.
      • Dang H.
      • Fares H.
      • Vatamaniuk O.K.
      ).

      PC Transport Mediated by Full-size ABC Transporters

      The finding that vacuoles isolated from the S. pombe hmt1 mutant exposed to Cd contain high levels of PCs, suggests the existence of an Hmt1-independent mechanism for accumulation of PCs into vacuoles (Fig. 3F) (
      • Sooksa-Nguan T.
      • Yakubov B.
      • Kozlovskyy V.I.
      • Barkume C.M.
      • Howe K.J.
      • Thannhauser T.W.
      • Rutzke M.A.
      • Hart J.J.
      • Kochian L.V.
      • Rea P.A.
      • Vatamaniuk O.K.
      ). PC uptake into vacuole preparations shows biochemical properties characteristic of ABC-type transporters (
      • Salt D.E.
      • Rauser W.E.
      ,
      • Ortiz D.F.
      • Ruscitti T.
      • McCue K.F.
      • Ow D.W.
      ), as confirmed in the present study (see “Results” section). The S. pombe genome encodes a relatively small number of ABC transporters (11 transporters), compared with 29 in S. cerevisiae and >100 in A. thaliana (
      • Rea P.A.
      ,
      • Iwaki T.
      • Giga-Hama Y.
      • Takegawa K.
      ,
      • Higgins C.F.
      ,
      • Decottignies A.
      • Goffeau A.
      ). Of the 11 S. pombe ABC transporters, only Hmt1, Abc2, Abc3, and Abc4 are targeted to the vacuolar membrane (Fig. 3D) (
      • Iwaki T.
      • Giga-Hama Y.
      • Takegawa K.
      ) and deletion of all vacuolar ABC transporters caused Cd hypersensitivity (Figs. 1F and 2) and abolished transport and accumulation of PCs into vacuoles (Fig. 3F and supplemental Fig. S3). Systematic characterization of different abc deletion mutants led us to identify Abc2 as a major contributor, among the full-size ABC transporters, to Cd tolerance (Fig. 2). Expression of Abc2 in the abc1–4 hmt1 mutant partially restores Cd tolerance and PC accumulation into vacuoles (Fig. 3, E and F). Plants do not have orthologs of the half-size ABC transporter Hmt1, which has hampered the discovery of plant PC transporters. However, and in contrast to Hmt1, plants and other organisms that produce PCs have Abc2 homologues (MRP/ABCC subfamily of ATP binding cassette transporters, supplemental Fig. S6).

      Substrate Specificity of S. pombe ABC Transporters

      In a recent analysis of the 11 S. pombe ABC transporters, Abc2 was found to be required for accumulation of red pigments inside vacuoles (
      • Iwaki T.
      • Giga-Hama Y.
      • Takegawa K.
      ). In a broad analysis, single and multiple ABC mutants were exposed to more than 20 different compounds, including antibiotics, metal ions and oxidizing agents. The Cd sensitivity of a abc2 abc4 hmt1 mutant was noted but not investigated further (
      • Iwaki T.
      • Giga-Hama Y.
      • Takegawa K.
      ). Here we show that Abc2, as other members of the MRP/ABCC family, mediates accumulation of GS-bimane conjugates (Fig. 3C) and, more interestingly, PC accumulation in vacuoles (Fig. 3F). Abc2 is a homologue of S. cerevisiae YCF1, a glutathione-S-conjugate pump with broad substrate specificity (
      • Iwaki T.
      • Giga-Hama Y.
      • Takegawa K.
      ,
      • Li Z.S.
      • Lu Y.P.
      • Zhen R.G.
      • Szczupka M.
      • Thiele D.J.
      • Rea P.A.
      ). YCF1 does not mediate PC uptake (
      • Li Z.S.
      • Lu Y.P.
      • Zhen R.G.
      • Szczupka M.
      • Thiele D.J.
      • Rea P.A.
      ), but based on the broad substrate specificity of Abc2 (Fig. 3), it will be interesting to determine whether, in addition to PCs, Abc2 also mediates transport of GS2-Cd complexes. The great plasticity of ABC transporters might not be surprising considering the variety of molecules that need to be mobilized and detoxified by the limited number of ABC transporters present in S. pombe. In addition, we cannot rule out at this point a possible interaction between ABC transporters, which has been documented to modify the kinetic properties of ABC transporters (
      • Mo W.
      • Zhang J.T.
      ,
      • Trompier D.
      • Alibert M.
      • Davanture S.
      • Hamon Y.
      • Pierres M.
      • Chimini G.
      ,
      • Yang Y.
      • Liu Y.
      • Dong Z.
      • Xu J.
      • Peng H.
      • Liu Z.
      • Zhang J.T.
      ) and could explain why Hmt1 and Abc2 alone do not restore the vacuolar PC content to wild type levels (Fig. 3F). It also remains unclear whether post-transcriptional regulation plays a role in cadmium tolerance by regulating the trafficking and turnover of Hmt1 and Abc2.

      Mechanism Mediating Heavy Metal Tolerance by Hmt1

      A key remaining question is the chemical nature of the Hmt1 substrate in vivo. Hmt1 requires GSH to confer Cd tolerance (
      • Prévéral S.
      • Gayet L.
      • Moldes C.
      • Hoffmann J.
      • Mounicou S.
      • Gruet A.
      • Reynaud F.
      • Lobinski R.
      • Verbavatz J.M.
      • Vavasseur A.
      • Forestier C.
      ), but analyses of the peptide content of vacuolar HMWCs in S. pombe have failed to detect significant amounts of GSH inside vacuoles (
      • Reese R.N.
      • Winge D.R.
      ,
      • Wu J.S.
      • Sung H.Y.
      • Juang R.H.
      ,
      • Plocke D.J.
      • Kägi J.H.
      ). Moreover, vacuolar vesicles isolated from S. pombe overexpressing Hmt1 did not show significant uptake rates of GSH or GS2-Cd (
      • Ortiz D.F.
      • Ruscitti T.
      • McCue K.F.
      • Ow D.W.
      ), suggesting that the GSH requirement of Hmt1 may not be related to GS2-Cd transport. It is possible that Hmt1 may have an additional yet unidentified substrate, which is highly toxic when it accumulates in the cytosol of hmt1 mutants.
      The closest homologue to Hmt1 in S. pombe is Atm1 (supplemental Fig. S6), a mitochondrial half-size ABC transporter that mediates export of FeS clusters from the mitochondrial matrix to the cytosol (
      • Iwaki T.
      • Giga-Hama Y.
      • Takegawa K.
      ,
      • Broderick J.B.
      ). The function of Atm1 in S. cerevisiae also requires GSH (
      • Sipos K.
      • Lange H.
      • Fekete Z.
      • Ullmann P.
      • Lill R.
      • Kispal G.
      ). Therefore, it is tempting to speculate that Hmt1 may transport CdS clusters from the cytosol into vacuoles (Fig. 5E) and that either GSH is required to synthesize these clusters or, as recently determined for FeS clusters in plants and bacteria, GSH and/or PC2 help to stabilize these clusters (
      • Iwema T.
      • Picciocchi A.
      • Traore D.A.
      • Ferrer J.L.
      • Chauvat F.
      • Jacquamet L.
      ,
      • Rouhier N.
      • Unno H.
      • Bandyopadhyay S.
      • Masip L.
      • Kim S.K.
      • Hirasawa M.
      • Gualberto J.M.
      • Lattard V.
      • Kusunoki M.
      • Knaff D.B.
      • Georgiou G.
      • Hase T.
      • Johnson M.K.
      • Jacquot J.P.
      ). Transport of CdS-PC clusters by Hmt1 could explain why the abc2 single mutant still accumulates PCs in the vacuole (Fig. 3F). In addition, active detoxification of CdS clusters by Hmt1 in the abc2 single mutant could also explain why the PC content in this mutant remained unaffected (Fig. 5E and supplemental Fig. S5A) and why abc mutants are not Cd hypersensitive (Figs. 1F and 2). On the other hand, accumulation of CdS clusters in the cytosol is expected to be highly toxic, impairing the activity FeS-containing enzymes and PC synthesis (
      • Vande Weghe J.G.
      • Ow D.W.
      ). Impairment of PC synthesis explains the decreased PC content in cell extracts obtained from hmt1 and the abc1–4hmt1 quintuple mutant (supplemental Fig. S5A). Moreover, formation of HMWC around CdS cores is a spontaneous process that takes place in the vacuole where all the components are readily available (
      • Wu J.S.
      • Sung H.Y.
      • Juang R.H.
      ). In hmt1 and abc mutants, traces of HMWC were detected in cell extracts, but not in purified vacuoles (Fig. 4), suggesting that HMWCs are also formed in the cytosol, impairing PC synthesis, and explaining the extreme Cd sensitivity of the hmt1, abc2 hmt1, and abc1–4hmt1mutants, even though they retain the ability to synthesize PCs (supplemental Fig. S5A).
      In conclusion, we have determined that Abc2, a full-size ABC transporter of the MRP/ABCC family, mediates accumulation of PCs in vacuoles, and we have demonstrated that Hmt1 and Abc2 have distinct and also overlapping functions in Cd detoxification. These findings modify the original model of Cd tolerance mediated by Hmt1 (Fig. 5E) and present the possibility to identify the long-sought PC transporters in other organisms. Tissue-specific expression of PC transporters may be useful for bioremediation of soils and waters contaminated with heavy metals and to exclude heavy metal accumulation in edible tissues of plants used for livestock and human nutrition.

      Note Added in Proof

      A report describing the independent identification of the related AtABCC1 and AtABCC2 as vacuolar phytochelatin uptake transporters in Arabidopsis thaliana is in press (Song, W.-Y., Park, J., Mendoza-Cózatl, D., Suter-Grotemeyer, M., Shim, D., Hörtensteiner, S., Geisler, M., Weder, B., Rea, P., Rentsch, D., Schroeder, J. I., Lee, Y., and Martinoia, E. (2010) Proc. Natl. Acad. Sci. U.S.A., in press).

      Acknowledgments

      We thank Kaoru Takegawa for providing the S. pombe abc1abc2abc3abc4 mutant strain, Angus Murphy for providing an abc2 abc3 hmt1 mutant, and Maja Schellenberg for technical assistance in transport experiments.

      REFERENCES

        • Ogunseitan O.A.
        • Schoenung J.M.
        • Saphores J.D.
        • Shapiro A.A.
        Science. 2009; 326: 670-671
        • Satarug S.
        • Garrett S.H.
        • Sens M.A.
        • Sens D.A.
        Environ. Health Perspect. 2010; 118: 182-190
        • Ryan J.A.
        • Scheckel K.G.
        • Berti W.R.
        • Brown S.L.
        • Casteel S.W.
        • Chaney R.L.
        • Hallfrisch J.
        • Doolan M.
        • Grevatt P.
        • Maddaloni M.
        • Mosby D.
        Environ. Sci. Technol. 2004; 38: 18A-24A
        • Guo Y.
        • Huo X.
        • Li Y.
        • Wu K.
        • Liu J.
        • Huang J.
        • Zheng G.
        • Xiao Q.
        • Yang H.
        • Wang Y.
        • Chen A.
        • Xu X.
        Sci. Total. Environ. 2010; 408: 3113-3117
        • Mendoza-Cózatl D.
        • Loza-Tavera H.
        • Hernández-Navarro A.
        • Moreno-Sánchez R.
        FEMS Microbiol. Rev. 2005; 29: 653-671
        • Clemens S.
        Biochimie. 2006; 88: 1707-1719
        • Verbruggen N.
        • Hermans C.
        • Schat H.
        Curr. Opin. Plant Biol. 2009; 12: 364-372
        • Zenk M.H.
        Gene. 1996; 179: 21-30
        • Grill E.
        • Winnacker E.L.
        • Zenk M.H.
        Science. 1985; 230: 674-676
        • Mutoh N.
        • Hayashi Y.
        Biochem. Biophys. Res. Commun. 1988; 151: 32-39
        • Hayashi Y.
        • Isobe M.
        • Mutoh N.
        • Nakagawa C.W.
        • Kawabata M.
        Methods Enzymol. 1991; 205: 348-358
        • Clemens S.
        • Schroeder J.
        • Degenkolb T.
        Eur. J. Biochem. 2001; 268: 3640-3643
        • Vatamaniuk O.K.
        • Bucher E.A.
        • Ward J.T.
        • Rea P.A.
        J. Biol. Chem. 2001; 276: 20817-20820
        • Ha S.B.
        • Smith A.P.
        • Howden R.
        • Dietrich W.M.
        • Bugg S.
        • O'Connell M.J.
        • Goldsbrough P.B.
        • Cobbett C.
        Plant Cell. 1999; 11: 1153-1164
        • Vatamaniuk O.K.
        • Mari S.
        • Lu Y.P.
        • Rea P.A.
        Proc. Natl. Acad. Sci., U.S.A. 1999; 96: 7110-7115
        • Clemens S.
        • Kim E.J.
        • Neumann D.
        • Schroeder J.I.
        EMBO J. 1999; 18: 3326-3333
        • Vögeli-Lange R.
        • Wagner G.J.
        Plant Physiol. 1990; 92: 1086-1093
        • Salt D.E.
        • Rauser W.E.
        Plant Physiol. 1995; 107: 1293-1301
        • Ortiz D.F.
        • Ruscitti T.
        • McCue K.F.
        • Ow D.W.
        J. Biol. Chem. 1995; 270: 4721-4728
        • Van Belleghem F.
        • Cuypers A.
        • Semane B.
        • Smeets K.
        • Vangronsveld J.
        • d'Haen J.
        • Valcke R.
        New Phytol. 2007; 173: 495-508
        • Gong J.M.
        • Lee D.
        • Schroeder J.I.
        Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 10118-10123
        • Chen A.
        • Komives E.A.
        • Schroeder J.I.
        Plant Physiol. 2006; 141: 108-120
        • Mendoza-Cózatl D.G.
        • Butko E.
        • Springer F.
        • Torpey J.W.
        • Komives E.A.
        • Kehr J.
        • Schroeder J.I.
        Plant J. 2008; 54: 249-259
        • Reese R.N.
        • Winge D.R.
        J. Biol. Chem. 1988; 263: 12832-12835
        • Wu J.S.
        • Sung H.Y.
        • Juang R.H.
        Biochem. Mol. Biol. Int. 1995; 36: 1169-1175
        • Ortiz D.F.
        • Kreppel L.
        • Speiser D.M.
        • Scheel G.
        • McDonald G.
        • Ow D.W.
        EMBO J. 1992; 11: 3491-3499
        • Speiser D.M.
        • Ortiz D.F.
        • Kreppel L.
        • Scheel G.
        • McDonald G.
        • Ow D.W.
        Mol. Cell. Biol. 1992; 12: 5301-5310
        • Salt D.E.
        • Wagner G.J.
        J. Biol. Chem. 1993; 268: 12297-12302
        • Shigaki T.
        • Barkla B.J.
        • Miranda-Vergara M.C.
        • Zhao J.
        • Pantoja O.
        • Hirschi K.D.
        J. Biol. Chem. 2005; 280: 30136-30142
        • Sánchez-Fernández R.
        • Davies T.G.
        • Coleman J.O.
        • Rea P.A.
        J. Biol. Chem. 2001; 276: 30231-30244
        • Rea P.A.
        Annu. Rev. Plant. Biol. 2007; 58: 347-375
        • Howden R.
        • Goldsbrough P.B.
        • Andersen C.R.
        • Cobbett C.S.
        Plant Physiol. 1995; 107: 1059-1066
        • Sooksa-Nguan T.
        • Yakubov B.
        • Kozlovskyy V.I.
        • Barkume C.M.
        • Howe K.J.
        • Thannhauser T.W.
        • Rutzke M.A.
        • Hart J.J.
        • Kochian L.V.
        • Rea P.A.
        • Vatamaniuk O.K.
        J. Biol. Chem. 2009; 284: 354-362
        • Chen S.
        • Sánchez-Fernández R.
        • Lyver E.R.
        • Dancis A.
        • Rea P.A.
        J. Biol. Chem. 2007; 282: 21561-21571
        • Kushnir S.
        • Babiychuk E.
        • Storozhenko S.
        • Davey M.W.
        • Papenbrock J.
        • De Rycke R.
        • Engler G.
        • Stephan U.W.
        • Lange H.
        • Kispal G.
        • Lill R.
        • Van Montagu M.
        Plant Cell. 2001; 13: 89-100
        • Teschner J.
        • Lachmann N.
        • Schulze J.
        • Geisler M.
        • Selbach K.
        • Santamaria-Araujo J.
        • Balk J.
        • Mendel R.R.
        • Bittner F.
        Plant Cell. 2010; 22: 468-480
        • Kim D.Y.
        • Bovet L.
        • Kushnir S.
        • Noh E.W.
        • Martinoia E.
        • Lee Y.
        Plant Physiol. 2006; 140: 922-932
        • Vatamaniuk O.K.
        • Bucher E.A.
        • Sundaram M.V.
        • Rea P.A.
        J. Biol. Chem. 2005; 280: 23684-23690
        • Schwartz M.S.
        • Benci J.L.
        • Selote D.S.
        • Sharma A.K.
        • Chen A.G.
        • Dang H.
        • Fares H.
        • Vatamaniuk O.K.
        PLoS One. 2010; 5: e9564
        • Prévéral S.
        • Gayet L.
        • Moldes C.
        • Hoffmann J.
        • Mounicou S.
        • Gruet A.
        • Reynaud F.
        • Lobinski R.
        • Verbavatz J.M.
        • Vavasseur A.
        • Forestier C.
        J. Biol. Chem. 2009; 284: 4936-4943
        • Forsburg S.L.
        • Rhind N.
        Yeast. 2006; 23: 173-183
        • Kennedy P.J.
        • Vashisht A.A.
        • Hoe K.L.
        • Kim D.U.
        • Park H.O.
        • Hayles J.
        • Russell P.
        Toxicol. Sci. 2008; 106: 124-139
        • Chen D.
        • Toone W.M.
        • Mata J.
        • Lyne R.
        • Burns G.
        • Kivinen K.
        • Brazma A.
        • Jones N.
        • Bähler J.
        Mol. Biol. Cell. 2003; 14: 214-229
        • Okazaki K.
        • Okazaki N.
        • Kume K.
        • Jinno S.
        • Tanaka K.
        • Okayama H.
        Nucleic Acids Res. 1990; 18: 6485-6489
        • Vatamaniuk O.K.
        • Mari S.
        • Lu Y.P.
        • Rea P.A.
        J. Biol. Chem. 2000; 275: 31451-31459
        • Nagy R.
        • Grob H.
        • Weder B.
        • Green P.
        • Klein M.
        • Frelet-Barrand A.
        • Schjoerring J.K.
        • Brearley C.
        • Martinoia E.
        J. Biol. Chem. 2009; 284: 33614-33622
        • Iwaki T.
        • Giga-Hama Y.
        • Takegawa K.
        Microbiology. 2006; 152: 2309-2321
        • Li Z.S.
        • Lu Y.P.
        • Zhen R.G.
        • Szczupka M.
        • Thiele D.J.
        • Rea P.A.
        Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 42-47
        • Vande Weghe J.G.
        • Ow D.W.
        Mol. Microbiol. 2001; 42: 29-36
        • Lu Y.P.
        • Li Z.S.
        • Rea P.A.
        Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 8243-8248
        • Higgins C.F.
        Annu. Rev. Cell. Biol. 1992; 8: 67-113
        • Decottignies A.
        • Goffeau A.
        Nat. Genet. 1997; 15: 137-145
        • Mo W.
        • Zhang J.T.
        Expert. Opin. Drug. Metab. Toxicol. 2009; 5: 1049-1063
        • Trompier D.
        • Alibert M.
        • Davanture S.
        • Hamon Y.
        • Pierres M.
        • Chimini G.
        J. Biol. Chem. 2006; 281: 20283-20290
        • Yang Y.
        • Liu Y.
        • Dong Z.
        • Xu J.
        • Peng H.
        • Liu Z.
        • Zhang J.T.
        J. Biol. Chem. 2007; 282: 8821-8830
        • Plocke D.J.
        • Kägi J.H.
        Eur. J. Biochem. 1992; 207: 201-205
        • Broderick J.B.
        Nat. Chem. Biol. 2007; 3: 243-244
        • Sipos K.
        • Lange H.
        • Fekete Z.
        • Ullmann P.
        • Lill R.
        • Kispal G.
        J. Biol. Chem. 2002; 277: 26944-26949
        • Iwema T.
        • Picciocchi A.
        • Traore D.A.
        • Ferrer J.L.
        • Chauvat F.
        • Jacquamet L.
        Biochemistry. 2009; 48: 6041-6043
        • Rouhier N.
        • Unno H.
        • Bandyopadhyay S.
        • Masip L.
        • Kim S.K.
        • Hirasawa M.
        • Gualberto J.M.
        • Lattard V.
        • Kusunoki M.
        • Knaff D.B.
        • Georgiou G.
        • Hase T.
        • Johnson M.K.
        • Jacquot J.P.
        Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 7379-7384