Drosophila ABC Transporter, DmHMT-1, Confers Tolerance to Cadmium
2008; Elsevier BV; Volume: 284; Issue: 1 Linguagem: Inglês
10.1074/jbc.m806501200
ISSN1083-351X
AutoresThanwalee Sooksa-nguan, Bakhtiyor Yakubov, V. Kozlovskyy, Caitlin M. Barkume, Kevin Howe, Theodore W. Thannhauser, Michael A. Rutzke, Jonathan J. Hart, Leon V. Kochian, Philip A. Rea, Olena K. Vatamaniuk,
Tópico(s)Heavy Metal Exposure and Toxicity
ResumoHalf-molecule ATP-binding cassette transporters of the HMT-1 (heavy metal tolerance factor 1) subfamily are required for Cd2+ tolerance in Schizosaccharomyces pombe, Caenorhabditis elegans, and Chlamydomonas reinhardtii. Based on studies of S. pombe, it has been proposed that SpHMT-1 transports heavy metal·phytochelatin (PC) complexes into the vacuolysosomal compartment. PCs are glutathione derivatives synthesized by PC synthases (PCS) in plants, fungi, and C. elegans in response to heavy metals. Our previous studies in C. elegans, however, suggested that HMT-1 and PCS-1 do not necessarily act in concert in metal detoxification. To further explore this inconsistency, we have gone on to test whether DmHMT-1, an HMT-1 from a new source, Drosophila, whose genome lacks PCS homologs, functions in heavy metal detoxification. In so doing, we show that heterologously expressed DmHMT-1 suppresses the Cd2+ hypersensitivity of S. pombe hmt-1 mutants and localizes to the vacuolar membrane but does not transport Cd·PC complexes. Crucially, similar analyses of S. pombe hmt-1 mutants extend this finding to show that SpHMT-1 itself either does not transport Cd·PC complexes or is not the principal Cd·PC/apoPC transporter. Consistent with this discovery and with our previous suggestion that HMT-1 and PCS-1 do not operate in a simple linear metal detoxification pathway, we demonstrate that, unlike PCS-deficient cells, which are hypersensitive to several heavy metals, SpHMT-1-deficient cells are hypersensitive to Cd2+, but not to Hg2+ or As3+. These findings significantly change our current understanding of the function of HMT-1 proteins and invoke a PC-independent role for these transporters in Cd2+ detoxification. Half-molecule ATP-binding cassette transporters of the HMT-1 (heavy metal tolerance factor 1) subfamily are required for Cd2+ tolerance in Schizosaccharomyces pombe, Caenorhabditis elegans, and Chlamydomonas reinhardtii. Based on studies of S. pombe, it has been proposed that SpHMT-1 transports heavy metal·phytochelatin (PC) complexes into the vacuolysosomal compartment. PCs are glutathione derivatives synthesized by PC synthases (PCS) in plants, fungi, and C. elegans in response to heavy metals. Our previous studies in C. elegans, however, suggested that HMT-1 and PCS-1 do not necessarily act in concert in metal detoxification. To further explore this inconsistency, we have gone on to test whether DmHMT-1, an HMT-1 from a new source, Drosophila, whose genome lacks PCS homologs, functions in heavy metal detoxification. In so doing, we show that heterologously expressed DmHMT-1 suppresses the Cd2+ hypersensitivity of S. pombe hmt-1 mutants and localizes to the vacuolar membrane but does not transport Cd·PC complexes. Crucially, similar analyses of S. pombe hmt-1 mutants extend this finding to show that SpHMT-1 itself either does not transport Cd·PC complexes or is not the principal Cd·PC/apoPC transporter. Consistent with this discovery and with our previous suggestion that HMT-1 and PCS-1 do not operate in a simple linear metal detoxification pathway, we demonstrate that, unlike PCS-deficient cells, which are hypersensitive to several heavy metals, SpHMT-1-deficient cells are hypersensitive to Cd2+, but not to Hg2+ or As3+. These findings significantly change our current understanding of the function of HMT-1 proteins and invoke a PC-independent role for these transporters in Cd2+ detoxification. The adverse health effects of heavy metals such as cadmium (Cd2+), mercury (Hg2+), and lead (Pb2+) from food and air are well established (1Hyman M.H. Altern. Ther. Health Med. 2004; 10: 70-75PubMed Google Scholar, 2Waalkes M.P. J. Inorg Biochem. 2000; 79: 241-244Crossref PubMed Scopus (788) Google Scholar, 3Vallee B.L. Ulmer D.D. Annu. Rev. Biochem. 1972; 41: 91-128Crossref PubMed Scopus (1253) Google Scholar, 4Stadtman E.R. Free Radic. Biol. Med. 1990; 9: 315-325Crossref PubMed Scopus (1033) Google Scholar). Despite this knowledge, exposure to heavy metals continues, and has even increased in some areas, due to their sustained production and emission into the environment. At the cellular level, the toxicity of heavy metals results from the displacement of endogenous cofactors from their cellular binding sites, the oxidation of essential enzymes and other proteins, and promotion of the formation of reactive oxygen species (3Vallee B.L. Ulmer D.D. Annu. Rev. Biochem. 1972; 41: 91-128Crossref PubMed Scopus (1253) Google Scholar, 4Stadtman E.R. Free Radic. Biol. Med. 1990; 9: 315-325Crossref PubMed Scopus (1033) Google Scholar). The variety of ways by which heavy metals exert their effects places demands on a wide range of distinct cellular detoxification mechanisms in which ATP-binding cassette (ABC) 3The abbreviations used are: ABC transporters, ATP-binding cassette transporters; HMT-1, heavy metal tolerance factor 1; DmHMT-1, D. melanogaster heavy metal tolerance factor 1; CeHMT-1, C. elegans heavy metal tolerance factor 1; SpHMT-1, S. pombe heavy metal tolerance factor 1; PC, phytochelatin; SpPCS-1, S. pombe phytochelatin synthase 1; TMD, transmembrane domain; NBD, nucleotide-binding domain; NTE, hydrophobic N-terminal extension; ESI-MS, electrospray ionization mass spectrometry; LC-MALDI-MS, tandem liquid chromatography matrix-assisted laser desorption ionization mass spectrometry; TOF, time of flight; ICP-AES, inductively coupled plasma-atomic emission spectrometry; MCB, monochlorobimane; bimane-GS, bimane-S-glutathione; EMM, Edinburgh minimal medium; HM, homogenization medium; MES, 3-(N-morpholino)-2-hydroxypropanesulfonic acid; RP-HPLC, reversed-phase high-performance liquid chromatography; ATM, ABC transporters of the mitochondrion; GFP, green fluorescent protein; EGFP, enhanced GFP. transporters are clearly implicated (5Vatamaniuk O.K. Bucher E.A. Sundaram M.V. Rea P.A. J. Biol. Chem. 2005; 280: 23684-23690Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 6Li Z.S. Lu Y.P. Zhen R.G. Szczypka M. Thiele D.J. Rea P.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 42-47Crossref PubMed Scopus (507) Google Scholar, 7Ortiz D.F. Kreppel L. Speiser D.M. Scheel G. McDonald G. Ow D.W. EMBO J. 1992; 11: 3491-3499Crossref PubMed Scopus (340) Google Scholar, 8Ghosh M. Shen J. Rosen B.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5001-5006Crossref PubMed Scopus (345) Google Scholar, 9Hanikenne M. Matagne R.F. Loppes R. FEMS Microbiol. Lett. 2001; 196: 107-111Crossref PubMed Google Scholar). The ABC transporter family is one of the largest families of membrane proteins. Although 60 ABC transporter family members are known in Caenorhabditis elegans, 49 in humans, 57 in Drosophila, 103 in Arabidopsis, 30 in Saccharomyces cerevisiae, and 11 in Schizosaccharomyces pombe (10Iwaki T. Giga-Hama Y. Takegawa K. Microbiology. 2006; 152: 2309-2321Crossref PubMed Scopus (35) Google Scholar, 11Sheps J.A. Ralph S. Zhao Z. Baillie D.L. Ling V. Genome Biol. 2004; 5: R15Crossref PubMed Google Scholar, 12Sanchez-Fernandez R. Davies T.G.E. Coleman J.O.D. Rea P.A. J. Biol. Chem. 2001; 276: 30231-30244Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar, 13Rea P.A. Annu. Rev. Plant Biol. 2007; 58: 347-375Crossref PubMed Scopus (377) Google Scholar), the exact role played by the many that are implicated in heavy metal detoxification remains to be determined. What is known is that ABC transporters mediate the Mg·ATP-energized transmembrane transport of a wide range of substrates, reside on different cellular membranes, and, although functionally diverse, share a common architecture. Canonical, "full-molecule" ABC transporters consist of four domains: two transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs) that contain the Walker A and B boxes and the ABC signature motif (14Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3386) Google Scholar). "Half-molecule" ABC transporters contain a single TMD and NBD. Some members of either the full- or half-molecule subfamilies of ABC proteins possess a hydrophobic N-terminal extension (NTE). The NTE encompasses five to six transmembrane spans (the TMD0 domain) and a cytosolic linker sequence (L0) contiguous with the TMD or NBD (5Vatamaniuk O.K. Bucher E.A. Sundaram M.V. Rea P.A. J. Biol. Chem. 2005; 280: 23684-23690Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 13Rea P.A. Annu. Rev. Plant Biol. 2007; 58: 347-375Crossref PubMed Scopus (377) Google Scholar). Among ABC transporters, the structure of HMT-1 (heavy metal tolerance factor 1) proteins is unique: they are the only half-molecule ABC proteins with an NTE. This domain organization of HMT-1 proteins is conserved across species and distinguishes the HMT-1 protein subfamily from other members of ABC transporter superfamily (5Vatamaniuk O.K. Bucher E.A. Sundaram M.V. Rea P.A. J. Biol. Chem. 2005; 280: 23684-23690Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 7Ortiz D.F. Kreppel L. Speiser D.M. Scheel G. McDonald G. Ow D.W. EMBO J. 1992; 11: 3491-3499Crossref PubMed Scopus (340) Google Scholar, 9Hanikenne M. Matagne R.F. Loppes R. FEMS Microbiol. Lett. 2001; 196: 107-111Crossref PubMed Google Scholar). The first HMT-1-like protein identified, SpHMT-1, was isolated from S. pombe in mutant screens for genes involved in phytochelatin (PC)-mediated heavy metal tolerance (7Ortiz D.F. Kreppel L. Speiser D.M. Scheel G. McDonald G. Ow D.W. EMBO J. 1992; 11: 3491-3499Crossref PubMed Scopus (340) Google Scholar). PCs are small, cysteine-rich peptides with the general structure γ-(EC)nXaa, where n = 2–11. PCs are synthesized in the presence of heavy metals from glutathione (GSH) and related thiols by PC synthases (PCS), bind heavy metals with high affinity, and facilitate heavy metal sequestration into the vacuole, a lysosome-like compartment of plant and fungal cells (15Zenk M.H. Gene (Amst.). 1996; 179: 21-30Crossref PubMed Scopus (912) Google Scholar, 16Grill E. Laffler S. Winnacker E.-L. Zenk M.H. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6838-6842Crossref PubMed Google Scholar). It has been suggested that, in plants, metal·PC complexes are transported into vacuoles by unidentified ABC transporter(s) (17Salt D.E. Rauser W.E. Plant Physiol. 1995; 107: 1293-1301Crossref PubMed Scopus (331) Google Scholar). Based on in vitro transport assays, it has been proposed that, in S. pombe, SpHMT-1 is a vacuolar membrane Cd·PC and/or apoPC transporter that functions downstream of PC formation in the PCS-dependent pathway (18Ortiz D.F. Ruscitti T. McCue K.F. Ow D.W. J. Biol. Chem. 1995; 270: 4721-4728Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar). However, it remains to be determined directly if the transport of PCs is the mechanism by which SpHMT-1 alleviates Cd2+ toxicity in vivo. Indeed, several considerations indicate that the function of HMT-1 in metal detoxification is more complex than previously thought. First, studies of the HMT-1-like protein from C. elegans, CeHMT-1, yielded findings that suggest an alternate and/or auxiliary role for HMT-1 in heavy metal detoxification, which is not obligatorily dependent on the upstream synthesis of PCs (5Vatamaniuk O.K. Bucher E.A. Sundaram M.V. Rea P.A. J. Biol. Chem. 2005; 280: 23684-23690Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Second, the Chlamydomonas reinhardtii SpHMT-1 homolog, CrCDS1, confers heavy metal tolerance, yet localizes to the mitochondrion, an organelle that does not directly participate in intracellular Cd·PC sequestration (9Hanikenne M. Matagne R.F. Loppes R. FEMS Microbiol. Lett. 2001; 196: 107-111Crossref PubMed Google Scholar). Third, genes encoding HMT-1 homologs have not been detected in the genomes of vascular plants, which utilize the PC-dependent pathway (12Sanchez-Fernandez R. Davies T.G.E. Coleman J.O.D. Rea P.A. J. Biol. Chem. 2001; 276: 30231-30244Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar, 13Rea P.A. Annu. Rev. Plant Biol. 2007; 58: 347-375Crossref PubMed Scopus (377) Google Scholar). Fourth, ABC transporters with an HMT-1-type domain organization have been identified in the genomes of organisms that do not have PCS genes. Examples are the fly, Drosophila melanogaster, HMT-1 (DmHMT-1 alias CG4225), and its counterparts in mammals, including human MTABC3 and mouse ABCB6 (5Vatamaniuk O.K. Bucher E.A. Sundaram M.V. Rea P.A. J. Biol. Chem. 2005; 280: 23684-23690Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Evidently, these HMT-1 proteins do not ordinarily transport metal·PC complexes and/or apoPCs, because these are substances they would never encounter in vivo. To further our understanding of the role of HMT-1-type transporters, we have sought to determine whether the HMT-1 from Drosophila is involved in metal detoxification. If it is, this would imply that HMT-1 proteins from different species share a conserved role in heavy metal detoxification, but one that does not depend on the synthesis of PCs. The results presented here establish the need for revision of the role that HMT-1 proteins have been considered to play in the detoxification of heavy metals, invoke a specific requirement for HMT-1 of S. pombe in the detoxification of Cd2+, but not other heavy metals while at the same time explain why some of the organisms that engage in PC-dependent metal detoxification lack strict HMT-1 homologs. Yeast Strains and Growth Conditions—The S. pombe strains used in these studies were the wild type strain YF016 (h- leu 1–32, ura 4-C190T ade7::ura4) and its isogenic hmt-1Δ mutant (h- leu 1–32, ura 4-C190T ade7::ura4; hmt-1::URA4) (19Iwaki T. Goa T. Tanaka N. Takegawa K. Mol. Genet. Genomics. 2004; 271: 197-207Crossref PubMed Scopus (31) Google Scholar), the wild type strain Sp286 (h+/h+ ade6-M210/ade6-M216 ura4-D18/ura4-D18 leu1–32/leu1–32) and its isogenic pcs-1Δ mutant (h+/h+ ade6-M210/ade6-M216). Cells were grown at 30 °C in Edinburgh minimal medium (EMM), which in addition to leucine, adenine (225 mg/liter each), 2% (w/v) dextrose, and in the case of YF016 and Sp286 cells, uracil (225 mg/liter), contained: 14.7 mm potassium hydrogen phthalate, 15.5 mm Na2HPO4, 93.5 mm NH4Cl, 0.26 m MgCl2·6H2O, 4.99 mm CaCl2·2H2O, 0.67 m KCl, 14.l mm Na2SO4, 80.9 mm boric acid, 23.7 mm MnSO4, 13.9 mm ZnSO4·7H2O, 7.4 mm FeCl2·6H2O, 2.47 mm molybdic acid, 6.02 mm KI, 1.60 mm CuSO4·5H2O, 47.6 mm citric acid, 4.20 mm pantothenic acid, 8l.2 mm nicotinic acid, 55.5 mm inositol, and 40.8 μm biotin. S. pombe transformants were selected for leucine prototrophy in EMM. For the assessment of Cd2+ tolerance, the EMM growth media were supplemented with CdCl2 at the concentrations indicated. Isolation and Heterologous Expression of dm-hmt-1—The cDNA corresponding to dm-hmt-1 was obtained from the Drosophila Genomics Resource Center, Indiana University, Bloomington, IN. Primers for amplification of the open reading frame for dm-hmt-1 were designed to generate Xho1 and Not1 restriction sites at the 5′- and 3′-termini, respectively, of the dm-hmt-1 amplification product. The sequences of the two primers yielding the 2.6-kb dm-hmt-1 amplification product were 5′-CGGCTCGAGATGCTGTACTGCCCGCCCAACG-3′ and 5′-ATAGTTTAGCGGCCGCCTAGCGTGCTCCCCCA-3′. The resulting cDNA (GenBank™ accession number ACE60575) was subcloned into the Xho1 and Not1 restriction sites of the S. pombe-Escherichia coli shuttle vector, pTN197 (19Iwaki T. Goa T. Tanaka N. Takegawa K. Mol. Genet. Genomics. 2004; 271: 197-207Crossref PubMed Scopus (31) Google Scholar), to place dm-hmt-1 under the control of the thiamine-repressible promoter of the nmt1 gene. The resulting pTN197-dm-hmt-1 construct, or pTN197 vector lacking the dm-hmt-1 insert, was expressed in S. pombe hmt-1Δ cells. To permit direct comparisons with isogenic wild-type YF016 cells grown under identical conditions, the pTN197 vector was expressed in YF016 cells. Transformation of S. pombe—S. pombe cells were transformed using a standard lithium acetate procedure (20Okazaki K. Okazaki N. Kume K. Jinno S. Tanaka K. Okayama H. Nucleic Acids Res. 1990; 18: 6485-6489Crossref PubMed Scopus (391) Google Scholar). Transformed cells were selected for leucine prototrophy in EMM medium as described above. Isolation of Intact Vacuoles—For the isolation of intact vacuoles, YF016/pTN197, hmt-1Δ/pTN197, or hmt-1Δ/DmHMT-1 cells were subjected to cell wall digestion, disruption, and fractionation by differential centrifugation. 200-ml volumes of stationary phase cultures were diluted into 1.5 liters of EMM medium containing supplements and grown for 4–6 h at 30 °C to an A600 nm of ∼0.6 after which time CdCl2 (500 μm) was added to the cultures to activate PC production. Thereafter the cells were cultured in the presence of CdCl2 for an additional 18 h at 30 °C to an A600 of ∼1.2, collected by centrifugation, and used for the isolation of intact vacuoles by a modification of the procedure described in Ref. 18Ortiz D.F. Ruscitti T. McCue K.F. Ow D.W. J. Biol. Chem. 1995; 270: 4721-4728Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar. Briefly, the sedimented cells were washed in water and harvested by centrifugation at 3,000 × g for 5 min. After resuspension in 20 mm 2-mercaptoethanol and 100 mm Tris-HCl (pH 9.4), the cells were incubated for 20 min at 30 °C with gentle shaking. The cells were then pelleted, resuspended in 100 ml of digestion medium containing 1.2 m sorbitol, 10 mm 2-mercaptoethanol, 20 mm potassium phosphate, pH 7.5, and converted to spheroplasts by the addition of 50 mg of Zymolyase 20T (ICN) and 100 mg of lysing enzymes from Trichoderma harzianum (Sigma-Aldrich). The suspension was incubated for 2 h at 30 °C with gentle shaking and pelleted by centrifugation at 3,000 × g for 5 min. The spheroplasts were washed free of digestion medium resuspension in 50 ml of ice-cold homogenization medium (HM) consisting of 1.6 m sorbitol, 10 mm MES-Tris (pH 6.9), 0.5 mm MgCl2, 5 mm 2-mercaptoethanol, 1 mm phenylmethylsulfonyl fluoride, and 1 μg/ml each of leupeptin, aprotinin, and pepstatin. The pelleted spheroplasts were lysed in the same medium (20 ml) by homogenization in a 50-ml glass Dounce homogenizer. The crude lysate was cleared of cell debris and unbroken cells by centrifugation at 4,000 × g at 4 °C for 10 min. The pellet was resuspended in another 20 ml of homogenization medium, homogenized again, and recentrifuged. Partially purified vacuoles (the P13,000 fraction) were collected by centrifugation of the supernatant at 13,000 × g at 4 °C for 30 min, resuspended in HM, layered onto a Percoll step gradient (18%/30% (v/v) prepared in HM), and pelleted at 68,320 × gat4°C for 1 h. The resulting vacuolar pellet was resuspended in HM and layered on a cushion of 50% (v/v) of Percoll, prepared in HM and re-pelleted at 68,320 × g for 1 h. The pellet containing purified vacuoles was washed free of Percoll by three rounds of resuspension in suspension medium containing 1.6 m sorbitol, 100 mm KCl, 10 mm MES-Tris, pH 6.9, 5 mm MgCl2, and protease inhibitors, and centrifuged at 4 °C at 13,000 rpm for 10 min in an Eppendorf microcentrifuge. The final vacuolar preparation was used immediately or stored at -80 °C. Assessment of Integrity of Vacuole Preparations—The integrity of the vacuoles prepared in this way was assessed by testing their ability to retain the fluorescent glutathione S-conjugate of monochlorobimane (MCB), bimane-GS (6Li Z.S. Lu Y.P. Zhen R.G. Szczypka M. Thiele D.J. Rea P.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 42-47Crossref PubMed Scopus (507) Google Scholar, 21Sarry J.E. Chen S. Collum R.P. Liang S. Peng M. Lang A. Naumann B. Dzierszinski F. Yuan C.X. Hippler M. Rea P.A. FEBS J. 2007; 274: 4287-4305Crossref PubMed Scopus (28) Google Scholar). MCB is a membrane-permeant, non-fluorescent compound that is specifically conjugated with GSH by cytosolic glutathione S-transferases to generate the intensely fluorescent, membrane-impermeant product bimane-GS that is actively transported into and sequestered within the vacuole of intact cells. 75-ml volumes of stationary phase S. pombe cell cultures were diluted into 500 ml of EMM medium containing MCB (150 μm) and grown for 20 h at 30 °C, after which time the cells were harvested and converted to spheroplasts for the purification of intact vacuoles as described above. Cells, spheroplasts, and vacuoles from this source were examined without fixation by fluorescent microscopy. Enzyme Assays—The purity of the vacuolar fractions was evaluated by marker enzyme assays. α-Mannosidase activity, a vacuolar membrane marker, was employed to enumerate enrichment of the partially purified (P13,000) and final vacuolar fractions. Cytochrome c oxidase and glucose-6-phosphate dehydrogenase activity were employed to assess contamination of these fractions with mitochondria and cytosolic components, respectively. α-Mannosidase was determined using p-nitrophenyl-α-d-mannopyranoside as substrate (22Roberts C.J. Raymond C.K. Yamashiro C.T. Stevens T.H. Methods Enzymol. 1991; 194: 644-661Crossref PubMed Scopus (287) Google Scholar). Glucose-6-phosphate dehydrogenase was assayed by measuring the rate of glucose-6-phosphate-dependent NADPH formation (22Roberts C.J. Raymond C.K. Yamashiro C.T. Stevens T.H. Methods Enzymol. 1991; 194: 644-661Crossref PubMed Scopus (287) Google Scholar). The activity of cytochrome c oxidase activity was assayed using a colorimetric assay based on the decrease in absorbance of ferrocytochrome c caused by its oxidation to ferricytochrome c by cytochrome c oxidase (23Poyton R.O. Goehring B. Droste M. Sevarino K.A. Allen L.A. Zhao X.J. Methods Enzymol. 1995; 260: 97-116Crossref PubMed Scopus (51) Google Scholar). Measurement of PC Content—The PC contents of the isolated intact vacuole preparations were estimated by a combination of reverse-phase (RP)-HPLC and thiol quantitation after reaction with Ellman reagent (24Vatamaniuk O.K. Mari S. Lu Y.P. Rea P.A. J. Biol. Chem. 2000; 275: 31451-31459Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). Aliquots of vacuoles (10–20 μg of protein) were made 5% (w/v) with 5-sulfosalicylic acid, protein was pelleted by centrifugation, and aliquots of the supernatant (50 μl) were loaded onto an Econosphere C18, 150 × 4.6-mm RP-HPLC column (Alltech). The column was developed with a linear gradient of water/0.05% (v/v) phosphoric acid, 17% (v/v) acetonitrile/0.05% (v/v) phosphoric acid at a flow rate of 1 ml/min. For the quantitation of PCs, thiols were estimated spectrophotometrically at 412 nm by reacting aliquots (500 μl) of the column fractions with 0.8 mm 5,5′-dithiobis(2-nitrobenzoic acid) (500 μl) dissolved in 250 mm phosphate buffer, pH 7.6 (25Ellman G.L. Arch. Biochem. Biophys. 1959; 82: 70-77Crossref PubMed Scopus (21624) Google Scholar). Calibration was with GSH. Individual PC fractions were identified on the basis of their co-migration with PC standards synthesized in vitro by purified AtPCS1-FLAG (26Vatamaniuk O.K. Mari S. Lu Y.P. Rea P.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7110-7115Crossref PubMed Scopus (340) Google Scholar) and by mass spectrometry as described below. ESI-MS and Tandem LC-MALDI-MS—Intact vacuoles were subjected to LC-MALDI-MS and ESI-MS analyses for the identification of PCs. The MALDI analysis utilized an LC Packings UltiMate nano-LC system. Mobile phase A consisted of 0.1% trifluoroacetic acid in water, and the mobile phase B consisted of 0.1% (w/v) trifluoroacetic acid in 80% acetonitrile (v/v). Injections (6.4 μl) of the PC samples dissolved in 0.1% trifluoroacetic acid were loaded for 5 min onto a trapping column (C18 Pep-Map100, 300 μmID × 5 mm, 5-μm particle size, 100-Å pore size) in 20 μl/min mobile phase A. Thereafter, a linear 40-min gradient of 0–55% B was directed through the trap column onto an analytical column (C18 PepMap 100, 75 μm inner diameter × 15 cm, 3-μm particle size) at a flow rate of 250 nl/min. The column effluent was directed to an LC Packings Probot fraction collector where it was mixed at a constant 705 nl/min flow rate with 7.5 mg/ml α-cyano-4-hydroxycinammic acid and 20 fmol/μl [Glu]1-fibrinopeptide B dissolved in 2% (w/w) ammonium citrate. This mixture was spotted onto MALDI plates at 20-s collections/fraction. The MALDI plates were analyzed in an Applied Biosystems/MDX Sciex 4700 MALDI TOF/TOF Proteomics Analyzer operated in positive ion mode. An m/z range from 400 to 4000 was scanned for each fraction with internal calibration at an m/z of 1570.677 corresponding to the GluFib added to the matrix solution. PCs were detected by integrating all fractions for representative m/z values to produce ion current chromatograms across each plate. PC2 in the vacuolar extracts was identified as an m/z 540.2 species (theoretical mean isotopic mass [M+H]+ of PC2 (γ-Gly-Cys)2-Gly is 540.14). The ESI analyses were performed using a PerkinElmer 200 micro LC system. Mobile phase A consisted of 0.1% (v/v) formic acid in water, and mobile phase B consisted of 0.1% formic acid in 90% (w/v) acetonitrile. After injection of a 10-μl sample, the column (Vydac C18 MassSpec, 1-mm inner diameter × 150 mm, 5-μm particle size, 300-Å pore size) was developed with 100% A (0–2 min); a linear gradient to 60% B (2–30 min); and 60% B (30–35 min) at a flow rate of 100 μl/min. The column effluent was directed to an Applied Biosystems/MDX Sciex API 150 single quadrupole mass spectrometer with a turbo ion spray source. The instrument was operated in positive ion mode with the settings optimized for GSH by scanning in the m/z range 300–1600. PCs were detected by extracting the ion currents for their representative m/z values from the total ion current for each separation. The presence of PC3 and PC4 was inferred from [M+H]+ m/z ratios of 772.2 and 1004.2, respectively (theoretical mean isotopic mass [M+H]+ of PC3 (γ-Gly-Cys)3-Gly is 772.19; that of PC4 (γ-Gly-Cys)4-Gly is 1004.2). Analyses of Cadmium Content of Isolated Intact Vacuoles by ICP-AES—Aliquots of the intact vacuole fractions (70 μl) were placed in 20.0-ml quartz tubes and digested with 0.25 ml of a 50/50 mixture of concentrated nitric acid and perchloric acid at 120 °C until dry before a second 0.25 ml of a 50/50 mixture of concentrated nitric acid, and perchloric acid was added and heated at 220 °C until dry. The ash was dissolved in 15.0 ml of 2% nitric acid and analyzed on an axially viewed ICP trace analyzer emission spectrometer (model ICAP 61E trace analyzer, Thermo Electron, Waltham, MA). To minimize matrix effects, short depth of field optics were employed (U.S. Patent No. 6,122050). Protein Estimations—Protein was estimated by using the dye-binding method (27Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217548) Google Scholar). Chemicals—All of the general reagents were obtained from Fisher, Research Organics, Inc., Invitrogen, or Sigma-Aldrich. Identification and Cloning of dm-hmt-1—dm-hmt-1 (Drosophila melanogaster heavy metal tolerance factor 1 (GenBank™ accession number ACE60575) encoding a 97.9-kDa polypeptide, DmHMT-1, was identified by systematic domain comparisons among the half-molecule ABC transporters in sequence databases by scanning for proteins with an HMT-1-specific organization: the presence of a single TMD, containing six transmembrane spans and an NBF, containing Walker A and B boxes (sequences GPSGAGKS and IVLLD, respectively), separated by an ABC signature motif (sequence LSGGEKQRVAIARTL), and an NTE consisting of an ∼200-residue TMD0 domain containing five hydrophilicity minima and an ∼50 residue L0 domain. DmHMT-1, which shares 55% sequence similarity (37% sequence identity) to SpHMT-1 and 57% sequence similarity (44% sequence identity) to CeHMT-1, possesses a 244-amino acid residue NTE, consisting of a 192-amino acid TMD0 encompassing five hydrophilicity minima and a 54-amino acid residue L0 domain, oriented in tandem with the TMD and NBF domains (Fig. 1). The presence of the NTE distinguishes DmHMT-1 from its closest homologs, the ATMs (ABC transporters of the mitochondrion), which possess a mitochondrial-targeting signal peptide instead of an NTE domain, are implicated in iron homeostasis, and localize to the inner-mitochondrial membrane (28Lill R. Kispal G. Res. Microbiol. 2001; 152: 331-340Crossref PubMed Scopus (63) Google Scholar). Phylogenetic analysis of the sequences of representative HMT-1 and ATM subfamily members from yeast, Arabidopsis, C. elegans, Drosophila, and mammals demonstrated that the HMTs form a common subcluster, distinct from that of the ATMs (Fig. 2). As is evident from Fig. 2, DmHMT-1, CeHMT-1, and MTABC3 group together within the HMT-1 subcluster, but are distinct from SpHMT-1, which might imply evolutionary and possibly functional divergence of the former three HMTs from SpHMT1. The cDNA corresponding to the predicted open reading frame of dm-hmt-1 was isolated by PCR from a cDNA clone obtained from the Drosophila Genomics Resource Center. After confirming the fidelity of the 2.6-kb amplification product by sequencing, it was used for the experiments described below. Heterologously Expressed DmHMT-1 Partially Suppresses the Cd2+ Hypersensitivity of S. pombe hmt-1Δ Mutants—All HMT-1-like proteins characterized to date have been isolated from organisms possessing PC synthase genes and have been shown to contribute to the alleviation of Cd2+ toxicity. Based on studies in S. pombe, they have been implicated in the vacuolysosomal sequestration of Cd·PC complexes. Because the Drosophila genome does not possess PC synthase homologs, the question of whether DmHMT-1 confers heavy metal tolerance was intriguing. If dm-hmt-1 encodes a protein that is functionally equivalent to or has significant functional overlap with SpHMT-1, its heterologous expression in a Cd2+-hypersensitive S. pombe hmt-1 mutant strain (hmt-1Δ) should alleviate the hypersen
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