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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
Institutes of 1Pharmacology and Toxicology and 2Neuropathology, University of Göttingen, Göttingen; 3Department of Biochemistry, Faculty of Chemistry, University of Kaiserslautern, Kaiserslautern, Germany; and 4Fraunhofer-Institute of Toxicology and Experimental Medicine, Hannover, Germany
Submitted 8 December 2006 ; accepted in final form 21 December 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIAL AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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LoVo; rat hepatocytes; lysosomes; endosomes; glycosylation; copper tolerance
In an attempt to identify novel ABC transporter genes expressed in rat hepatocytes, we previously isolated a cDNA comprising the full open-reading frame of a novel ABC half-transporter. Since corresponding mRNA was found to be expressed in all rat tissues examined (lung, testis, spleen, intestine, skeletal muscle, brain, kidney, liver, and heart), the transporter was termed Umat (ubiquitously expressed mammalian ABC half-transporter) (20). Particularly high half-transporter mRNA expression was observed in rat testis (20). The corresponding human half-transporter (31), cloned on the basis of a partial cDNA sequence (1), has been assigned the symbol ABCB6, in accordance with the human genome nomenclature committee. Since the deduced rat and human protein sequences share 88% amino acid identity, they are presumed to represent orthologs. Thus the rat gene is further referred to as rat Abcb6 (rAbcb6). Interestingly, a variant sequence of rAbcb6 was isolated as shown in Ref. 12 and was demonstrated to be overexpressed during hepatocarcinogenesis (12).
Several extensively characterized ABC transporters, such as the human MDR proteins, are primarily found in the plasma membrane. Nevertheless, many other ABC transporters are situated in intracellular membranes or membranous networks surrounding intracellular compartments. Comparison of rAbcb6 to other ABC transporters revealed that rAbcb6 is related to the fission yeast heavy metal tolerance factor 1 (HMT1) half-transporter protein (34), with 44% identity over a 624-amino acid overlap. HMT1 was found to reside in the Schizosaccharomyces pombe vacuolar membrane and to confer tolerance toward heavy metal toxicity, presumably by sequestration of metal complexes with thiol-bearing peptides (phytochelatins) into the vacuolar interior (34, 35). Caenorhabditis elegans HMT-1 (CeHMT-1) (40) represents a further protein related to rAbcb6, with 55% identity over a 591-amino acid overlap. Recently, expression of the CeHMT-1 gene has been shown to be required for cadmium tolerance in the nematode Caenorhabditis elegans, indicating that comparable mechanisms of alleviating heavy metal toxicity exist in fission yeast, as well as in animals (40). Finally, rAbcb6 exhibits sequence similarity to the human ABCB7 protein (9), with 41% identity over a 617-amino acid overlap, and to the ABC transporter in mitochondria 1 (Atm1p) half-transporter found in Saccharomyces cerevisiae (27), with 39% identity over a 612-amino acid overlap. Human ABCB7 and yeast Atm1p have been localized to mitochondria (9, 27) and appear to be involved in assembly of cytosolic iron-sulfur cluster-containing proteins (4, 22). Mutation of the human ABCB7 gene has been shown to be responsible for X-linked sideroblastic anemia and cerebellar ataxia (2). Taken together, although rAbcb6-related half-transporters in other species appear to be situated primarily intracellularly, the precise distribution differs between individual transporters.
A major aim of the present study was to clarify the subcellular distribution of rAbcb6. The knowledge of rAbcb6 localization would be expected to provide an important basis for further resolution of its physiological function.
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MATERIAL AND METHODS |
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TOPABSTRACTMATERIAL AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Messenger RNA expression analyses. Total RNA was isolated from cultured rat hepatocytes or human cell lines, according to Ref. 8. For Northern blot analyses, 20 µg of total RNA per lane were separated through 1% formaldehyde/agarose gels. To check for equal loading of lanes, gels were stained with ethidium bromide. RNA was blotted onto Hybond N membranes (GE Healthcare, Freiburg, Germany), and the resulting blots were hybridized to a rAbcb6-/human ABCB6-specific 32P-labeled oligonucleotide probe, as described previously (43). The oligonucleotide probe (5'-GGATGCAGCCAGAGCTGATG-3') (20) corresponds to a sequence conserved in both rAbcb6 and human ABCB6 mRNA.
For detection of rAbcb6 and human ABCB6 mRNA expression by reverse transcription (RT)-PCR, 1 µg of total cellular LoVo RNA was subjected to RT and subsequent PCR of cDNA using the Titan One Tube RT-PCR Kit (Roche, Mannheim, Germany) and the following primers specific for human ABCB6 mRNA at 0.5 µM each: 5'-CAGCGGCTACGTGAGCCA-3' (hB6for); 5'-GCCGTTCCATGGTCTGAGGCTTA-3' (hB6rev). RT was performed at 50°C for 30 min and terminated at 94°C for 2 min. Cycling conditions for PCR were as follows: denaturation at 94°C for 15 s, annealing at 60°C for 30 s, and elongation at 68°C for 2.5 min for 10 cycles, followed by 25 cycles of denaturation at 94°C for 15 s, annealing at 60°C for 30 s, and elongation at 68°C for 2.5 min + 5 s/cycle. Final extension was performed at 68°C for 7 min.
rAbcb6- and organelle marker-expression plasmids. A PCR product representing the entire coding sequence of rAbcb6 cDNA, but lacking the stop codon, was cloned into the TOPO TA cloning site of the pcDNA3.1/V5/His vector (Invitrogen, Karlsruhe, Germany), in frame with the COOH-terminal V5 epitope and the polyhistidine tag encoding region, to generate the expression vector pcDNA3.1/rAbcb6/V5/His. rAbcb6 cDNA containing the native stop codon was cloned into the same recipient vector to obtain an expression vector for rAbcb6 without an epitope tag (pcDNA3.1/rAbcb6). Alternatively, rAbcb6 cDNA was amplified with primers introducing restriction enzyme cleavage sites and, following digestion, was ligated into pEGFP-N1 (BD Biosciences, Palo Alto, CA), leading to the expression plasmid prAbcb6-enhanced green fluorescent protein (EGFP). The organelle marker plasmid pDsRed-Mito, yielding a DsRed fusion protein directed to mitochondria via the targeting sequence of cytochrome-c oxidase subunit VIII, was constructed as described previously (28). The plasmid pDsRed-Lyso, which led to expression of DsRed targeted to lysosomes/late endosomes, was constructed accordingly (28) and yielded a protein (Lamp1-DsRed) comprising the rat lysosome-associated membrane protein (Lamp) 1 [including the cytoplasmic tail with the late endosomal/lysosomal targeting sequence (32)] fused to COOH-terminal DsRed.
Transfection of LoVo cells.
LoVo cells, 
50% confluent, were transiently transfected with pcDNA3.1/rAbcb6/V5/His or transfected with control vector without rAbcb6 cDNA insert, employing the SuperFect transfection reagent (Qiagen, Hilden, Germany), according to the manufacturer's protocol. In experiments involving transient LoVo transfection with plasmids yielding fluorescent proteins, the Fugene reagent (Roche) was used, as described previously (36). For stable transfection, LoVo cells were transfected with the pcDNA3.1/rAbcb6/V5/His construct or with the corresponding plasmid yielding rAbcb6 protein without a tag, employing the Effectene transfection reagent (Qiagen). Stably transfected cells were selected and maintained with 300 µg/ml G418 (Calbiochem, Merck Biosciences, Darmstadt, Germany) added to the growth medium. A construct bearing the β-galactosidase gene in-frame with the V5/His epitope coding region (pcDNA3.1/V5/His-TOPO/lacZ, Invitrogen) was used to generate stably transfected control LoVo cell lines.
Isolation of subcellular fractions.
For isolation of crude membrane fractions from LoVo cells, all steps were performed at 4°C in the presence of 1 mM of the protease inhibitor Pefabloc SC (Roche). Crude membrane fractions were obtained, applying a procedure according to Ref. 39. Cells were homogenized in sucrose buffer TES (20 mM Tris, 1 mM EDTA, 254 mM sucrose, pH 7.4; total volume 20 ml) by 10 strokes in a 30-ml Teflon pestle homogenizer, and differential centrifugation was subsequently performed as outlined in Fig. 1. The postnuclear supernatant resulting from centrifugation at 650 g for 10 min (SN1) was subjected to centrifugation at 10,000 g for 10 min, yielding a pellet enriched in mitochondria (see 
Fig. 3A, fractions 1a and 1b). The resulting supernatant SN2 was further centrifuged at 18,000 g for 30 min. The collected supernatant (SN3) was centrifuged at 48,000 g for 30 min, and the pellet was resuspended in TES (see Fig. 3A, fractions 3a and 3b). The supernatant thereof (SN4) was centrifuged at 205,000 g for 75 min, and the resulting pellet was resuspended in TES (see Fig. 3A, fractions 4a and 4b). The pellet obtained after centrifugation of supernatant SN2 at 18,000 g was resuspended in TES, layered onto a cushion of 20 mM Tris, 1 mM EDTA, 1.12 M sucrose, pH 7.4, and centrifuged at 100,000 g for 1 h. The fraction collected from the interphase was diluted in 10 ml TES and pelleted at 100,000 g for 1 h. The resulting fraction (see Fig. 3A, fractions 2a and 2b), enriched in the lysosomal/late endosomal marker LAMP1, was resuspended in TES. Isolated membrane fractions were stored in liquid nitrogen.
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Immunoblot analysis. Subcellular fractions of LoVo cells were subjected to electrophoresis through SDS-polyacrylamide gels and were transferred to polyvinylidene difluoride membranes (Millipore, Eschborn, Germany) by semidry blotting using a continuous transfer buffer system, as described previously (21). The following primary antibodies for detection of V5-tagged rAbcb6 or organelle markers, respectively, were purchased and used in dilutions indicated in the figure legends: monoclonal anti-V5 (Invitrogen); monoclonal anti-cytochrome-c oxidase subunit IV (20E8-C12, Molecular Probes, Leiden, The Netherlands); monoclonal anti-human LAMP1 (BD Biosciences, San Diego, CA); monoclonal anti-EEA1 (early endosome antigen 1); monoclonal anti-BiP/GRP78 (immunoglobulin heavy chain-binding protein/glucose-regulated protein); monoclonal anti-GM130; monoclonal anti-annexin II; monoclonal anti-PECI (peroxisomal monofunctional delta3, delta2-enoyl-CoA isomerase) (all BD Biosciences). Secondary, peroxidase-conjugated antibodies directed against primary polyclonal antibodies (goat anti-rabbit IgG, Sigma no. A0545, diluted 1:10,000) or against monoclonal mouse antibodies (goat anti-mouse IgG, Sigma no. A9309, diluted 1:4,000) were employed for visualization of immunoreactive proteins in conjunction with the enhanced chemiluminescence detection kit (GE Healthcare).
Antibody against rAbcb6. rAbcb6-specific polyclonal antisera were produced in rabbits by BioScience (Göttingen, Germany), employing the synthetic peptide (CAPGLRPQSYTLHVNEEDQ), which represented a region unique to rAbcb6, but which was extended by an additional cysteine at the NH2-terminus. For affinity purification of antibodies, the rAbcb6-specific peptide was coupled to an N-hydroxysuccinimide-activated sepharose high-performance matrix, packed in HiTrap columns (GE Healthcare), and the peptide-binding antibody was adsorbed and eluted as described in Ref. 7.
Immunoprecipitation.
LoVo cells were harvested 3 days after transient transfection. Following homogenization, samples were cleared of nuclei by centrifugation at 650 g, and the postnuclear fraction was used for immunoprecipitation. Aliquots containing 300 µg protein (in a volume of 50 µl) were incubated with an equal volume of lysis buffer (300 mM NaCl, 100 mM HEPES, 2% Triton X-100, 4 mM Pefabloc, pH 7.4) for 1 h at 4°C. Subsequent to centrifugation of samples at 14,000 g for 30 min, the resulting supernatant was incubated for 2 h at 4°C under agitation, either with the polyclonal antibody against rAbcb6 (1:100) or with the corresponding preimmune serum obtained from the rabbit before immunization (1:100). A suspension of preswollen protein A sepharose (GE Healthcare), containing 3.75 mg sepharose/50 µl, was added to the samples in an equal volume, and precipitation of immune complexes was performed at 4°C for 90 min under agitation. Subsequently, samples were spun for 1 min at 10,000 g, supernatants were discarded, and the sepharose pellet was washed three times with 10 mM NaCl, 50 mM HEPES, 0.1% Triton X-100, pH 7.4; twice with 150 mM NaCl, 50 mM HEPES, 0.1% Triton X-100, pH 7.4; and once with 150 mM NaCl, 50 mM HEPES, 0.1% Triton X-100, 0.1% SDS, pH 7.4. Finally, protein bound to the sepharose was eluted by incubation with 2x Laemmli sample buffer (25) for 5 min at 95°C. Following centrifugation at 10,000 g for 30 s, 10 µl of each supernatant were resolved by SDS-polyacrylamide gel electrophoresis with gels containing 7.5% acrylamide and blotted onto polyvinylidene difluoride membranes, as described above. Immunoprecipitated protein was detected in Western blots using V5 antibody (1: 5,000) as the primary antibody.
Immunofluorescence analyses and fluorescent protein expression analyses.
LoVo cells stably transfected with rAbcb6 or rAbcb6-V5 expression vectors were grown on coverslips to 70% confluence. Primary rat hepatocytes were cultured on coverslips for 2 days. Rat liver tissue sections were prepared as described above. Specimens were washed with PBS and subsequently fixed in prechilled methanol for 10 min at –20°C. All further incubation steps were performed at 20°C. Cells were permeabilized with 0.1% Triton X-100 in PBS for 5 min and blocked with 10% normal goat serum in PBS for 30 min. Specimens were then briefly rinsed in PBS and subsequently incubated for 1 h with the primary antibody, diluted in 0.5% BSA/1.5% normal goat serum in PBS (the dilution being 1:50 to 1:100 for the unpurified peptide-specific antiserum against rAbcb6, 1:10 for the purified rAbcb6 antibody, 1:10 for the monoclonal V5-antibody, 1:25 for monoclonal anti-human-LAMP1, and 1:200–1:300 for monoclonal anti-rat-Lamp1; Stressgen, Ann Arbor, MI). After rinsing in PBS, specimens were incubated for 1 h with appropriate secondary antibodies. Fluorescein isothiocyanate-conjugated affinity-purified anti-rabbit IgG from goat (Roche), diluted 1:500, or Alexa Fluor 488-conjugated goat anti-rabbit IgG (Invitrogen), diluted 1:500–1:2,000, was used for the detection of polyclonal primary antibodies. Tetraethylrhodamine isothiocyanate-conjugated anti-mouse IgG from goat (Sigma no. T6653), diluted 1:50, or Alexa Fluor 546-conjugated goat anti-mouse IgG1 (Invitrogen), diluted 1:2,000, was employed for the detection of primary monoclonal antibodies. After washing in PBS, the specimens were mounted in glycerol or in mounting medium (Vectashield, Vector Laboratories, Burlingame, CA) and examined with a Zeiss fluorescence microscope. Fluorescence of rAbcb6-EGFP and of red fluorescent organelle marker proteins (Mito-DsRed and Lamp1-DsRed) was observed 2–3 days after transfection via fluorescence microscopy in unfixed LoVo cells that had been grown on Lab-Tek II chamber slides (Nalgene Nunc, Kamstrup, Denmark) and transfected with red or green fluorescent protein expression vectors, as described previously (36).
MTT toxicity assay. LoVo cells were seeded into 96-well microtiter plates at 5,000 cells/well. Following preincubation for 24 h, cells were incubated with dilutions of transition metal salts (CuSO4, CoCl2, or ZnSO4) in the culture medium for 48 h. After addition of 25 µl/well of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution in PBS (5 mg/ml) and further incubation for 4 h at 37°C, cells were lysed by addition of 100 µl 20% SDS in 0.02 N HCl for 18 h at 20°C. MTT conversion was subsequently measured in a microplate reader by determining absorption at 550 nm.
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RESULTS |
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TOPABSTRACTMATERIAL AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Detection of rAbcb6-V5 fusion protein in subcellular fractions of transiently transfected LoVo cells.
LoVo cells were used as recipient cells for transient and stable transfection with a vector expressing a rAbcb6 fusion protein (rAbcb6-V5). This fusion protein comprised rAbcb6 extended by a COOH-terminal peptide containing the viral V5 epitope and terminal histidine residues (His6). Employing a commercially available monoclonal antibody directed against the V5-epitope, we were able to probe for the fusion protein in immunoblots of subcellular fractions obtained by differential centrifugation (as outlined in Fig. 1 and in the MATERIAL AND METHODS section). In transiently transfected LoVo cells, substantial rAbcb6-V5 protein expression was observed within 3 days after transfection (Fig. 3A). The rAbcb6-V5 fusion protein exhibited an electrophoretic mobility corresponding to 80–97 kDa (Fig. 3B). No substantial immunoreactivity was found in the postnuclear pellet sedimented at 10,000 g (Fig. 3A, fraction 1b), which was enriched in mitochondria, as demonstrated by the presence of the cytochrome-c oxidase subunit IV (Fig. 3B). However, major immunoreactivity against V5 was observed in the postmitochondrial pellet purified on a sucrose cushion (Fig. 3, A and B, fraction 2b). This fraction was enriched in human LAMP1, a marker of late endosomes and lysosomes, and in annexin II, a protein recruited to endosomal vesicles and to actin assembly sites at the plasma membrane (38) (Fig. 3B). The fractions 3b and 4b had been sedimented at 48,000 g and 205,000 g, respectively, and thus contained lighter particles than fractions 1b and 2b. Nevertheless, only fraction 3b exhibited slight immunoreactivity against the V5-epitope compared with samples of control-transfected cells (Fig. 3A).
To further define the subcellular localization of the epitope-tagged rAbcb6 protein in transiently transfected LoVo cells, enrichment of lysosomes was performed according to Ref. 33, and rAbcb6-V5 immunoreactivity was compared with organelle marker immunoreactivity, using a panel of antibodies against organelle markers (Fig. 4). The rAbcb6-V5 fusion protein was found to be concentrated in the fractions 1 and 4, representing the postmitochondrial supernatant (fraction 1) and the purified lysosomal membrane fraction (fraction 4). However, the mitochondrial marker cytochrome-c oxidase, the peroxisomal marker PECI, the Golgi marker GM130, or the endoplasmic reticulum marker BiP/GRP78 were not coenriched with rAbcb6-V5. Rather, major rAbcb6-V5 immunoreactivity was found in the fractions enriched in EEA1 (a marker of early endosomes), in LAMP1 (lysosomal/late endosomal marker), or in annexin II (Fig. 4).
V5-epitope-tagged rAbcb6 represents an N-glycosylated protein.
In immunoblots of cell fractions obtained from prAbcb6-V5-transfected LoVo cells, we found immunoreactive protein (V5-tagged rAbcb6) to exhibit an electrophoretic mobility corresponding to 80–97 kDa. Immunoreactivity presented either multiple protein bands (Fig. 3A) and/or a smeared broad region (Fig. 4) rather than a single sharply focused band. This is a pattern typically observed with glycosylated proteins (23). We, therefore, examined whether the rAbcb6-V5-fusion protein concentrated in the fraction enriched with lysosomal membranes (Fig. 4, fraction 4) might be N-glycosylated. Digestion with protein N-glycosidase F led to a shift and focusing in gel mobility of the fusion protein (Fig. 5), resulting in a protein band corresponding to an electrophoretic mobility of 77–80 kDa, and most likely representing the unglycosylated core protein. Taking into account that the V5-epitope tag of the rAbcb6 fusion protein would be expected to add up to 5 kDa to the core protein, the electrophoretic mobility of the unglycosylated, untagged rAbcb6 would be estimated to correspond to an even lower molecular mass. This would differ from the molecular mass calculated from the deduced rAbcb6 amino acid sequence, amounting to 93.3 kDa (20). A similar discrepancy has been observed for other ABC half-transporters, e.g., for Abcb9, and may be due to aberrant behavior of partly hydrophobic proteins in an aqueous environment (42). Nevertheless, the deglycosylation experiments support the conclusion that rAbcb6-V5 was indeed expressed in LoVo cells as an N-glycosylated protein.
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160 µM CuSO4 for control LoVo cells and at 210–220 µM CuSO4 for rAbcb6- or rAbcb6-V5-expressing cells (Fig. 10A), resulting in 1.4-fold tolerance toward CuSO4 in the transporter-transfected cells. On the other hand, transfection of rAbcb6/rAbcb6-V5 did not lead to tolerance toward the other transition metal salts CoCl2 (Fig. 10C) or ZnSO4 (data not shown). Thus expression of rAbcb6 or rAbcb6-V5 in the LoVo cell line, which we had found to be devoid of endogenous human ABCB6 mRNA, specifically conferred tolerance toward copper-dependent toxicity.
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DISCUSSION |
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TOPABSTRACTMATERIAL AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Intracellular localization of rAbcb6. The rAbcb6 protein belongs to a subgroup of related ABC half-transporters within the [MDR/TAP (transporter associated with antigen processing)] cluster of ABC proteins, which includes the Saccharomyces cerevisiae Atm1p, human ABCB7, the fission yeast HMT1 protein, and CeHMT-1. Although the members of this group reside in intracellular membranes, the subcellular localization is heterogenous: human ABCB7 and Atm1p are associated with the mitochondrial inner membrane (9, 27), whereas HMT1 is targeted to the vacuolar membrane (34, 35). Heterologous expression of CeHMT-1 in S. pombe revealed a vacuolar membrane localization also for CeHMT-1 (40). Our findings indicating the lysosomal/late endosomal distribution of rAbcb6 are in line with the vacuolar membrane localization of HMT1, since the yeast vacuole is regarded as being analogous to lysosomes in higher eukaryotic cells (41).
Interestingly, previous studies concerning human ABCB6 (also termed MTABC3) have suggested mitochondrial localization (31) or a mitochondrial outer membrane localization (24). However, the results presented for rAbcb6 in the present study challenge the notion that mitochrondria represent the general target organelle for mammalian Abcb6. Subcellular fractionation, as well as fluorescence microscopy of transfected LoVo cells expressing either rAbcb6-V5 or rAbcb6-EGFP fusion proteins, pointed to a vesicular (lysosomal/endosomal) distribution. This was not due to the choice of the epitope tag, since differently tagged rAbcb6 proteins were colocalized with lysosomal/late endosomal markers (human LAMP1/rat Lamp1-DsRed). Moreover, this localization was not confined only to the LoVo cell system (that was devoid of endogenous human ABCB6 expression). Transfected TM3 cells that express endogenous murine Abcb6 displayed a comparable pattern of rAbcb6-EGFP distribution. Finally, a consistent pattern of endogenous rAbcb6 localization (with rat Lamp1 colocalization) was observed in primary rat hepatocyte cultures and in rat liver tissue sections. The presence of putative lysosomal/endosomal sorting sequences (as reviewed by Ref. 6) within the rAbcb6 primary amino acid sequence is in accordance with lysosomal/endosomal distribution. In particular, a tyrosine-based putative sorting signal of the YXXØ type (in which Ø represents an amino acid with a bulky/hydrophobic side chain) resides within the COOH-terminal region of rAbcb6 (YAEM). Its functional relevance, however, remains to be investigated in further studies.
Deglycosylation experiments performed with V5-tagged rAbcb6 support the conclusion that rAbcb6 is an N-glycosylated integral membrane protein. Extensive glycosylation is typical of lysosomal/late endosomal membrane proteins such as the Lamp proteins, and a lysosomal luminal coat of carbohydrates has been proposed to protect lysosomal membrane proteins against degradation by proteases (18). As lysosomes may fuse with different cellular membranes [endosomes, autophagosomes, plasma membrane (30)], it is conceivable that, in the course of membrane trafficking, a fraction of rAbcb6 might appear in the plasma membrane. However, the fluorescence microscopy procedures performed in the present study with fixed and living transfected LoVo cells or with fixed rat hepatocyte/rat liver specimens are in line with a primarily intracellular (vesicular) localization.
Implications for rAbcb6 function. Several rAbcb6-related half-transporters are involved in metal homeostasis or metal tolerance (exemplified by the participation of Atm1p and ABCB7 in maturation of cytosolic Fe/S cluster proteins, or by the contribution of HMT1 or CeHMT-1 to heavy metal tolerance). Considering that HMT1 appears to confer tolerance toward heavy metal toxicity by transporting phytochelatin-metal complexes into the yeast vacuole (34, 35), it is tempting to speculate that rAbcb6, associated with lysosomal/late endosomal membranes, may have a similar function in translocation of transition metal complexes into the interior of acidic vesicles.
Several transition metals (e.g., iron, copper, zinc, cobalt) are required to fulfill essential cellular functions as trace elements. However, in amounts exceeding those to maintain cellular functions, metal-dependent toxicity may occur. The tendency of iron or copper to participate in redox reactions is the basis for their presence in enzymes catalyzing single-electron transfer reactions. However, Fe2+-/Cu+-dependent redox-reactivity may also contribute to the generation of reactive oxygen species. Therefore, homeostasis of these transition metals is connected to redox homeostasis and must be tightly controlled, e.g., by the presence of iron- or copper-binding molecules and by the activity of membrane-situated transporters.
In the present study, we investigated whether expression of rAbcb6 following transfection of LoVo cells, a cell line we found to be devoid of endogenous human ABCB6 mRNA expression, might confer tolerance to particular transition metals. Interestingly, LoVo clones expressing rAbcb6 exhibited tolerance toward copper (applied as CuSO4). By contrast, we were not able to observe a comparable tolerance toward salts of other biologically important transition metals such as zinc (ZnSO4) or cobalt (CoCl2), indicating a rAbcb6-dependent mechanism of specific cellular protection against toxic effects of copper.
On the one hand, tolerance of LoVo cells toward copper might be explained by sequestration of a copper-binding complex, e.g., involving amino acids or peptides, into endosomes/lysosomes. Thus rAbcb6 may participate in intracellular copper trafficking. Numerous reports exist that establish a link between lysosomal function and cellular trafficking/detoxification of copper. Several transition metals, including nickel, palladium, platinum, copper, silver, and gold, are accumulated in lysosomes (5) and may, in part, be detoxified via lysosomes. During copper overload, hepatocellular excretion into the bile via secretory lysosomes appears to represent a major pathway of detoxification (15). Noteworthy, an ATP-dependent transport mechanism specific for copper in the presence of glutathione has been characterized previously on isolated hepatocyte lysosomes (17). However, to our knowledge, the molecular identity of this transporter has not yet been resolved. Known proteins contributing to copper trafficking include Ctr1, which is involved in copper uptake at the plasma membrane (26), and the Wilson disease protein ATP7B, a P-type ATPase, that seems to play a role in biliary copper excretion, possibly by mediating copper transport into the lumen of late endosomes (16). Finally, a novel transporter has been described (Ctr2) that appears to enable transport of copper out of lysosomes (37). Mediation of the translocation of copper or a copper complex into the lysosomal interior (as hypothesized for rAbcb6) would be expected to contribute not only to copper sequestration, but also to the establishment of copper stores, which might be used for mobilization of copper required for the assembly of copper-containing enzymes.
Alternatively, tolerance of LoVo cells toward copper toxicity might reflect a mechanism other than direct transport of a copper complex by rAbcb6. Heterologous expression of human ABCB6 protein in mutant Saccharomyces cerevisiae cells that were partially defective in Atm1p function resulted in reversal of the mutant phenotype (31), leading particularly to an alleviation of mitochondrial iron overload and of mitochondrial DNA damage, implying a role of ABCB6 in iron homeostasis (31). Intriguingly, yeast cells exhibiting disruption of the Atm1 gene have been reported to be more sensitive to copper toxicity (2). Therefore, mitochondrial Atm1p protein and vesicular rAbcb6, although situated in different intracellular compartments, may fulfill related functions. Notably, multiple connections exist between iron and copper metabolism (reviewed by Refs. 3, 13). For example, the plasma cupro-enzyme ceruloplasmin catalyzes the oxidation of ferrous iron to Fe3+ (a prerequisite for iron binding to transferrin), and is thus involved in iron mobilization out of storage compartments, e.g., from the liver. Both iron and copper ions have the ability to participate in one-electron redox reactions. In particular, both Fe2+ and Cu+ may lead to the generation of highly reactive hydroxyl radicals by transformation of hydrogen peroxide via the Fenton reaction and may, therefore, contribute to oxidative stress. Thus rAbcb6 might prevent copper-dependent toxicity indirectly by alteration of iron homeostasis and/or by preventing damage that would result from the iron- or copper-dependent generation of reactive oxygen species.
Further studies are required to clarify the exact nature of rAbcb6 substrates. However, a function in transition metal homeostasis would also imply a pivotal role in the regulation of the cellular redox status. Taken together, results presented in our study demonstrate that rAbcb6 is a glycosylated protein targeted to vesicular membranes belonging to lysosomal or endosomal compartments and are in line with the involvement of rAbcb6 in cellular transition metal homeostasis.
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ACKNOWLEDGMENTS |
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Present address of C. Ziemann: Fraunhofer-Institute of Toxicology and Experimental Medicine, Hannover, Germany.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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-rAbcb6). Immunoprecipitated rAbcb6-V5 was detected in Western blots by employing the antibody directed against the V5-epitope tag.
(C-V5) denote control cells transfected with the control pcDNA3.1/V5/His-TOPO/lacZ expression vector; 
represent cells transfected with the vector expressing native rAbcb6; 
represent cells transfected with the vector expressing the epitope-tagged rAbcb6 (rAbcb6-V5). A: n = 11 (C-V5, rAbcb6), n = 7 (rAbcb6-V5). Significantly different from C-V5 values: *P < 0.05; **P < 0.01; ***P < 0.001; Student's t-test for unpaired values. B: comparison of MTT conversion at 200 µM CuSO4 between nontransfected parental cells (LoVo-P), control-transfected cells (C-V5), and cells transfected with a rAbcb6 expression plasmid (rAbcb6/rAbcb6-V5). n = 3 (LoVo-P); n = 11 (C-V5 and rAbcb6); n = 7 (rAbcb6-V5). ***Significantly different from LoVo-P and C-V5, Student's t-test for unpaired values, P < 0.001. C: comparison of sensitivity toward CoCl2. n = 5 (Co-V5); n = 4 (rAbcb6); n = 3 (rAbcb6-V5).