The Experts below are selected from a list of 8259 Experts worldwide ranked by ideXlab platform
Laura M. Garrick - One of the best experts on this subject based on the ideXlab platform.
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A role for divalent Metal Transporter (DMT1) in mitochondrial uptake of iron and manganese.
Scientific reports, 2018Co-Authors: Natascha A. Wolff, Laura M. Garrick, Michael D Garrick, Andrew J. Ghio, Lin Zhao, Frank ThévenodAbstract:Much of iron and manganese metabolism occurs in mitochondria. Uptake of redox-active iron must be tightly controlled, but little is known about how Metal ions enter mitochondria. Recently, we established that the divalent Metal Transporter 1 (DMT1) is present in the outer mitochondrial membrane (OMM). Therefore we asked if it mediates Fe2+ and Mn2+ influx. Mitochondria were isolated from HEK293 cells permanently transfected with inducible rat DMT1 isoform 1 A/+IRE (HEK293-rDMT1). Fe2+-induced quenching of the dye PhenGreen™SK (PGSK) occurred in two phases, one of which reflected OMM DMT1 with stronger Fe2+ uptake after DMT1 overexpression. DMT1-specific quenching showed an apparent affinity of ~1.5 µM for Fe2+and was blocked by the DMT1 inhibitor CISMBI. Fe2+ influx reflected an imposed proton gradient, a response that was also observed in purified rat kidney cortex (rKC) mitochondria. Non-heme Fe accumulation assayed by ICPOES and stable 57Fe isotope incorporation by ICPMS were increased in HEK293-rDMT1 mitochondria. HEK293-rDMT1 mitochondria displayed higher 59Fe2+ and 54Mn2+ uptake relative to controls with 54Mn2+ uptake blocked by the DMT1 inhibitor XEN602. Such transport was defective in rKC mitochondria with the Belgrade (G185R) mutation. Thus, these results support a role for DMT1 in mitochondrial Fe2+ and Mn2+ acquisition.
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Mitochondria represent another locale for the divalent Metal Transporter 1 (DMT1)
Channels (Austin Tex.), 2014Co-Authors: Natascha A. Wolff, Laura M. Garrick, Michael D Garrick, Lin Zhao, Frank ThévenodAbstract:The divalent Metal Transporter (DMT1) is well known for its roles in duodenal iron absorption across the apical enterocyte membrane, in iron efflux from the endosome during transferrin-dependent cellular iron acquisition, as well as in uptake of non-transferrin bound iron in many cells. Recently, using multiple approaches, we have obtained evidence that the mitochondrial outer membrane is another subcellular locale of DMT1 expression. While iron is of vital importance for mitochondrial energy metabolism, its delivery is likely to be tightly controlled due to iron's damaging redox properties. Here we provide additional support for a role of DMT1 in mitochondrial iron acquisition by immunofluorescence colocalization with mitochondrial markers in cells and isolated mitochondria, as well as flow cytometric quantification of DMT1-positive mitochondria from an inducible expression system. Physiological consequences of mitochondrial DMT1 expression are discussed also in consideration of other DMT1 substrates, su...
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Evidence for mitochondrial localization of divalent Metal Transporter 1 (DMT1)
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 2014Co-Authors: Natascha A. Wolff, Laura M. Garrick, Michael D Garrick, Andrew J. Ghio, Lin Zhao, Robert A. Fenton, Frank ThévenodAbstract:In mammalian cells, mitochondria receive most incoming iron, yet no entry pathway for iron at the outer mitochondrial membrane (OMM) has been characterized. Our results show that the divalent Metal Transporter 1 (DMT1) occurs in the OMM. Immunoblots detected DMT1 in mitochondria from a pneumocyte cell model in their OMM. Using the split-ubiquitin yeast 2-hybrid system, we found that cytochrome c oxidase subunit II (COXII) and the translocase of OMM 6-kDa subunit (Tom6) homologue interact with DMT1. COXII coimmunoprecipitates with DMT1. There are 4 DMT1 isoforms that differ at the N and C termini. Using HEK293 cells that inducibly express all of the 4 ends of DMT1, we found all of them in the OMM, as detected by immunoblots after cell fractionation, and in isolated mitochondria, as detected by immunofluorescence. Immunoblot analysis of purified cell fractions from rat renal cortex confirmed and extended these results to the kidney, which expressed high levels of DMT1. Immunogold labeling detected DMT1 colocalization in mitochondria with the voltage-dependent anion-selective channel protein-1, which is expressed in the OMM. We suggest that DMT1 not only exports iron from endosomes, but also serves to import the Metal into the mitochondria.
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Divalent Metal Transporter 1 (DMT1) contributes to neurodegeneration in animal models of Parkinson's disease.
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Julio Salazar, Lin Zhao, Natalia Mena, Stéphane Hunot, Annick Prigent, Daniel Alvarez-fischer, Miguel Arredondo, Charles Duyckaerts, Véronique Sazdovitch, Laura M. GarrickAbstract:Dopaminergic cell death in the substantia nigra (SN) is central to Parkinson's disease (PD), but the neurodegenerative mechanisms have not been completely elucidated. Iron accumulation in dopaminergic and glial cells in the SN of PD patients may contribute to the generation of oxidative stress, protein aggregation, and neuronal death. The mechanisms involved in iron accumulation also remain unclear. Here, we describe an increase in the expression of an isoform of the divalent Metal Transporter 1 (DMT1/Nramp2/Slc11a2) in the SN of PD patients. Using the PD animal model of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication in mice, we showed that DMT1 expression increases in the ventral mesencephalon of intoxicated animals, concomitant with iron accumulation, oxidative stress, and dopaminergic cell loss. In addition, we report that a mutation in DMT1 that impairs iron transport protects rodents against parkinsonism-inducing neurotoxins MPTP and 6-hydroxydopamine. This study supports a critical role for DMT1 in iron-mediated neurodegeneration in PD.
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comparison of mammalian cell lines expressing distinct isoforms of divalent Metal Transporter 1 in a tetracycline regulated fashion
Biochemical Journal, 2006Co-Authors: Michael D Garrick, Jerome A Roth, Lin Zhao, Prasad N. Paradkar, Hung-chieh Kuo, Steven T. Singleton, Farida Vargas, Jaime J Smith, Laura M. GarrickAbstract:DMT1 (divalent Metal Transporter; also known as SLC11A2, DCT1 or Nramp2) is responsible for ferrous iron uptake in the duodenum, iron exit from endosomes during the transferrin cycle and some transferrin-independent iron uptake in many cells. Four protein isoforms differ by starting in exon 1A or 2 and ending with alternative peptides encoded by mRNA that contains or lacks an IRE (iron responsive element; ±IRE). We have compared 1A/+IRE and 2/−IRE DMT1 during regulated ectopic expression. HEK-293-F (human embryonic kidney-293-fast growing variant) cells were stably transfected with each construct expressed from a tetracycline-regulated CMV promoter. Reverse transcriptase-PCR analysis showed that construct expression responded to doxycycline. Immunofluorescence staining of cells, using antibodies specific for DMT1 isoforms, confirmed an increase in expression in the plasma membrane and cytosolic vesicles after doxycycline treatment, but with isoform specific distributions. Immunoblotting also revealed stimulation of expression. Nevertheless, both DMT1 isoforms performed similarly in assays for functional properties based on 54Mn2+ and 59Fe2+ uptake. Mn incorporation after doxycycline treatment was ∼10-fold greater than that of untreated cells, while expression in the untreated cells was ∼5-fold greater than in the untransfected cells. Uptake of Mn depended on addition of doxycycline, with half maximal response at ∼1 nM doxycycline. Doxycycline-stimulated Mn and Fe uptake was linear with time for 10 min but not over longer periods. Transport exhibited a pH optimum at ∼5.5 and dependence on incubation temperature and Mn or Fe concentration. The new cell lines should prove useful for research on Metal homoeostasis, toxicological studies and efforts to identify distinctive properties of the isoforms.
Michael G. Caparon - One of the best experts on this subject based on the ideXlab platform.
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a perr regulated Metal Transporter pmta is an interface between oxidative stress and Metal homeostasis in streptococcus pyogenes
Molecular Microbiology, 2007Co-Authors: Audrey Brenot, Benjamin F. Weston, Michael G. CaparonAbstract:In Streptococcus pyogenes, mutation of the peroxide sensor PerR results in avirulence despite producing hyper-resistance to peroxide stress. To understand the basis of this effect, global transcription profiling was conducted revealing one highly downregulated gene (czcD), and five highly upregulated genes in the mutant. Of the latter, only pmtA contained a binding site for PerR, while phtY, phtD, lsp and rpsN2 harboured an AdcR motif and were regulated by AdcR, a repressor of an ABC-type Metal Transporter. Furthermore, only the PerR-regulated pmtA (PerR-regulated Metal Transporter A), a putative Metal Transporter, contributed to resistance against peroxide stress, while AdcR and the other AdcR-regulated genes did not. However, overexpression of pmtA resulted in upregulation of several AdcR-regulated genes, suggesting that the AdcR regulon is sensitive to PerR regulation of Metal homeostasis. Finally, examination of S. pyogenes following murine subcutaneous infection revealed that while pmtA was not upregulated in a late infection, the AdcR-regulated genes were. Taken together, these data suggest that PerR has a greater impact on the transcriptome than can be predicted by its binding sites and that pmtA functions to link Metal homeostasis and oxidative stress responses.
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A PerR‐regulated Metal Transporter (PmtA) is an interface between oxidative stress and Metal homeostasis in Streptococcus pyogenes
Molecular microbiology, 2007Co-Authors: Audrey Brenot, Benjamin F. Weston, Michael G. CaparonAbstract:In Streptococcus pyogenes, mutation of the peroxide sensor PerR results in avirulence despite producing hyper-resistance to peroxide stress. To understand the basis of this effect, global transcription profiling was conducted revealing one highly downregulated gene (czcD), and five highly upregulated genes in the mutant. Of the latter, only pmtA contained a binding site for PerR, while phtY, phtD, lsp and rpsN2 harboured an AdcR motif and were regulated by AdcR, a repressor of an ABC-type Metal Transporter. Furthermore, only the PerR-regulated pmtA (PerR-regulated Metal Transporter A), a putative Metal Transporter, contributed to resistance against peroxide stress, while AdcR and the other AdcR-regulated genes did not. However, overexpression of pmtA resulted in upregulation of several AdcR-regulated genes, suggesting that the AdcR regulon is sensitive to PerR regulation of Metal homeostasis. Finally, examination of S. pyogenes following murine subcutaneous infection revealed that while pmtA was not upregulated in a late infection, the AdcR-regulated genes were. Taken together, these data suggest that PerR has a greater impact on the transcriptome than can be predicted by its binding sites and that pmtA functions to link Metal homeostasis and oxidative stress responses.
Craig P. Smith - One of the best experts on this subject based on the ideXlab platform.
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Divalent Metal Transporter 1 in the kidney proximal tubule is expressed in late endosomes/lysosomal membranes: implications for renal handling of protein-Metal complexes
American journal of physiology. Renal physiology, 2006Co-Authors: Marouan Abouhamed, Natascha A. Wolff, Frank Thévenod, Wei Liu, Jakub Gburek, Blazej Torchalski, Andreas Wilhelm, Erik Ilso Christensen, Craig P. SmithAbstract:The H+-coupled polyligand transport protein divalent Metal Transporter 1 (DMT1) plays a key role in mammalian iron homeostasis. It has a widespread pattern of expression including tissues associate...
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divalent Metal Transporter 1 in the kidney proximal tubule is expressed in late endosomes lysosomal membranes implications for renal handling of protein Metal complexes
American Journal of Physiology-renal Physiology, 2006Co-Authors: Marouan Abouhamed, Natascha A. Wolff, Frank Thévenod, Wei Liu, Jakub Gburek, Blazej Torchalski, Andreas Wilhelm, Erik Ilso Christensen, Craig P. SmithAbstract:The H+-coupled polyligand transport protein divalent Metal Transporter 1 (DMT1) plays a key role in mammalian iron homeostasis. It has a widespread pattern of expression including tissues associate...
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Differential expression of divalent Metal Transporter DMT1 (Slc11a2) in the spermatogenic epithelium of the developing and adult rat testis.
American journal of physiology. Cell physiology, 2004Co-Authors: Kathleen P. Griffin, Donald T. Ward, Wei Liu, Gavin Stewart, Ian D. Morris, Craig P. SmithAbstract:Iron is essential for male fertility, and disruptions in iron balance lead to impairment of testicular function. The divalent Metal Transporter DMT1 is a key modulator of transferrin- and non-transferrin-bound iron homeostasis. As a first step in determining the role of DMT1 in the testis, we have characterized the pattern of DMT1 expression in the developing and adult rat testis. Northern blot analysis and RT-PCR of testis polyadenylated RNA revealed the presence of iron-responsive element (IRE) and non-IRE transcripts. Semiquantitative immunoblotting of immature and adult rat testis uncovered the expression of two distinct DMT1 protein species. Immunohistochemistry showed that DMT1 was widespread throughout each seminiferous tubule and was expressed in the intracellular compartment. In the adult rat testis, DMT1 was immunolocalized to both the Sertoli and germ cells. In contrast to the immature testis, expression was dependent on the stage of the spermatogenic cycle. DMT1 was not detected on any plasma membranes in either the developing or the adult testis, suggesting that DMT1 is not primarily responsible for translocating iron across this epithelium. Our data suggest an important role for DMT1 in intracellular iron handling during spermatogenesis and imply that germ cells have a need for a precisely targeted and timed supply of iron. We suggest that DMT1 may, as it does in other tissues, play a role in transporting iron between intracellular compartments and thus may play an important role in male fertility.
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Cellular localization of divalent Metal Transporter DMT-1 in rat kidney
American journal of physiology. Renal physiology, 2001Co-Authors: Carole J. Ferguson, Donald T. Ward, Craig P. Smith, Mark Wareing, R. Green, Daniela RiccardiAbstract:We have demonstrated that the kidney plays an important role in iron balance and that metabolically significant reabsorption of this ion occurs in the loop of Henle and the collecting ducts [Wareing M, Ferguson CJ, Green R, Riccardi D, and Smith CP. J Physiol (Lond) 524: 581-586, 2000]. To test the possibility that the divalent Metal Transporter DMT1 (Gunshin H, Mackenzie B, Berger UV, Gunshin Y, Romero MF, Boron WF, Nussberger S, Gollan JL, and Hediger MA. Nature 388: 482-488, 1997) could represent the apical route for iron entry in the kidney, we raised and affinity-purified an anti-DMT-1 polyclonal antibody and determined DMT-1 distribution in rat kidney by Western analysis, immunofluorescence, and confocal microscopy. The strongest DMT1-specific (i.e., peptide-protectable) immunoreactivity was found in the collecting ducts, in both principal and intercalated cells. Thick ascending limbs of Henle's loop and, more intensely, distal convoluted tubules exhibited apical immunostaining. Considerable intracellular DMT-1 immunoreactivity was seen throughout the nephron, particularly in S3 segments. The described distribution of DMT-1 protein is in agreement with our previous identification of nephron sites of iron reabsorption, suggesting that DMT-1 provides the molecular mechanism for apical iron entry in the distal nephron but not in the proximal tubule. Basolateral iron exit may be facilitated by a different system.
Audrey Brenot - One of the best experts on this subject based on the ideXlab platform.
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a perr regulated Metal Transporter pmta is an interface between oxidative stress and Metal homeostasis in streptococcus pyogenes
Molecular Microbiology, 2007Co-Authors: Audrey Brenot, Benjamin F. Weston, Michael G. CaparonAbstract:In Streptococcus pyogenes, mutation of the peroxide sensor PerR results in avirulence despite producing hyper-resistance to peroxide stress. To understand the basis of this effect, global transcription profiling was conducted revealing one highly downregulated gene (czcD), and five highly upregulated genes in the mutant. Of the latter, only pmtA contained a binding site for PerR, while phtY, phtD, lsp and rpsN2 harboured an AdcR motif and were regulated by AdcR, a repressor of an ABC-type Metal Transporter. Furthermore, only the PerR-regulated pmtA (PerR-regulated Metal Transporter A), a putative Metal Transporter, contributed to resistance against peroxide stress, while AdcR and the other AdcR-regulated genes did not. However, overexpression of pmtA resulted in upregulation of several AdcR-regulated genes, suggesting that the AdcR regulon is sensitive to PerR regulation of Metal homeostasis. Finally, examination of S. pyogenes following murine subcutaneous infection revealed that while pmtA was not upregulated in a late infection, the AdcR-regulated genes were. Taken together, these data suggest that PerR has a greater impact on the transcriptome than can be predicted by its binding sites and that pmtA functions to link Metal homeostasis and oxidative stress responses.
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A PerR‐regulated Metal Transporter (PmtA) is an interface between oxidative stress and Metal homeostasis in Streptococcus pyogenes
Molecular microbiology, 2007Co-Authors: Audrey Brenot, Benjamin F. Weston, Michael G. CaparonAbstract:In Streptococcus pyogenes, mutation of the peroxide sensor PerR results in avirulence despite producing hyper-resistance to peroxide stress. To understand the basis of this effect, global transcription profiling was conducted revealing one highly downregulated gene (czcD), and five highly upregulated genes in the mutant. Of the latter, only pmtA contained a binding site for PerR, while phtY, phtD, lsp and rpsN2 harboured an AdcR motif and were regulated by AdcR, a repressor of an ABC-type Metal Transporter. Furthermore, only the PerR-regulated pmtA (PerR-regulated Metal Transporter A), a putative Metal Transporter, contributed to resistance against peroxide stress, while AdcR and the other AdcR-regulated genes did not. However, overexpression of pmtA resulted in upregulation of several AdcR-regulated genes, suggesting that the AdcR regulon is sensitive to PerR regulation of Metal homeostasis. Finally, examination of S. pyogenes following murine subcutaneous infection revealed that while pmtA was not upregulated in a late infection, the AdcR-regulated genes were. Taken together, these data suggest that PerR has a greater impact on the transcriptome than can be predicted by its binding sites and that pmtA functions to link Metal homeostasis and oxidative stress responses.
Michael D Garrick - One of the best experts on this subject based on the ideXlab platform.
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A role for divalent Metal Transporter (DMT1) in mitochondrial uptake of iron and manganese.
Scientific reports, 2018Co-Authors: Natascha A. Wolff, Laura M. Garrick, Michael D Garrick, Andrew J. Ghio, Lin Zhao, Frank ThévenodAbstract:Much of iron and manganese metabolism occurs in mitochondria. Uptake of redox-active iron must be tightly controlled, but little is known about how Metal ions enter mitochondria. Recently, we established that the divalent Metal Transporter 1 (DMT1) is present in the outer mitochondrial membrane (OMM). Therefore we asked if it mediates Fe2+ and Mn2+ influx. Mitochondria were isolated from HEK293 cells permanently transfected with inducible rat DMT1 isoform 1 A/+IRE (HEK293-rDMT1). Fe2+-induced quenching of the dye PhenGreen™SK (PGSK) occurred in two phases, one of which reflected OMM DMT1 with stronger Fe2+ uptake after DMT1 overexpression. DMT1-specific quenching showed an apparent affinity of ~1.5 µM for Fe2+and was blocked by the DMT1 inhibitor CISMBI. Fe2+ influx reflected an imposed proton gradient, a response that was also observed in purified rat kidney cortex (rKC) mitochondria. Non-heme Fe accumulation assayed by ICPOES and stable 57Fe isotope incorporation by ICPMS were increased in HEK293-rDMT1 mitochondria. HEK293-rDMT1 mitochondria displayed higher 59Fe2+ and 54Mn2+ uptake relative to controls with 54Mn2+ uptake blocked by the DMT1 inhibitor XEN602. Such transport was defective in rKC mitochondria with the Belgrade (G185R) mutation. Thus, these results support a role for DMT1 in mitochondrial Fe2+ and Mn2+ acquisition.
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Mitochondria represent another locale for the divalent Metal Transporter 1 (DMT1)
Channels (Austin Tex.), 2014Co-Authors: Natascha A. Wolff, Laura M. Garrick, Michael D Garrick, Lin Zhao, Frank ThévenodAbstract:The divalent Metal Transporter (DMT1) is well known for its roles in duodenal iron absorption across the apical enterocyte membrane, in iron efflux from the endosome during transferrin-dependent cellular iron acquisition, as well as in uptake of non-transferrin bound iron in many cells. Recently, using multiple approaches, we have obtained evidence that the mitochondrial outer membrane is another subcellular locale of DMT1 expression. While iron is of vital importance for mitochondrial energy metabolism, its delivery is likely to be tightly controlled due to iron's damaging redox properties. Here we provide additional support for a role of DMT1 in mitochondrial iron acquisition by immunofluorescence colocalization with mitochondrial markers in cells and isolated mitochondria, as well as flow cytometric quantification of DMT1-positive mitochondria from an inducible expression system. Physiological consequences of mitochondrial DMT1 expression are discussed also in consideration of other DMT1 substrates, su...
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Evidence for mitochondrial localization of divalent Metal Transporter 1 (DMT1)
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 2014Co-Authors: Natascha A. Wolff, Laura M. Garrick, Michael D Garrick, Andrew J. Ghio, Lin Zhao, Robert A. Fenton, Frank ThévenodAbstract:In mammalian cells, mitochondria receive most incoming iron, yet no entry pathway for iron at the outer mitochondrial membrane (OMM) has been characterized. Our results show that the divalent Metal Transporter 1 (DMT1) occurs in the OMM. Immunoblots detected DMT1 in mitochondria from a pneumocyte cell model in their OMM. Using the split-ubiquitin yeast 2-hybrid system, we found that cytochrome c oxidase subunit II (COXII) and the translocase of OMM 6-kDa subunit (Tom6) homologue interact with DMT1. COXII coimmunoprecipitates with DMT1. There are 4 DMT1 isoforms that differ at the N and C termini. Using HEK293 cells that inducibly express all of the 4 ends of DMT1, we found all of them in the OMM, as detected by immunoblots after cell fractionation, and in isolated mitochondria, as detected by immunofluorescence. Immunoblot analysis of purified cell fractions from rat renal cortex confirmed and extended these results to the kidney, which expressed high levels of DMT1. Immunogold labeling detected DMT1 colocalization in mitochondria with the voltage-dependent anion-selective channel protein-1, which is expressed in the OMM. We suggest that DMT1 not only exports iron from endosomes, but also serves to import the Metal into the mitochondria.
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Mouse divalent Metal Transporter 1 is a copper Transporter in HEK293 cells.
Biometals : an international journal on the role of metal ions in biology biochemistry and medicine, 2013Co-Authors: Miguel Arredondo, Maria Jose Mendiburo, Sebastián Flores, Steven T. Singleton, Michael D GarrickAbstract:Divalent Metal Transporter 1 (DMT1) is an apical Fe Transporter in the duodenum and is involved in endosomal Fe export. Four protein isoforms have been described for DMT1, two from mRNA with an iron responsive element (IRE) and two from mRNA without it. The sets of two begin in exon 1A or 2. We have characterized copper transport using mouse 2/−IRE DMT1 during regulated ectopic expression. HEK293 cells carrying a TetR:Hyg element were stably transfected with pDEST31 containing a 2/−IRE construct. 64Cu1+ incorporation in doxycycline treated cells exhibited 18.6 and 30.0-fold increases in Cu content, respectively when were exposed to 10 and 100 μM of extracellular Cu. Cu content was ~4-fold above that of parent cells or cells carrying just the vector. 64Cu uptake in transfected cells pre-incubated with 5 μM of Cu-His revealed a Vmax and Km of 11.98 ± 0.52 pmol mg protein−1 min−1 and 2.03 ± 0.03 μM, respectively. Doxycycline-stimulated Cu uptake was linear with time. The rates of apical Cu uptake decreased and transepithelial transport increased when intracellular Cu increased. The optimal pH for Cu transport was 6.5; uptake of Cu was temperature dependent. Silver does not inhibit Cu uptake in cells carrying the vector. In conclusion, Cu uptake in HEK293 cells that over-expressed the 2/−IRE isoform of DMT1 Transporter supports our earlier contention that DMT1 transports Cu as Cu1+.
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comparison of mammalian cell lines expressing distinct isoforms of divalent Metal Transporter 1 in a tetracycline regulated fashion
Biochemical Journal, 2006Co-Authors: Michael D Garrick, Jerome A Roth, Lin Zhao, Prasad N. Paradkar, Hung-chieh Kuo, Steven T. Singleton, Farida Vargas, Jaime J Smith, Laura M. GarrickAbstract:DMT1 (divalent Metal Transporter; also known as SLC11A2, DCT1 or Nramp2) is responsible for ferrous iron uptake in the duodenum, iron exit from endosomes during the transferrin cycle and some transferrin-independent iron uptake in many cells. Four protein isoforms differ by starting in exon 1A or 2 and ending with alternative peptides encoded by mRNA that contains or lacks an IRE (iron responsive element; ±IRE). We have compared 1A/+IRE and 2/−IRE DMT1 during regulated ectopic expression. HEK-293-F (human embryonic kidney-293-fast growing variant) cells were stably transfected with each construct expressed from a tetracycline-regulated CMV promoter. Reverse transcriptase-PCR analysis showed that construct expression responded to doxycycline. Immunofluorescence staining of cells, using antibodies specific for DMT1 isoforms, confirmed an increase in expression in the plasma membrane and cytosolic vesicles after doxycycline treatment, but with isoform specific distributions. Immunoblotting also revealed stimulation of expression. Nevertheless, both DMT1 isoforms performed similarly in assays for functional properties based on 54Mn2+ and 59Fe2+ uptake. Mn incorporation after doxycycline treatment was ∼10-fold greater than that of untreated cells, while expression in the untreated cells was ∼5-fold greater than in the untransfected cells. Uptake of Mn depended on addition of doxycycline, with half maximal response at ∼1 nM doxycycline. Doxycycline-stimulated Mn and Fe uptake was linear with time for 10 min but not over longer periods. Transport exhibited a pH optimum at ∼5.5 and dependence on incubation temperature and Mn or Fe concentration. The new cell lines should prove useful for research on Metal homoeostasis, toxicological studies and efforts to identify distinctive properties of the isoforms.