Factors associated with nuclear localization of the ire isoform of dmt1 (nramp2/dct1)

M. D. Garrick, C. Horbinski, K. G. Dolan, L. Feng, J. R. Burdo, James Connor, D. Higgins, J. A. Roth

Research output: Contribution to journalArticle

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Abstract

DMT1 (Divalent Metal Transporter) is the metal transporter responsible for ferrous iron uptake into enterocytes and exit from endosomes. It also transports other metals like Mn2 and Ni2. Two mRNA isoforms of DMT1 have or lack a 3'iron responsive element (IRE) and encode proteins identical for 543 residues but differing in their C-terminal 18 or 25 âmino acid residues (+IRE or -IRE, respectively). We prepared affinity purified rabbit antibodies with these peptides: 545-SISKVLLSEDTSGGNTK-561 (+IRE) and 547GLTARPEIYLLNTVDAVSLVSR-568 (-IRE) to learn the tissue-specific distribution and cellular localization of the isoforms via immunofluorescent confocal microscopy and immunoblot analyses. In rat pheocnromocytoma (PC12) cells and cultured sympathetic neurons the -IRE form localized predominantly within the nucleus while the +IRE form appeared in cytosolic vesicles as expected. Specificity of the antibodies was confirmed when the immunizing peptides inhibited antibody staining with no cross inhibition by the opposite peptide and after ectopic expression of rat -IRE DMT1 in HEK293T cells. Immunoblots on fractions from PC 12 cells also support a nuclear location for the -IRE form and indicate that it is -67 kD with the cytosolic -IRE form -65 kD; while the +IRE form, absent from the nuclear fraction, is also -65 kD. Immunoafftnity purified antibody directed against a peptide from the 4th extracellular domain integrates ±IRE immunofluorescence results in both PC 12 cells and sympathetic neurons and confirms that some DMT1 is nuclear. The immunoblots together with the 3rd antibody argue that entire -IRE isoform may be in nucleus although we cannot yet exclude loss of a large N-terminal portion compensated by extensive derivatization. Nuclear localization is also observed in sympathetic neurons from Belgrade rats where the mutation, gly 185 to arg, inactivates Fe2 transport. Thus the G185R mutation does not alter the unexpected nuclear localization. A survey of ±IRE distribution in a wide range of cell types revealed that neuronal cells have the most striking nuclear localization of the -IRE form, although it is found to a lesser degree in the nuclei of certain nonneuronal cells. Probably the two DMT1 isoforms have different functions given that they should be regulated differently and appear in different subcellular comparments. Future investigations should ask whether the -IRE form in the nucleus sequesters a metal, transports a metal or transmits information on metal status.

Original languageEnglish (US)
JournalBlood
Volume96
Issue number11 PART I
StatePublished - 2000

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Protein Isoforms
Iron
Metals
Antibodies
Neurons
Rats
Peptides
RNA Isoforms
Mutation
Antibody Specificity
Enterocytes
Confocal microscopy
Endosomes
PC12 Cells
Tissue Distribution
Confocal Microscopy
Fluorescent Antibody Technique

All Science Journal Classification (ASJC) codes

  • Hematology

Cite this

Garrick, M. D., Horbinski, C., Dolan, K. G., Feng, L., Burdo, J. R., Connor, J., ... Roth, J. A. (2000). Factors associated with nuclear localization of the ire isoform of dmt1 (nramp2/dct1). Blood, 96(11 PART I).
Garrick, M. D. ; Horbinski, C. ; Dolan, K. G. ; Feng, L. ; Burdo, J. R. ; Connor, James ; Higgins, D. ; Roth, J. A. / Factors associated with nuclear localization of the ire isoform of dmt1 (nramp2/dct1). In: Blood. 2000 ; Vol. 96, No. 11 PART I.
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abstract = "DMT1 (Divalent Metal Transporter) is the metal transporter responsible for ferrous iron uptake into enterocytes and exit from endosomes. It also transports other metals like Mn2 and Ni2. Two mRNA isoforms of DMT1 have or lack a 3'iron responsive element (IRE) and encode proteins identical for 543 residues but differing in their C-terminal 18 or 25 {\^a}mino acid residues (+IRE or -IRE, respectively). We prepared affinity purified rabbit antibodies with these peptides: 545-SISKVLLSEDTSGGNTK-561 (+IRE) and 547GLTARPEIYLLNTVDAVSLVSR-568 (-IRE) to learn the tissue-specific distribution and cellular localization of the isoforms via immunofluorescent confocal microscopy and immunoblot analyses. In rat pheocnromocytoma (PC12) cells and cultured sympathetic neurons the -IRE form localized predominantly within the nucleus while the +IRE form appeared in cytosolic vesicles as expected. Specificity of the antibodies was confirmed when the immunizing peptides inhibited antibody staining with no cross inhibition by the opposite peptide and after ectopic expression of rat -IRE DMT1 in HEK293T cells. Immunoblots on fractions from PC 12 cells also support a nuclear location for the -IRE form and indicate that it is -67 kD with the cytosolic -IRE form -65 kD; while the +IRE form, absent from the nuclear fraction, is also -65 kD. Immunoafftnity purified antibody directed against a peptide from the 4th extracellular domain integrates ±IRE immunofluorescence results in both PC 12 cells and sympathetic neurons and confirms that some DMT1 is nuclear. The immunoblots together with the 3rd antibody argue that entire -IRE isoform may be in nucleus although we cannot yet exclude loss of a large N-terminal portion compensated by extensive derivatization. Nuclear localization is also observed in sympathetic neurons from Belgrade rats where the mutation, gly 185 to arg, inactivates Fe2 transport. Thus the G185R mutation does not alter the unexpected nuclear localization. A survey of ±IRE distribution in a wide range of cell types revealed that neuronal cells have the most striking nuclear localization of the -IRE form, although it is found to a lesser degree in the nuclei of certain nonneuronal cells. Probably the two DMT1 isoforms have different functions given that they should be regulated differently and appear in different subcellular comparments. Future investigations should ask whether the -IRE form in the nucleus sequesters a metal, transports a metal or transmits information on metal status.",
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Garrick, MD, Horbinski, C, Dolan, KG, Feng, L, Burdo, JR, Connor, J, Higgins, D & Roth, JA 2000, 'Factors associated with nuclear localization of the ire isoform of dmt1 (nramp2/dct1)', Blood, vol. 96, no. 11 PART I.

Factors associated with nuclear localization of the ire isoform of dmt1 (nramp2/dct1). / Garrick, M. D.; Horbinski, C.; Dolan, K. G.; Feng, L.; Burdo, J. R.; Connor, James; Higgins, D.; Roth, J. A.

In: Blood, Vol. 96, No. 11 PART I, 2000.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Factors associated with nuclear localization of the ire isoform of dmt1 (nramp2/dct1)

AU - Garrick, M. D.

AU - Horbinski, C.

AU - Dolan, K. G.

AU - Feng, L.

AU - Burdo, J. R.

AU - Connor, James

AU - Higgins, D.

AU - Roth, J. A.

PY - 2000

Y1 - 2000

N2 - DMT1 (Divalent Metal Transporter) is the metal transporter responsible for ferrous iron uptake into enterocytes and exit from endosomes. It also transports other metals like Mn2 and Ni2. Two mRNA isoforms of DMT1 have or lack a 3'iron responsive element (IRE) and encode proteins identical for 543 residues but differing in their C-terminal 18 or 25 âmino acid residues (+IRE or -IRE, respectively). We prepared affinity purified rabbit antibodies with these peptides: 545-SISKVLLSEDTSGGNTK-561 (+IRE) and 547GLTARPEIYLLNTVDAVSLVSR-568 (-IRE) to learn the tissue-specific distribution and cellular localization of the isoforms via immunofluorescent confocal microscopy and immunoblot analyses. In rat pheocnromocytoma (PC12) cells and cultured sympathetic neurons the -IRE form localized predominantly within the nucleus while the +IRE form appeared in cytosolic vesicles as expected. Specificity of the antibodies was confirmed when the immunizing peptides inhibited antibody staining with no cross inhibition by the opposite peptide and after ectopic expression of rat -IRE DMT1 in HEK293T cells. Immunoblots on fractions from PC 12 cells also support a nuclear location for the -IRE form and indicate that it is -67 kD with the cytosolic -IRE form -65 kD; while the +IRE form, absent from the nuclear fraction, is also -65 kD. Immunoafftnity purified antibody directed against a peptide from the 4th extracellular domain integrates ±IRE immunofluorescence results in both PC 12 cells and sympathetic neurons and confirms that some DMT1 is nuclear. The immunoblots together with the 3rd antibody argue that entire -IRE isoform may be in nucleus although we cannot yet exclude loss of a large N-terminal portion compensated by extensive derivatization. Nuclear localization is also observed in sympathetic neurons from Belgrade rats where the mutation, gly 185 to arg, inactivates Fe2 transport. Thus the G185R mutation does not alter the unexpected nuclear localization. A survey of ±IRE distribution in a wide range of cell types revealed that neuronal cells have the most striking nuclear localization of the -IRE form, although it is found to a lesser degree in the nuclei of certain nonneuronal cells. Probably the two DMT1 isoforms have different functions given that they should be regulated differently and appear in different subcellular comparments. Future investigations should ask whether the -IRE form in the nucleus sequesters a metal, transports a metal or transmits information on metal status.

AB - DMT1 (Divalent Metal Transporter) is the metal transporter responsible for ferrous iron uptake into enterocytes and exit from endosomes. It also transports other metals like Mn2 and Ni2. Two mRNA isoforms of DMT1 have or lack a 3'iron responsive element (IRE) and encode proteins identical for 543 residues but differing in their C-terminal 18 or 25 âmino acid residues (+IRE or -IRE, respectively). We prepared affinity purified rabbit antibodies with these peptides: 545-SISKVLLSEDTSGGNTK-561 (+IRE) and 547GLTARPEIYLLNTVDAVSLVSR-568 (-IRE) to learn the tissue-specific distribution and cellular localization of the isoforms via immunofluorescent confocal microscopy and immunoblot analyses. In rat pheocnromocytoma (PC12) cells and cultured sympathetic neurons the -IRE form localized predominantly within the nucleus while the +IRE form appeared in cytosolic vesicles as expected. Specificity of the antibodies was confirmed when the immunizing peptides inhibited antibody staining with no cross inhibition by the opposite peptide and after ectopic expression of rat -IRE DMT1 in HEK293T cells. Immunoblots on fractions from PC 12 cells also support a nuclear location for the -IRE form and indicate that it is -67 kD with the cytosolic -IRE form -65 kD; while the +IRE form, absent from the nuclear fraction, is also -65 kD. Immunoafftnity purified antibody directed against a peptide from the 4th extracellular domain integrates ±IRE immunofluorescence results in both PC 12 cells and sympathetic neurons and confirms that some DMT1 is nuclear. The immunoblots together with the 3rd antibody argue that entire -IRE isoform may be in nucleus although we cannot yet exclude loss of a large N-terminal portion compensated by extensive derivatization. Nuclear localization is also observed in sympathetic neurons from Belgrade rats where the mutation, gly 185 to arg, inactivates Fe2 transport. Thus the G185R mutation does not alter the unexpected nuclear localization. A survey of ±IRE distribution in a wide range of cell types revealed that neuronal cells have the most striking nuclear localization of the -IRE form, although it is found to a lesser degree in the nuclei of certain nonneuronal cells. Probably the two DMT1 isoforms have different functions given that they should be regulated differently and appear in different subcellular comparments. Future investigations should ask whether the -IRE form in the nucleus sequesters a metal, transports a metal or transmits information on metal status.

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Garrick MD, Horbinski C, Dolan KG, Feng L, Burdo JR, Connor J et al. Factors associated with nuclear localization of the ire isoform of dmt1 (nramp2/dct1). Blood. 2000;96(11 PART I).