Abstract
Divalent metal ion transporter (DMT1) is the major transporter for iron entrance into mammalian cells and iron exit from endosomes during the transferrin cycle. Four major mRNA isoforms correspond to four protein isoforms, differing at 5′/3′ and N-/C-termini, respectively. Isoforms are designated 1A versus 1B reflecting where transcription starts or +iron responsive element (+IRE) versus −IRE reflecting the presence/absence of an IRE in the 3′ end of the mRNA. These differences imply regulation at transcriptional and posttranscriptional levels. Many proteins are degraded by a ubiquitination-dependent mechanism. Two different ubiquitin ligases (E3s) appear to be involved in DMT1 ubiquitination: Parkin or neuronal precursor cell-expressed developmentally downregulated 4 (Nedd4) family E3s which often utilize Nedd4 family interacting protein-1 and -2 (Ndfip1 and 2) to ubiquitinate their substrate proteins. Prior data suggest that Parkin ubiquitinates 1B DMT1 but not 1A DMT1 while Nedd4/Ndfips ligate ubiquitin to DMT1 in the duodenum where 1A/+IRE DMT1 predominates. Our assay for whether these systems target DMT1 depends on two HEK293 cell lines that express permanently transfected 1A/+IRE DMT1 or 1B/−IRE DMT1 after induction by doxycycline. Transient transfection with a Parkin construct before induction diminishes 1B/−IRE DMT1 detected by immune-blots but not 1A/+IRE DMT1. Mutant Parkin serves as a control that does not affect DMT1 levels. Thus DMT1 regulation in an isoform specific fashion can occur by ubiquitination and the events involved have implications for DMT1 function and disease processes.
Similar content being viewed by others
Abbreviations
- DMT1:
-
Divalent metal ion transporter
- IRE:
-
Iron responsive element
- PD:
-
Parkinson’s disease
- NF-κB:
-
Nuclear factor-κB
- HIFs:
-
Hypoxia inducible factors
- IRPs:
-
Iron regulatory proteins
- miRNA:
-
MicroRNA
- Mn:
-
Manganese
- wt:
-
Wild type
- Nedd4 :
-
Neuronal precursor cell-expressed developmentally downregulated 4
- WWP2:
-
WW domain-containing protein 2
- PY:
-
PPxY motifs
- Ndfip1 and Ndfip2:
-
Nedd4 family interacting proteins 1 and 2
References
Andolfo I, Falco LD, Asci R, Russo R, Colucci S, Gorrese M, Zollo M, Iolascon A (2010) Regulation of divalent metal transporter 1 (DMT1) non-IRE isoform by the microRNA Let-7d in erythroid cells. Haematologica 95(8):1244–1252. doi:10.3324/haematol.2009.020685
Chung KKK, Zhang Y, Lim KL, Tanaka Y, Huang H, Gao J, Ross CA, Dawson VL, Dawson TM (2001) Parkin ubiquitinates the [alpha]-synuclein-interacting protein, synphilin-1: implications for Lewy-body formation in Parkinson disease. Nat Med 7(10):1144–1150
Etlinger JD, Goldberg AL (1977) A soluble ATP-dependent proteolytic system responsible for the degradation of abnormal proteins in reticulocytes. Proc Natl Acad Sci USA 74(1):54–58
Foot NJ, Dalton HE, Shearwin-Whyatt LM, Dorstyn L, Tan S-S, Yang B, Kumar S (2008) Regulation of the divalent metal ion transporter DMT1 and iron homeostasis by a ubiquitin-dependent mechanism involving Ndfips and WWP2. Blood 112(10):4268–4275. doi:10.1182/blood-2008-04-150953
Foot NJ, Leong YA, Dorstyn LE, Dalton HE, Ho K, Zhao L, Garrick MD, Yang B, Hiwase D, Kumar S (2011) Ndfip1-deficient mice have impaired DMT1 regulation and iron homeostasis. Blood 117(2):638–646. doi:10.1182/blood-2010-07-295287
Galy B, Ferring-Appel D, Kaden S, Gröne H-J, Hentze MW (2008) Iron regulatory proteins are essential for intestinal function and control key iron absorption molecules in the duodenum. Cell Metab 7(1):79–85
Garrick M (2011) Human iron transporters. Genes Nutr 6(1):45–54. doi:10.1007/s12263-010-0184-8
Garrick MD, Garrick LM (2009) Cellular iron transport. Biochim Biophys Acta 1790(5):309–325
Garrick MD, Dolan KG, Ghio A, Horbinski C, Higgins D, Porubcin M, Moore EG, Hainsworth LN, Umbreit JN, Conrad ME, Feng L, Lis A, Roth JE, Singleton S, Garrick LM (2003) DMT1 (divalent metal transporter 1): a mammalian transporter for multiple metals. Biometals 16:41–54
Garrick MD, Kuo H-C, Vargas F, Singleton S, Zhao L, Smith JJ, Paradkar P, Roth JA, Garrick LM (2006a) Comparison of mammalian cell lines expressing distinct isoforms of divalent metal transporter 1 in a tetracycline-regulated fashion. Biochem J 398(3):539–546. doi:10.1042/bj20051987
Garrick MD, Singleton ST, Vargas F, Kuo H-C, Zhao L, Knöpfel M, Davidson T, Costa M, Paradkar P, Roth JA, Garrick LM (2006b) DMT1: Which metals does it transport? Biol Res 39:79–85
Ghosh A, Roy A, Liu X, Kordower JH, Mufson EJ, Hartley DM, Ghosh S, Mosley RL, Gendelman HE, Pahan K (2007) Selective inhibition of NF-κB activation prevents dopaminergic neuronal loss in a mouse model of Parkinson’s disease. Proc Natl Acad Sci USA 104(47):18754–18759. doi:10.1073/pnas.0704908104
Gunshin H, Mackenzie B, Berger UV, Gunshin Y, Romero MF, Boron WF, Nussberger S, Gollan JL, Hediger MA (1997) Cloning and characterization of a mammalian proton-coupled metal-ion transporter. Nature 388(6641):482–488
Higashi Y, Asanuma M, Miyazaki I, Hattori N, Mizuno Y, Ogawa N (2004) Parkin attenuates manganese-induced dopaminergic cell death. J Neurochem 89(6):1490–1497
Hirsch EC, Brandel JP, Galle P, Javoyagid F, Agid Y (1991) Iron and aluminum increase in the substantia-nigra of patients with Parkinsons-disease—an X-ray-microanalysis. J Neurochem 56(2):446–451
Howitt J, Putz U, Lackovic J, Doan A, Dorstyn L, Cheng H, Yang B, Chan-Ling T, Silke J, Kumar S, Tan S-S (2009) Divalent metal transporter 1 (DMT1) regulation by Ndfip1 prevents metal toxicity in human neurons. Proc Natl Acad Sci USA 106(36):15489–15494. doi:10.1073/pnas.0904880106
Hubert N, Hentze MW (2002) Previously uncharacterized isoforms of divalent metal transporter (DMT)-1: implications for regulation and cellular function. Proc Natl Acad Sci USA 99(19):12345–12350
Hunot S, Brugg B, Ricard D, Michel PP, Muriel M-P, Ruberg M, Faucheux BA, Agid Y, Hirsch EC (1997) Nuclear translocation of NF-κB is increased in dopaminergic neurons of patients with Parkinson disease. Proc Natl Acad Sci USA 94(14):7531–7536
Kitada T, Asakawa S, Hattori N, Matsumine H, Yamamura Y, Minoshima S, Yokochi M, Mizuno Y, Shimizu N (1998) Mutations in the Parkin gene cause autosomal recessive juvenile parkinsonism. Nature 392(6676):605–608
Lee PL, Gelbart T, West C, Halloran C, Beutler E (1998) The human Nramp2 gene: characterization of the gene structure, alternative splicing, promoter region and polymorphisms. Blood Cells Mol Dis 24(9):199–215
Lis A, Paradkar PN, Singleton S, Kuo HC, Garrick MD, Roth JA (2005) Hypoxia induces changes in expression of isoforms of the divalent metal transporter (DMT1) in rat pheochromocytoma (PC12) cells. Biochem Pharmacol 69(11):1647–1655
Lodish H, Berk A, Matsudaira P, Kaiser C, Krieger M, Scott M, Zipursky S, Darnell J (2004) Molecular cell biology, 5th edn. W.H. Freeman, New York, pp 66–72
Mastrogiannaki M, Matak P, Keith B, Simon C, Vaulont S, Peyssonnaux C (2009) HIF-2α, but not HIF-1α, promotes iron absorption in mice. J Clin Investig 119(5):1159–1166. doi:10.1172/JCI38499
Paradkar PN, Roth JA (2006a) Nitric oxide transcriptionally down-regulates specific isoforms of divalent metal transporter (DMT1) via NF-κB. J Neurochem 96(6):1768–1777
Paradkar PN, Roth JA (2006b) Post-translational and transcriptional regulation of DMT1 during P19 embryonic carcinoma cell differentiation by retinoic acid. Biochem J 394(1):173–183. doi:10.1042/bj20051296
Roth JA, Singleton S, Feng J, Garrick M, Paradkar PN (2010) Parkin regulates metal transport via proteasomal degradation of the 1B isoforms of divalent metal transporter 1. J Neurochem 113:454–464. doi:10.1111/j.1471-4159.2010.06607.x
Salazar J, Mena N, Hunot S, Prigent A, Alvarez-Fischer D, Arredondo M, Duyckaerts C, Sazdovitch V, Zhao L, Garrick LM, Nuñez MT, Garrick MD, Raisman-Vozari R, Hirsch EC (2008) Divalent metal transporter 1 (DMT1) contributes to neurodegeneration in animal models of Parkinson’s disease. Proc Natl Acad Sci USA 105(47):18578–18583. doi:10.1073/pnas.0804373105
Shah YM, Matsubara T, Ito S, Yim S-H, Gonzalez FJ (2009) Intestinal hypoxia-inducible transcription factors are essential for iron absorption following iron deficiency. Cell Metab 9(2):152–164
Tchernitchko D, Bourgeois M, Martin ME, Beaumont C (2002) Expression of the two mRNA isoforms of the iron transporter Nrmap2/DMTI in mice and function of the iron responsive element. Biochem J 363(3):449–455
Acknowledgments
We thank Mr. Fabian Pirrman for technical assistance preparing DNA constructs.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Garrick, M.D., Zhao, L., Roth, J.A. et al. Isoform specific regulation of divalent metal (ion) transporter (DMT1) by proteasomal degradation. Biometals 25, 787–793 (2012). https://doi.org/10.1007/s10534-012-9522-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10534-012-9522-1