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Intracellular iron trafficking: role of cytosolic ligands

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Abstract

Iron acquired by cells is delivered to mitochondria for metabolic processing via pathways comprising undefined chemical forms. In order to assess cytosolic factors that affect those iron delivery pathways, we relied on microscopy and flow-cytometry for monitoring iron traffic in: (a) K562 erythroleukemia cells labeled with fluorescent metal-sensors targeted to either cytosol or mitochondria and responsive to changes in labile iron and (b) permeabilized cells that retained metabolically active mitochondria accessible to test substrates. Iron supplied to intact cells as transferrin–Fe(III) or Fe(II)-salts evoked concurrent metal ingress to cytosol and mitochondria. With either supplementation modality, iron ingress into cytosol was mostly absorbed by preloaded chelators, but ingress into mitochondria was fully inhibited only by some chelators, indicating different cytosol-to-mitochondria delivery mechanisms. Iron ingress into cytosol or mitochondria were essentially unaffected by depletion of cytosolic iron ligands like glutathione or the hypothesized 2,5 dihydroxybenzoate (2,5-DHBA) siderophore/chaperone. These ligands also failed to affect mitochondrial iron ingress in permeabilized K562 cells suspended in cytosol-simulating medium. In such medium, mitochondrial iron uptake was >6-eightfold higher for Fe(II) versus Fe(III), showed saturable properties and submicromolar K1/2 corresponding to cytosolic labile iron levels. When measured in iron(II)-containing media, ligands like AMP, ADP or ATP, did not affect mitochondrial iron uptake whereas in iron(III)-containing media ADP and ATP reduced it and AMP stimulated it. Thus, cytosolic iron forms demonstrably contribute to mitochondrial iron delivery, are apparently not associated with DHBA analogs or glutathione but rather with resident components of the cytosolic labile iron pool.

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Abbreviations

AM:

Acetomethoxyl ester

BDH2:

Butyrate dehydrogenase2

BIPC:

Carboxy-bipyridyl

BSO:

Buthionine sulfoximine

CALG:

Calcein green

2,5-DHBA:

2,5-Dihydroxybenzoic acid

DMB:

5,5′-Dimethyl-BAPTA

DFO:

Desferrioxamine

DMEM:

Dulbecco’s modified eagle medium

DMSO:

Dimethyl-sulfoxide

DMT1:

Divalent metal transporter 1

FAS:

Ferrous ammonium sulfate

FeS:

Iron–sulfur cluster

F.U:

Fluorescence units

GSH:

Reduced glutathione

INT:

Iodonitrotetrazolium

JC1:

5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide

LIP:

Labile iron pool

MDR:

Multidrug resistance

MRP:

Multidrug resistance proteins

NTA:

Nitrilotriacetic acid

PBS:

Phosphate buffered saline

RPA:

Rhodamine B-[(1,10-phenanthrolin-5-yl) aminocarbonyl benzyl ester

SIH:

Salicyl isonicotinoyl hydrazide

TfFe:

Transferrin–iron

References

  • Andolfo I, De Falco L, 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 95:1244–1252

    Article  PubMed  CAS  Google Scholar 

  • Arosio P, Ingrassia R, Cavadini P (2009) Ferritins: a family of molecules for iron storage, antioxidation and more. Biochim Biophys Acta 1790:589–599

    Article  PubMed  CAS  Google Scholar 

  • Bao G, Clifton M, Hoette TM, Mori K, Deng SX, Qiu A, Viltard M, Williams D, Paragas N, Leete T, Kulkarni R, Li X, Lee B, Kalandadze A, Ratner AJ, Pizarro JC, Schmidt-Ott KM, Landry DW, Raymond KN, Strong RK, Barasch J (2010) Iron traffics in circulation bound to a siderocalin (Ngal)–catechol complex. Nat Chem Biol 6:602–609

    Article  PubMed  CAS  Google Scholar 

  • Breuer W, Epsztejn S, Cabantchik ZI (1995) Iron acquired from transferrin by K562 cells is delivered into a cytoplasmic pool of chelatable iron(II). J Biol Chem 271:24209–24215

    Google Scholar 

  • Breuer W, Shvartsman M, Cabantchik ZI (2008) Intracellular labile iron. Int J Biochem Cell Biol 40:350–354

    Article  PubMed  CAS  Google Scholar 

  • Crook TR, Souhami RL, Whyman GD, McLean AEM (1986) Glutathione depletion as a determinant of human leukemia cells to cyclophosphamide. Cancer Res 46:5035–5038

    PubMed  CAS  Google Scholar 

  • Devireddy LR, Hart DO, Goetz DH, Green MR (2010) A mammalian siderophore synthesized by an enzyme with a bacterial homolog involved in enterobactin production. Cell 141:1006–1017

    Article  PubMed  CAS  Google Scholar 

  • Epzstejn S, Kakhlon O, Glickstejn H, Breuer W, Cabantchik ZI (1997) Fluorescence analysis of the labile iron pool of mammalian cells. Anal Biochem 248:31–40

    Article  Google Scholar 

  • Guo K, Lukacik P, Papagrigoriou E, Meier M, Lee WH, Adamski J, Oppermann U (2006) Characterization of human DHRS6, an orphan short chain dehydrogenase/reductase enzyme: a novel, cytosolic type 2 R-beta-hydroxybutyrate dehydrogenase. J Biol Chem 281:10291–10297

    Article  PubMed  CAS  Google Scholar 

  • Hider RC, Kong XL (2011) Glutathione: a key component of the cytoplasmic labile iron pool. Biometals 24:1179–1187

    Article  PubMed  CAS  Google Scholar 

  • Kakhlon O, Cabantchik ZI (2002) The labile iron pool: characterization, measurement and participation in cellular processes. Free Radic Biol Med 33:1037–1046

    Article  PubMed  CAS  Google Scholar 

  • Kakhlon O, Manning H, Breuer W, Melamed-Book N, Lu C, Cortopassi G, Munnich A, Cabantchik ZI (2008) Cell functions impaired by frataxin deficiency are restored by drug-mediated iron relocation. Blood 112:5219–5227

    Article  PubMed  CAS  Google Scholar 

  • Klausner RD, Van Renswoude J, Ashwell G, Kempf C, Schechter AN, Dean A, Bridges KR (1983) Receptor-mediated endocytosis of transferrin in K562 cells. J Biol Chem 258:4715–4724

    PubMed  CAS  Google Scholar 

  • Lane DJR, Lawen A (2008) Non-transferrin iron reduction and uptake are regulated by transmembrane ascorbate cycling in K562 cells. J Biol Chem 283:12701–12708

    Article  PubMed  CAS  Google Scholar 

  • Lange H, Kispal G, Lill R (1999) Mechanism of iron transport to the site of heme synthesis inside yeast mitochondria. J Biol Chem 274:18989–18996

    Article  PubMed  CAS  Google Scholar 

  • Liuzzi JP, Aydemir F, Nam H, Knutson MD, Cousins RJ (2006) Zip14 (Slc39a14) mediates non-transferrin-bound iron uptake into cells. PNAS USA 103:13612–13617

    Article  PubMed  CAS  Google Scholar 

  • Machida K, Ohta Y, Osada H (2006) Suppression of apoptosis by cyclophilin D via stabilization of hexokinase II mitochondrial binding in cancer cells. J Biol Chem 281:14314–14320

    Article  PubMed  CAS  Google Scholar 

  • McKie AT (2005) A ferrireductase fills the gap in the transferrin cycle. Nat Genet 37:1159–1160

    Article  PubMed  CAS  Google Scholar 

  • Mühlenhoff U, Richhardt N, Gerber J, Lill R (2002) Characterization of iron–sulfur protein assembly in isolated mitochondria. A requirement for ATP, NADH, and reduced iron. J Biol Chem 277:29810–29816

    Article  PubMed  Google Scholar 

  • Mühlenhoff U, Molik S, Godoy JR, Uzarska MA, Richter N, Seubert A, Zhang Y, Stubbe J, Pierrel F, Herrero E, Lillig CH, Lill R (2010) Cytosolic monothiol glutaredoxins function in intracellular iron sensing and trafficking via their bound iron-sulfur cluster. Cell Metab 12:373–385

    Google Scholar 

  • Munujos P, Coll-Canti J, Gonzalez-Sastre F, Gella FJ (1993) Assay of succinate dehydrogenase activity by a colorimetric-continuous method using iodonitrotetrazolium chloride as electron acceptor. Anal Biochem 212:506–509

    Article  PubMed  CAS  Google Scholar 

  • Nishikawa M, Nojima S, Akiyama T, Sankawa U, Inoue K (1984) Interaction of digitonin and its analogs with membrane cholesterol. J Biochem (Tokyo) 96:1231–1239

    CAS  Google Scholar 

  • Paradkar PN, Zumbrennen KB, Paw BH, Ward DM, Kaplan J (2009) Regulation of mitochondrial iron import through differential turnover of mitoferrin 1 and mitoferrin 2. Mol Cell Biol 29:1007–1016

    Article  PubMed  CAS  Google Scholar 

  • Petrat F, Weisheit D, Lensen M, de Groot H, Sustmann R, Rauen U (2002) Selective determination of mitochondrial chelatable iron in viable cells with a new fluorescent sensor. Biochem J 362:137–147

    Article  PubMed  CAS  Google Scholar 

  • Rahman I, Kode A, Biswas SK (2006) Assay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling method. Nat Protoc 1:3159–3165

    Article  PubMed  CAS  Google Scholar 

  • Shalev O, Hebbel RP (1996) Extremely high affinity association of Fe(III) with the sickle cell red membrane. Blood 88:349–352

    PubMed  CAS  Google Scholar 

  • Shaw GC, Cope JJ, Li L, Corson K, Hersey C, Ackermann GE, Gwynn B, Lambert AJ, Wingert RA, Traver D, Trede NS, Barut BA, Zhou Y, Minet E, Donovan A, Brownlie A, Balzan R, Weiss MJ, Peters LL, Kaplan J, Zon LI, Paw BH (2006) Mitoferrin is essential for erythroid iron assimilation. Nature 440:96–100

    Article  PubMed  CAS  Google Scholar 

  • Sheftel AD, Lill R (2009) The power plant of the cell is also a smithy: the emerging role of mitochondria in iron homeostasis. Ann Med 41:82–89

    Article  PubMed  CAS  Google Scholar 

  • Sheftel AD, Zhang AS, Brown C, Shirihai OS, Ponka P (2007) Direct interorganellar transfer of iron from endosome to mitochondrion. Blood 110:125–132

    Article  PubMed  CAS  Google Scholar 

  • Shi H, Bencze KZ, Stemmler TL, Philpott CC (2008) A cytosolic iron chaperone that delivers iron to ferritin. Science 320:1207–1210

    Article  PubMed  CAS  Google Scholar 

  • Shvartsman M, Kikkeri R, Shanzer A, Cabantchik ZI (2007) Non-transferrin-bound iron reaches mitochondria by a chelator-inaccessible mechanism: Biological and clinical implications. Am J Physiol Cell Physiol 293:C1383–C1394

    Article  PubMed  CAS  Google Scholar 

  • Shvartsman M, Fibach E, Cabantchik ZI (2010) Transferrin-iron routing to the cytosol and mitochondria as studied by live and real-time fluorescence. Biochem J 429:185–193

    Article  PubMed  CAS  Google Scholar 

  • Wang J, Pantopoulos K (2011) Regulation of cellular iron metabolism. Biochem J 434:365–381

    Article  PubMed  CAS  Google Scholar 

  • Weaver J, Pollack S (1989) LoW-Mr iron isolated from guinea pig reticulocytes as AMP-Fe and ATP-Fe complexes. Biochem J 261:787–792

    Google Scholar 

  • Weaver J, Pollack S (1990) Two types of receptors for iron on mitochondria. Biochem J 271:463–466

    PubMed  CAS  Google Scholar 

  • Weaver J, Pollack S, Zhan H (1989) Low molecular weight iron from guinea pig reticulocytes isolated by Sephadex G25 chromatography. Eur J Haematol 43:321–327

    Article  PubMed  CAS  Google Scholar 

  • Weaver J, Zhan H, Pollack S (1990) Mitochondria have Fe(III) receptors. Biochem J 265:415–419

    PubMed  CAS  Google Scholar 

  • Zhan H, Gupta RK, Weaver J, Pollack S (1990) Iron bound to low MW ligands: interactions with mitochondria and cytosolic proteins. Eur J Haematol 44:124–130

    Google Scholar 

  • Zhao N, Gao J, Enns CA, Knutson MD (2010) ZRT/IRT-like protein 14 (ZIP14) promotes the cellular assimilation of iron from transferrin. J Biol Chem 285:32141–32150

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgment

We wish to thank Dr. N. Melamed-Book for assistance with confocal microscopy.

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Authors and Affiliations

  1. Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Edmond Safra Campus at Givat Ram, 91904, Jerusalem, Israel

    Maya Shvartsman & Z. Ioav Cabantchik

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  1. Maya Shvartsman
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  2. Z. Ioav Cabantchik
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Correspondence to Z. Ioav Cabantchik.

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Shvartsman, M., Ioav Cabantchik, Z. Intracellular iron trafficking: role of cytosolic ligands. Biometals 25, 711–723 (2012). https://doi.org/10.1007/s10534-012-9529-7

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