Abstract
The ZIP (ZRT-, IRT-like Protein) protein ZupT from Escherichia coli is a transporter with a broad substrate range. Phenotypic and transport analysis showed that ZupT, in addition to Zn(II), Fe(II) and Co(II) uptake, is also involved in transport of Mn(II) and Cd(II). Competition experiments with other substrate cations suggested that ZupT has a slight preference for Zn(II) and kinetic parameters for Zn(II) in comparison to Co(II) and Mn(II) transport support this observation. Metal uptake into cells by ZupT was optimum at near neutral pH and inhibited by ionophores. Bicarbonate or other ions did not influence metal-uptake via ZupT. Amino acid residues of ZupT contributing to substrate specificity were identified by site directed mutagenesis. ZupT with a H89A exchange lost Co(II) and Fe(II) transport activity, while the S117V mutant no longer transported Mn(II). ZupT with E152D was impaired in overall metal uptake but completely lost its ability to transport the substrates Zn(II) and Mn(II). These experimental findings expand our knowledge on the substrate specificity of ZupT and provide further insight into the function of ZupT as a bacterial member of the vastly distributed and important ZIP family.
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Abbreviations
- DIP:
-
2,2′-Dipyridyl
- TPEN:
-
N,N,N′,N′-Tetrakis-(2-pyridylmethyl)-ethylenediamine
- EDTA:
-
Ethylenediaminetetraacetic acid
- FCCP:
-
Carbonyl cyanide-p-trifluoromethoxyphenyl hydrazone
- CCCP:
-
Carbonyl cyanide m-chlorophenyl hydrazone
- ZIP:
-
ZRT1, IRT1-like Protein
- TMH:
-
Transmembrane helices
- d.w.:
-
Dry weight
- MES:
-
2-(N-morpholino)ethanesulfonic acid
References
Courville P, Urbankova E, Rensing C, Chaloupka R, Quick M, Cellier MF (2008) Solute carrier 11 cation symport requires distinct residues in transmembrane helices 1 and 6. J Biol Chem 283:9651–9658
Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 97:6640–6645
Eide D, Broderius M, Fett J, Guerinot ML (1996) A novel iron-regulated metal transporter from plants identified by functional expression in yeast. Proc Natl Acad Sci USA 93:5624–5628
Gaither LA, Eide DJ (2000) Functional expression of the human hZIP2 zinc transporter. J Biol Chem 275:5560–5564
Girijashanker K, He L, Soleimani M, Reed JM, Li H, Liu Z, Wang B, Dalton TP, Nebert DW (2008) Slc39a14 gene encodes ZIP14, a metal/bicarbonate symporter: similarities to the ZIP8 transporter. Mol Pharmacol 73:1413–1423
Grass G (2009) Supplemental Online Material for: amino acid residues important for metal uptake and transport kinetics of ZupT from Escherichia coli. https://sites.google.com/site/thegrasslab/Biometals. Accessed 2 Dec 2009
Grass G, Wong MD, Rosen BP, Smith RL, Rensing C (2002) ZupT is a Zn(II) uptake system in Escherichia coli. J Bacteriol 184:864–866
Grass G, Franke S, Taudte N, Nies DH, Kucharski LM, Maguire ME, Rensing C (2005a) The metal permease ZupT from Escherichia coli is a transporter with a broad substrate spectrum. J Bacteriol 187:1604–1611
Grass G, Otto M, Fricke B, Haney CJ, Rensing C, Nies DH, Munkelt D (2005b) FieF (YiiP) from Escherichia coli mediates decreased cellular accumulation of iron and relieves iron stress. Arch Microbiol 183:9–18
Grunewald M, Menaker D, Kanner BI (2002) Cysteine-scanning mutagenesis reveals a conformationally sensitive reentrant pore-loop in the glutamate transporter GLT-1. J Biol Chem 277:26074–26080
Guerinot ML (2000) The ZIP family of metal transporters. Biochim Biophys Acta 1465:190–198
He L, Girijashanker K, Dalton TP, Reed J, Li H, Soleimani M, Nebert DW (2006) ZIP8, member of the solute-carrier-39 (SLC39) metal-transporter family: characterization of transporter properties. Mol Pharmacol 70:171–180
Hudek L, Rai LC, Freestone D, Michalczyk A, Gibson M, Song YF, Ackland ML (2009) Bioinformatic and expression analyses of genes mediating zinc homeostasis in Nostoc punctiforme. Appl Environ Microbiol 75:784–791
Korshunova YO, Eide D, Clark WG, Guerinot ML, Pakrasi HB (1999) The IRT1 protein from Arabidopsis thaliana is a metal transporter with a broad substrate range. Plant Mol Biol 40:37–44
Liu Z, Li H, Soleimani M, Girijashanker K, Reed JM, He L, Dalton TP, Nebert DW (2008) Cd2+ versus Zn2+ uptake by the ZIP8 HCO3-dependent symporter: kinetics, electrogenicity and trafficking. Biochem Biophys Res Commun 365:814–820
Mergeay M, Nies D, Schlegel HG, Gerits J, Charles P, Van Gijsegem F (1985) Alcaligenes eutrophus CH34 is a facultative chemolithotroph with plasmid-bound resistance to heavy metals. J Bacteriol 162:328–334
Milon B, Wu Q, Zou J, Costello LC, Franklin RB (2006) Histidine residues in the region between transmembrane domains III and IV of hZip1 are required for zinc transport across the plasma membrane in PC-3 cells. Biochim Biophys Acta 1758:1696–1701
Outten CE, O’Halloran TV (2001) Femtomolar sensitivity of metalloregulatory proteins controlling zinc homeostasis. Science 292:2488–2492
Patzer SI, Hantke K (1998) The ZnuABC high-affinity zinc uptake system and its regulator Zur in Escherichia coli. Mol Microbiol 28:1199–1210
Rogers EE, Eide DJ, Guerinot ML (2000) Altered selectivity in an Arabidopsis metal transporter. Proc Natl Acad Sci USA 97:12356–12360
Sabri M, Houle S, Dozois CM (2009) Roles of the extraintestinal pathogenic Escherichia coli ZnuACB and ZupT zinc transporters during urinary tract infection. Infect Immun 77:1155–1164
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
Sonnhammer EL, von Heijne G, Krogh A (1998) A hidden Markov model for predicting transmembrane helices in protein sequences. Proc Int Conf Intell Syst Mol Biol 6:175–182
Tusnady GE, Simon I (2001) The HMMTOP transmembrane topology prediction server. Bioinformatics 17:849–850
Zhao H, Eide D (1996) The yeast ZRT1 gene encodes the zinc transporter protein of a high-affinity uptake system induced by zinc limitation. Proc Natl Acad Sci USA 93:2454–2458
Acknowledgements
This work was supported by the Deutsche Forschungsgemeinschaft by Grant GR2061/1-3 to G. G. and to N. T. Additional equipment for this project was purchased with The Nebraska Tobacco Settlement Biomedical Research Development Funds. We thank Grit Schleuder for skillful technical assistance, Dietrich H. Nies for support and Audrey L. Atkin for critically reading the manuscript.
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Taudte, N., Grass, G. Point mutations change specificity and kinetics of metal uptake by ZupT from Escherichia coli . Biometals 23, 643–656 (2010). https://doi.org/10.1007/s10534-010-9319-z
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DOI: https://doi.org/10.1007/s10534-010-9319-z