Advertisement

BioMetals

, Volume 31, Issue 2, pp 255–266 | Cite as

Functional analysis RaZIP1 transporter of the ZIP family from the ectomycorrhizal Zn-accumulating Russula atropurpurea

  • Tereza Leonhardt
  • Jan Sácký
  • Pavel Kotrba
Article

Abstract

A search of R. atropurpurea transcriptome for sequences encoding the transporters of the Zrt-, Irt-like Protein (ZIP) family, which are in eukaryotes integral to Zn supply into cytoplasm, allowed the identification of RaZIP1 cDNA with a predicted product belonging to ZIP I subfamily; it was subjected to functional studies in mutant Saccharomyces cerevisiae strains. The expression of RaZIP1, but not RaZIP1H208A or RaZIP1H232A mutants lacking conserved-among-ZIPs transmembrane histidyls, complemented Zn uptake deficiency in zrt1Δzrt2Δ yeasts. RaZIP1 substantially increased cellular Zn uptake in this strain and added to Zn sensitivity in zrc1Δcot1Δ mutant. The Fe uptake deficiency in ftr1Δ strain was not rescued and Mn uptake was insufficient for toxicity in Mn-sensitive pmr1Δ yeasts. By contrast, RaZIP1 increased Cd sensitivity in yap1Δ strain and conferred Cd transport activity in yeasts, albeit with substantially lower efficiency compared to Zn transport. In metal uptake assays, the accumulation of Zn in zrt1Δzrt2Δ strain remained unaffected by Cd, Fe, and Mn present in 20-fold molar excess over Zn. Immunofluorescence microscopy detected functional hemagglutinin-tagged HA::RaZIP1 on the yeast cell protoplast periphery. Altogether, these data indicate that RaZIP1 is a high-affinity plasma membrane transporter specialized in Zn uptake, and improve the understanding of the cellular and molecular biology of Zn in R. atropurpurea that is known for its ability to accumulate remarkably high concentrations of Zn.

Keywords

Russula atropurpurea Metal uptake Zinc transporter ZIP family Ectomycorrhizal fungi 

Notes

Acknowledgements

We thank Dr. Jan Borovička (Institute of Geology and Nuclear Physics Institute, Academy of Science of the Czech Republic) for the provision of characterized R. atropurpurea sporocarps and helpful discussions, and Prof. David Eide (University of Wisconsin-Madison) for the gift of CM100, CM34 and CM137 strains. This work was supported by the Specific University Research [MSMT No. 20/2014] and the Czech Science Foundation [16-15065S].

Supplementary material

10534_2018_85_MOESM1_ESM.tif (5.2 mb)
Fig. S1 Growth of S. cerevisiae pmr1Δ and ftr1Δ expressing RaZIP1. (A) Growth of transformants of Mn sensitive pmr1Δ strain and parental BY4741 strain on SD medium containing increasing Mn2+ concentrations. (B) Growth of transformants of Fe uptake deficient ftr1Δ strain and parental BY4741 strain on low-iron FLM medium without or with Fe2+ supplement. Diluted cultures of cells transformed with the empty p416GPD vector or with the same vector inserted with RaZIP1 were spotted on the agar plates. Supplementary material 1 (TIFF 5367 kb)
10534_2018_85_MOESM2_ESM.tif (9.1 mb)
Fig. S2 Immunofluorescence microscopy of S. cerevisiae zrt1Δzrt2Δ expressing HA::RaZIP1 and RaZIP1. Confocal images of the protoplasts probed with anti-HA FITC-conjugated antibody (green fluorescence), anti-mouse IgG-Alexa Fluor 633 antibody (red fluorescence), and DAPI staining nuclei and mitochondria (blue fluorescence) as indicated. Note that the anti-PMA1 antibody was not used to confirm that the red fluorescence in protoplasts incubated with anti-mouse IgG Alexa Fluor 633 antibody (Fig. 4) was not due to a non-specific binding of anti-mouse IgG to yeasts but due to its binding to primary mouse anti-PMA1 antibody. Bars in brighfield micrographs represent 10 µm. Supplementary material 2 (TIFF 9327 kb)
10534_2018_85_MOESM3_ESM.docx (13 kb)
Supplementary material 3 (DOCX 13 kb)

References

  1. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410.  https://doi.org/10.1016/S0022-2836(05)80360-2 CrossRefPubMedGoogle Scholar
  2. Borovička J, Řanda Z (2007) Distribution of iron, cobalt, zinc and selenium in macrofungi. Mycol Prog 6:249–259.  https://doi.org/10.1007/s11557-007-0544-y CrossRefGoogle Scholar
  3. Boch A, Trampczynska A, Simm C, Taudte N, Krämer U, Clemen S (2008). Loss of Zhf and the tightly regulated zinc-uptake system SpZrt1 in Schizosaccharomyces pombe reveals the delicacy of cellular zinc balance. FEMS Yeast Res 8:883–896.  https://doi.org/10.1111/j.1567-1364.2008.00414.x CrossRefPubMedGoogle Scholar
  4. Cohen CK, Garvin DF, Kochian LV (2004) Kinetic properties of a micronutrient transporter from Pisum sativum indicate a primary function in Fe uptake from the soil. Planta 218:784–792.  https://doi.org/10.1007/s00425-003-1156-7 CrossRefPubMedGoogle Scholar
  5. Colpaert JV, Wevers JHL, Krznaric E, Adriaensen K (2011) How metal-tolerant ecotypes of ectomycorrhizal fungi protect plants from heavy metal pollution. Ann Sci 68:17–24.  https://doi.org/10.1007/s13595-010-0003-9 CrossRefGoogle Scholar
  6. Dainty SJ, Kennedy C, Watt S, Bähler J, Whitehall SK (2008) Response of Schizosaccharomyces pombe to zinc deficiency. Eukaryot Cell 7:454–464.  https://doi.org/10.1128/EC.00408-07 CrossRefPubMedPubMedCentralGoogle Scholar
  7. Do E, Hu G, Caza M, Kronstad JW, Jung WH (2016) The ZIP family zinc transporters support the virulence of Cryptococcus neoformans. Med Mycol 54:605–615.  https://doi.org/10.1093/mmy/myw013 CrossRefPubMedPubMedCentralGoogle Scholar
  8. Drew D, Newstead S, Sonoda Y, Kim H, von Heijne G, Iwata S (2008) GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae. Nat Protoc 3:784–798.  https://doi.org/10.1038/nprot.2008.44 CrossRefPubMedPubMedCentralGoogle Scholar
  9. Dufner-Beattie J, Langmade SJ, Wang F, Eide D, Andrews GK (2003) Structure, function, and regulation of a subfamily of mouse zinc transporter genes. J Biol Chem 278:50142–50150.  https://doi.org/10.1074/jbc.M304163200 CrossRefPubMedGoogle Scholar
  10. Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797.  https://doi.org/10.1093/nar/gkh340 CrossRefPubMedPubMedCentralGoogle Scholar
  11. Eide DJ (2006) Zinc transporters and the cellular trafficking of zinc. Biochim Biophys Acta 1763:711–722.  https://doi.org/10.1016/j.bbamcr.2006.03.005 CrossRefPubMedGoogle Scholar
  12. 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 93:5624–5628CrossRefPubMedPubMedCentralGoogle Scholar
  13. Falandysz J, Borovička J (2013) Macro and trace mineral constituents and radionuclides in mushrooms: health benefits and risks. Appl Microbiol Biotechnol 97:477–501.  https://doi.org/10.1007/s00253-012-4552-8 CrossRefPubMedGoogle Scholar
  14. Fujishiro H, Yano Y, Takada Y, Tanihara M, Himeno S (2012) Roles of ZIP8, ZIP14, and DMT1 in transport of cadmium and manganese in mouse kidney proximal tubule cells. Metallomics 4:700–708.  https://doi.org/10.1039/c2mt20024d CrossRefPubMedGoogle Scholar
  15. Füzik T, Ulbrich P, Ruml T (2014) Efficient mutagenesis independent of ligation (EMILI). J Microbiol Methods 106:67–71.  https://doi.org/10.1016/j.mimet.2014.08.003 CrossRefPubMedGoogle Scholar
  16. Gadd GM, Rhee YJ, Stephenson K, Wei Z (2012) Geomycology: metals, actinides and biominerals. Environ Microbiol Rep 4:270–296.  https://doi.org/10.1111/j.1758-2229.2011.00283.x CrossRefPubMedGoogle Scholar
  17. Gaither LA, Eide DJ (2001) Eukaryotic zinc transporters and their regulation. Biometals 14:251–270.  https://doi.org/10.1023/A:1012988914300 CrossRefPubMedGoogle Scholar
  18. 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.  https://doi.org/10.1124/mol.107.043588 CrossRefPubMedPubMedCentralGoogle Scholar
  19. Guerinot ML (2000) The ZIP family of metal transporters. Biochim Biophys Acta 1465:90–198.  https://doi.org/10.1016/S0005-2736(00)00138-3 CrossRefGoogle Scholar
  20. Huynh C, Sacks DL, Andrews NW (2006) A Leishmania amazonensis ZIP family iron transporter is essential for parasite replication within macrophage phagolysosomes. J Exp Med 203:2363–2375.  https://doi.org/10.1084/jem.20060559 CrossRefPubMedPubMedCentralGoogle Scholar
  21. Kambe T, Hashimoto A, Fujimoto S (2014) Current understanding of ZIP and ZnT zinc transporters in human health and diseases. Cell Mol Life Sci 71:3281–3295.  https://doi.org/10.1007/s00018-014-1617-0 CrossRefPubMedGoogle Scholar
  22. Kiranmayi P, Tiwari A, Sagar KP, Haritha A, Maruthi Mohan P (2009) Functional characterization of tzn1 and tzn2-zinc transporter genes in Neurospora crassa. Biometals 22:411–420.  https://doi.org/10.1007/s10534-008-9177-0 CrossRefPubMedGoogle Scholar
  23. 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.  https://doi.org/10.1023/A:1026438615520 CrossRefPubMedGoogle Scholar
  24. Kumánovics A, Poruk KE, Osborn KA, Ward DM, Kaplan J (2006) YKE4 (YIL023C) encodes a bidirectional zinc transporter in the endoplasmic reticulum of Saccharomyces cerevisiae. J Biol Chem 281:22566–22574.  https://doi.org/10.1074/jbc.M604730200 CrossRefPubMedGoogle Scholar
  25. Leonhardt T, Sácký J, Šimek P, Šantrůček J, Kotrba P (2014) Metallothionein-like peptides involved in sequestration of Zn in the Zn-accumulating ectomycorrhizal fungus Russula atropurpurea. Metallomics 6:1693–1701.  https://doi.org/10.1039/c4mt00141a CrossRefPubMedGoogle Scholar
  26. Lin SJ, Culotta VC (1996) Suppression of oxidative damage by Saccharomyces cerevisiae ATX2, which encodes a manganese-trafficking protein that localizes to Golgi-like vesicles. Mol Cell Biol 16:6303–6312.  https://doi.org/10.1128/MCB.16.11.6303 CrossRefPubMedPubMedCentralGoogle Scholar
  27. Lin YF, Liang HM, Yang SY, Boch A, Clemens S, Chen CC, Wu JF, Huang JL, Yeh KC (2009) Arabidopsis IRT3 is a zinc-regulated and plasma membrane localized zinc/iron transporter. New Phytol 182:392–404.  https://doi.org/10.1111/j.1469-8137.2009.02766.x CrossRefPubMedGoogle Scholar
  28. MacDiarmid CW, Gaither LA, Eide DJ (2000) Zinc transporters that regulate vacuolar zinc storage in Saccharomyces cerevisiae. EMBO J 19:2845–2855.  https://doi.org/10.1093/emboj/19.12.2845 CrossRefPubMedPubMedCentralGoogle Scholar
  29. MacDiarmid CW, Milanick MA, Eide DJ (2002) Biochemical properties of vacuolar zinc transport systems of Saccharomyces cerevisiae. J Biol Chem 277:39187–39194.  https://doi.org/10.1074/jbc.M205052200 CrossRefPubMedGoogle Scholar
  30. MacDiarmid CW, Milanick MA, Eide DJ (2003) Induction of the ZRC1 metal tolerance gene in zinc-limited yeast confers resistance to zinc shock. J Biol Chem 278:15065–15072.  https://doi.org/10.1074/jbc.M300568200 CrossRefPubMedGoogle Scholar
  31. Milner MJ, Craft E, Yamaji N, Koyama E, Ma JF, Kochian LV (2012) Characterization of the high affinity Zn transporter from Noccaea caerulescens, NcZNT1, and dissection of its promoter for its role in Zn uptake and hyperaccumulation. New Phytol 195:113–123.  https://doi.org/10.1111/j.1469-8137.2012.04144.x CrossRefPubMedGoogle Scholar
  32. 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.  https://doi.org/10.1016/j.bbamem.2006.06.005
  33. Mitchell A, Chang HY, Daugherty L, Fraser M, Hunter S, Lopez R, McAnulla C, McMenamin C, Nuka G, Pesseat S, Sangrador-Vegas A, Scheremetjew M, Rato C, Yong SY, Bateman A, Punta M, Attwood TK, Sigrist CJA, Redaschi N, Rivoire C, Xenarios I, Kahn D, Guyot D, Bork P, Letunic I, Gough J, Oates M, Haft D, Huang H, Natale DA, Wu CH, Orengo C, Sillitoe I, Mi H, Thomas PD, Finn RD (2014) The InterPro protein families database: the classification resource after 15 years. Nucleic Acids Res 43:D213–D221.  https://doi.org/10.1093/nar/gku1243 CrossRefPubMedPubMedCentralGoogle Scholar
  34. Mizuno T, Usui K, Horie K, Nosaka S, Mizuno N, Obata H (2005) Cloning of three ZIP/Nramp transporter genes from a Ni hyperaccumulator plant Thlaspi japonicum and their Ni2+-transport abilities. Plant Physiol Biochem 43:793–801.  https://doi.org/10.1016/j.plaphy.2005.07.006 CrossRefPubMedGoogle Scholar
  35. Mumberg D, Müller R, Funk M (1995) Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene 156:119–122.  https://doi.org/10.1016/0378-1119(95)00037-7 CrossRefPubMedGoogle Scholar
  36. Nevo Y, Nelson N (2006) The NRAMP family of metal-ion transporters. Biochim Biophys Acta 1763:609–620.  https://doi.org/10.1016/j.bbamcr.2006.05.007 CrossRefPubMedGoogle Scholar
  37. Newstead S, Kim H, von Heijne G, Iwata S, Drew D (2007) High-throughput fluorescent-based optimization of eukaryotic membrane protein overexpression and purification in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 104:13936–13941.  https://doi.org/10.1073/pnas.0704546104 CrossRefPubMedPubMedCentralGoogle Scholar
  38. Pemberton LF (2014) Preparation of yeast cells for live-cell imaging and indirect immunofluorescence. In: Smith JS, Burke DJ (eds) Yeast Genetics: Methods and Protocols. Springer, New York, pp 79–90Google Scholar
  39. Rogers EE, Eide DJ, Guerinot ML (2000) Altered selectivity in an Arabidopsis metal transporter. Proc Natl Acad Sci USA 97:12356–12360.  https://doi.org/10.1073/pnas.210214197 CrossRefPubMedPubMedCentralGoogle Scholar
  40. Sácký J, Leonhardt T, Kotrba P (2016) Functional analysis of two genes coding for distinct cation diffusion facilitators of the ectomycorrhizal Zn-accumulating fungus Russula atropurpurea. Biometals 29:349–363.  https://doi.org/10.1007/s10534-016-9920-x CrossRefPubMedGoogle Scholar
  41. Stearman R, Yuan DS, Yamaguchi-Iwai Y, Klausner RD, Dancis A (1996) A permease-oxidase complex involved in high-affinity iron uptake in yeast. Science 271:1552–1557.  https://doi.org/10.1126/science.271.5255.1552 CrossRefPubMedGoogle Scholar
  42. Stephens BW, Cook DR, Grusak MA (2011) Characterization of zinc transport by divalent metal transporters of the ZIP family from the model legume Medicago truncatula. Biometals 24:51–58.  https://doi.org/10.1007/s10534-010-9373-6 CrossRefPubMedGoogle Scholar
  43. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol 30:2725–2729.  https://doi.org/10.1093/molbev/mst197 CrossRefPubMedPubMedCentralGoogle Scholar
  44. Tsirigos KD, Peters C, Shu N, Käll L, Elofsson A (2015) The TOPCONS web server for consensus prediction of membrane protein topology and signal peptides. Nucleic Acids Res 43:W401–W407.  https://doi.org/10.1093/nar/gkv485 CrossRefPubMedPubMedCentralGoogle Scholar
  45. Vert G, Briat JF, Curie C (2001) Arabidopsis irt2 gene encodes a root-periphery iron transporter. Plant J 26:181–189.  https://doi.org/10.1046/j.1365-313x.2001.01018.x CrossRefPubMedGoogle Scholar
  46. Vetter J, Siller I, Horváth Z (1997) Zinc content of sporocarps of basidiomycetous fungi. Mycologia 89:481–483.  https://doi.org/10.2307/3761041 CrossRefGoogle Scholar
  47. Vicentefranqueira R, Moreno MÁ, Leal F, Calera JA (2005) The zrfA and zrfB genes of Aspergillus fumigatus encode the zinc transporter proteins of a zinc uptake system induced in an acid, zinc-depleted environment. Eukaryot Cell 4:837–848.  https://doi.org/10.1128/EC.4.5.837-848.2005 CrossRefPubMedPubMedCentralGoogle Scholar
  48. Zhao H, Eide DJ (1996a) The yeast ZRT1 gene encodes the zinc transporter protein of a high-affinity uptake system induced by zinc limitation. Proc Natl Acad Sci USA 93:2454–2458CrossRefPubMedPubMedCentralGoogle Scholar
  49. Zhao H, Eide DJ (1996b) The ZRT2 gene encodes the low affinity zinc transporter in Saccharomyces cerevisiae. J Biol Chem 271:23203–23210CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Biochemistry and MicrobiologyUniversity of Chemistry and Technology, PraguePragueCzech Republic

Personalised recommendations