, 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 KotrbaEmail author


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.


Russula atropurpurea Metal uptake Zinc transporter ZIP family Ectomycorrhizal fungi 



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)


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Authors and Affiliations

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

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