Homeostatic mechanisms preventing the toxicity of free Zn ions in cells involve, among others, cytosolic Zn-binding ligands, particularly the cysteine-rich metallothioneins (MTs). Here we examined the Zn-binding peptides of Russula atropurpurea, an ectomycorrhizal fungus known for its ability to accumulate high amounts of Zn in its sporocarps. The Zn complexes and their peptide ligands were characterized using chromatography, electrophoresis after fluorescent labeling of cysteine residues, and tandem mass spectrometry. Functional complementation assays in Saccharomyces cerevisiae were used to obtain and characterize cDNA sequences. Zn-speciation analysis showed that nearly 80% of the Zn extracted from the sporocarps was associated with cysteine-containing peptides in a 5 kDa complex. Screening of an R. atropurpurea cDNA library for sequences encoding peptides capable of sequestering divalent heavy metals was conducted in the Cd-hypersensitive ycf1Δ yeast. This allowed identification of two cDNAs, RaZBP1 and RaZBP2, which protected the metal-sensitive yeast mutants against Cd and Zn, but not Co, Mn or Cu, toxicity. The corresponding RaZBP1 and RaZBP2 peptides consisting of 53 amino acid (AA) residues and sharing 77% identity showed only a limited sequence similarity to known MTs, particularly due to the absence of multiple Cys-AA-Cys motifs. Both RaZBPs were detected in a native Zn-complex of R. atropurpurea and the recombinant RaZBP1 was found associated with Zn and Cd in yeasts. Altogether, the results point to an important role of RaZBPs in the handling of a substantial portion of the Zn pool in R. atropurpurea.

• MeSH
• Basidiomycota metabolismus   MeSH
• intracelulární prostor metabolismus   MeSH
• kadmium metabolismus   MeSH
• metalothionein chemie   izolace a purifikace   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• mutace genetika   MeSH
• mykorhiza metabolismus   MeSH
• peptidy chemie   izolace a purifikace   metabolismus   MeSH
• Saccharomyces cerevisiae metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční analýza proteinů MeSH
• sekvenční seřazení MeSH
• zinek chemie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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.

• MeSH
• Basidiomycota genetika   růst a vývoj   MeSH
• biologický transport účinky léků   MeSH
• fungální proteiny genetika   metabolismus   MeSH
• mykorhiza chemie   genetika   MeSH
• proteiny přenášející kationty MeSH
• regulace genové exprese u rostlin MeSH
• rostlinné proteiny chemie   genetika   metabolismus   MeSH
• sekvence aminokyselin genetika   MeSH
• zinek chemie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Russula atropurpurea can accumulate remarkably high concentrations of Zn in its sporocarps. We have previously demonstrated that 40 % of the intracellular Zn in this species is sequestered by MT-like RaZBP peptides. To see what other mechanisms for the handling of the accumulated Zn are available to R. atropurpurea, we searched its transcriptome for cDNAs coding for transporters of the cation diffusion facilitator (CDF) family. The transcriptome search enabled us to identify RaCDF1 and RaCDF2, which were further subjected to functional studies in metal sensitive Saccharomyces cerevisiae. The expression of RaCDF1 and its translational fusion with green fluorescent protein (GFP) protected the yeasts against Zn and Co, but not Cd or Mn, toxicity and led to increased Zn accumulation in the cells. The GFP fluorescence, observed in the RaCDF1::GFP-expressing yeasts on tonoplasts, indicated that the RaCDF1-mediated Zn and Co tolerance was a result of vacuolar sequestration of the metals. The expression of RaCDF2 supported Zn, but not Mn, tolerance in the yeasts and reduced the cellular uptake of Zn, which is congruent with the proposed idea of the Zn-efflux function of RaCDF2, supported by the localization of GFP-derived fluorescence on the plasma membrane of the yeasts expressing functional RaCDF2::GFP. Contrarily, RaCDF2 increased the sensitivity to Co and Cd in the yeasts and significantly promoted Cd uptake, which suggested that it can act as a bidirectional metal transporter. The notion that RaCDF1 and RaCDF2 are genuine CDF transporters in R. atropurputrea was further reinforced by the fact that the RaCDF-associated metal tolerance and uptake phenotypes were lost upon the replacement of histidyl (in RaCDF1) and aspartyl (in RaCDF2), which are highly conserved in the second transmembrane domain and known to be essential for the function of CDF proteins.

• MeSH
• aktivace transkripce MeSH
• antifungální látky farmakologie   MeSH
• fungální proteiny chemie   genetika   metabolismus   MeSH
• fyziologická adaptace MeSH
• geny hub MeSH
• kobalt farmakologie   MeSH
• membránové transportní proteiny chemie   genetika   metabolismus   MeSH
• mikrobiální testy citlivosti MeSH
• mikrobiální viabilita MeSH
• mykorhiza genetika   metabolismus   MeSH
• regulace genové exprese u hub MeSH
• Saccharomyces cerevisiae účinky léků   růst a vývoj   MeSH
• sekvence aminokyselin MeSH
• zinek metabolismus   farmakologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH