Amanita muscaria is an ectomycorrhizal mushroom that commonly grows at metal-polluted sites. Sporocarps from the lead smelter-polluted area near Příbram (Central Bohemia, Czech Republic) showed elevated concentrations of Cd and Zn. Size exclusion chromatography of the cell extracts of the sporocarps from both polluted and unpolluted sites indicated that substantial part of intracellular Cd and Zn was sequestered in 6-kDa complexes, presumably with metallothionein(s) (MT). When the cultured mycelial isolates were compared, those from Příbram were more Cd-tolerant and accumulated slightly less Cd and Zn than those from the unpolluted site. The analysis of the available A.muscaria sequence data returned a 67-amino acid (AA) MT encoded by the AmMT1 gene. Weak Cd and Zn responsiveness of AmMT1 in the mycelia suggested its metal homeostasis function in A.muscaria, rather than a major role in detoxification. The AmMT1 belongs to a ubiquitous peptide group in the Agaricomycetes consisting of 60-70-AA MTs containing seven cysteinyl domains and a conserved histidyl, features observed also in a newly predicted, atypical 45-AA RaMT1 of the Zn-accumulator Russula bresadolae in which the C-terminal cysteinyl domains VI and VII are missing. Heterologous expression in metal-sensitive yeast mutants indicated that AmMT1 and RaMT1 encode functional peptides that can protect cells against Cd, Zn, and Cu toxicity. The metal protection phenotype observed in yeasts with mutant variants of AmMT1 and RaMT1 further indicated that the conserved histidyl seems to play a structural, not metal binding role, and the cysteinyls of the C-terminal domains VI and VII are important for Cu binding. The data provide an important insight into the metal handling of site-associated ectomycorrhizal species disturbed by excess metals and the properties of MTs common in Agaricomycetes.

Department of Analytical Chemistry University of Chemistry and Technology Prague Technická 5 166 28 Prague Czech Republic

Department of Biochemistry and Microbiology University of Chemistry and Technology Prague Technická 3 166 28 Prague 6 Czech Republic

Institute of Geology of the Czech Academy of Sciences Rozvojová 269 16500 Prague 6 Czech Republic; Nuclear Physics Institute of the Czech Academy of Sciences Hlavní 130 25068 Husinec Řež Czech Republic

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Translocation of elements and fractionation of Mg, Cu, Zn, and Cd stable isotopes in a penny bun mushroom (Boletus edulis) from western Czech Republic