Intracellular sequestration of cadmium and zinc in ectomycorrhizal fungus Amanita muscaria (Agaricales, Amanitaceae) and characterization of its metallothionein gene

• Published in: Fungal genetics and biology 2022-09, Vol.162, p.103717-103717, Article 103717
• Main Authors: Sácký, Jan, Chaloupecká, Anna, Kaňa, Antonín, Šantrůček, Jiří, Borovička, Jan, Leonhardt, Tereza, Kotrba, Pavel
• Format: Article
• Language: English
• Subjects: Amanita; Macrofungi; Metal accumulation; Metal binding peptides; Metal pollution; Russula
• ISSN: 1087-1845; 1096-0937
• DOI: 10.1016/j.fgb.2022.103717

[Display omitted] •Amanita muscaria contains elevated Cd and Zn that can intracellularly store bound with peptidaceous ligands.•Its metallothionein (MT) AmMT1 gene responds weakly to Cd and Zn, likely serving basal metal homeostasis, not detoxification, function.•AmMT1 plus its A. strobiliformis AsMT3, and Russula atropurpurea RaMT1 homologs were inspected in yeasts.•All MT homologs can protect yeasts against Cd, Zn and Cu toxicity, albeit with different efficiencies.•Mutagenesis indicated that C-terminal parts of these homologs contribute to Cu binding, while conserved histidyl has structural function. 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.