Fungal activity significantly contributes to the turnover of minerals and other nutrients in the environment. Some fungi exhibit a remarkable ability to accumulate heavy metals; however, the molecular basis of this process remains poorly understood. This study investigates the metal tolerance and uptake capabilities of Agaricus crocodilinus mycelium, focusing on cadmium (Cd) and zinc (Zn). The mycelium was isolated from a naturally growing sporocarp that showed a Cd concentration of 149 mg kg⁻¹ dry weight, roughly 100-fold higher than typical mushroom levels. Mycelial metal accumulation assays demonstrated a linear relationship for Cd uptake (R2 = 0.9977) but a logarithmic relationship for Zn uptake (R2 = 0.9965), suggesting unregulated Cd uptake and regulated Zn uptake. Comparison of the A. crocodilinus Zinc-regulated transporter/Iron-regulated transporter-like Protein (ZIP) gene AcZIP1, showed homology with known high-affinity Zn transporters in Agaricomycetes. Functional expression of AcZIP1 in Saccharomyces cerevisiae supported its role in Zn and Cd transport, with localization studies indicating its presence at the plasma membrane. Zn exposure reduced AcZIP1 expression in mycelium ~ 3-fold, while Cd had no significant effect. This study improves our understanding of the possible means of metal uptake in A. crocodilinus and other Agaricomycetes.

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

Institute of Geology of the Czech Academy of Sciences Rozvojová 269 Prague 6 16500 Czech Republic

Nuclear Physics Institute of the Czech Academy of Sciences Hlavní 130 Husinec Řež 25068 Czech Republic

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