The gilled mushroom Agaricus crocodilinus (Agaricaceae) analyzed in this study hyperaccumulated Cd and showed common Zn and very low Mn concentrations. To gain an insight into the handling of heavy metals in this saprotrophic species, its two genes of the cation diffusion facilitator (CDF) protein family were isolated, AcCDF1 and AcCDF2, encoding the membrane transporters of the Zn-CDF and Mn-CDF subfamilies, respectively. When expressed in the model, metal-sensitive yeast, AcCDF1 conferred marked Zn tolerance and promoted the intracellular accumulation of Zn. Green fluorescent protein (GFP) tagging of AcCDF1 visualized the functional protein predominantly in the tonoplast, indicating that AcCDF1 can mediate the transport of Zn into vacuoles, which are used for deposition of excess Zn in most fungi. AcCDF2 conferred a high degree of Mn tolerance to model yeast, in which the transport-active AcCDF2:GFP fusion was localized to the plasma membrane, suggesting a role in Mn export and thus reduced Mn accumulation. Furthermore, the AcCDF2 gene appeared to be Mn-inducible in A. crocodilinus, suggesting an Mn efflux function of AcCDF2. Neither AcCDFs nor the mutant AcCDF1 variants constructed to mimic transmembrane tetrahedral Cd transport sites manifested appreciable Cd-related phenotypes in yeast models, and further efforts are needed to elucidate the mechanism underlying Cd hyperaccumulation in A. crocodilinus.

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 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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Identification of two metallothioneins in Agaricus crocodilinus reveals gene duplication and domain expansion, a pattern conserved across fungal species