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

Agaricus crocodilinus (Agaricaceae), an edible saprotrophic mushroom, accumulates high concentrations of cadmium (Cd) in unpolluted environments. This study investigates whether this species has evolved mechanisms to store Cd complexed with metallothioneins (MTs), proteins that bind heavy metal ions via cysteinyl (Cys)–thiolate bonds, how these MTs originated, and how similar mechanisms are present in other fungal species. Size exclusion chromatography revealed that a substantial fraction of Cd in A. crocodilinus sporocarps was sequestered in a 3.4 kDa complex containing Cys-rich peptides. Screening a sporocarp cDNA expression library in a Cd-sensitive Saccharomyces cerevisiae strain identified two MT transcripts, AcMT1 and AcMT2, encoding 49-amino acid (AA) AcMT1 with 10 Cys and 32-AA AcMT2 with 7 Cys. The presence of AcMT2 in the 3.4 kDa Cd–peptide complex isolated from sporocarp was confirmed by mass spectrometry. In mycelial isolates exposed to heavy metals, AcMT1 was more strongly upregulated, while AcMT2 was more expressed under normal conditions. Sequence comparisons revealed that AcMT2 is closer to the ancestral gene, whereas AcMT1 is a more recent duplicate. Combined bioinformatic and functional evidence supports AcMT2 as a constitutively expressed MT involved in Cd binding in the sporocarp, while AcMT1, though more inducible in mycelia and more protective in yeast, appears to serve a transient detoxification role. Moreover, the gene duplication and domain rearrangement mechanism underlying this MT diversification was also identified in other Agaricales and Boletales species.