Arsenic (As) is a toxic metalloid widespread in the environment, and many organisms have evolved mechanisms to mitigate its toxic effects. Bioinformatic analyses revealed that acr3 genes are predominantly distributed in mushrooms, highlighting their evolutionary and functional importance in eukaryotic arsenic metabolism. In this study, two homologous genes, HbACR3 and HsACR3, from the mushrooms Hebeloma bulbiferum and Hebeloma sinapizans were identified and functionally characterized. Both encode 399-amino-acid membrane proteins showing 99% sequence identity to each other and substantial similarity to previously characterized ACR3-type arsenite transporters from plants, yeasts, and bacteria. Heterologous expression of HbACR3 and HsACR3 in a Saccharomyces cerevisiae arr3Δ mutant restored resistance to arsenite and arsenate and significantly reduced intracellular arsenic accumulation. Fluorescence microscopy of GFP-tagged HbACR3 and HsACR3 confirmed their localization to the plasma membrane, consistent with an efflux transport function. Exposure of H. bulbiferum and H. sinapizans mycelia to arsenate led to a significant but differential transcriptional upregulation of both genes. This work provides new insight into the evolution, distribution, and physiological significance of ACR3 transporters in eukaryotic arsenic homeostasis. KEY POINTS: Acr3 genes are widespread in fungi, indicating a key role in arsenic detoxification. HbACR3 and HsACR3 reduce cellular arsenic and confer As(III) tolerance. Arsenate exposure strongly induces HbACR3 and HsACR3 gene expression.

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

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

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 Czech Republic

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