Accumulation of Ag and Cu in Amanita strobiliformis and characterization of its Cu and Ag uptake transporter genes AsCTR2 and AsCTR3.

ABSTRACT

Macrofungi can accumulate in their sporocarps remarkably high concentrations of Cu and Ag. We have previously demonstrated that the non-essential Ag is in the ectomycorrhizal, Ag-hyperaccumulating Amanita strobiliformis sequestered by 3.4-kDa metallothioneins (MTs) produced as AsMT1a, 1b and 1c isoforms. Here, we describe two populations of wild-grown A. strobiliformis sporocarps, which showed certain correlation between the concentrations of accumulated Ag (284 ± 64 and 67 ± 15 mg kg(-1)) and Cu (76 ± 13 and 30 ± 12 mg kg(-1)), suggesting that an overlap may exist in the cell biology of Ag and Cu in this species. Metal speciation analysis revealed that the intracellular Cu in the sporocarps of both populations was, like Ag, associated with the 3.4-kDa MTs. A search of A. strobiliformis transcriptome for sequences encoding proteins of the Cu transporter (CTR) family identified four AsCTR cDNAs, which were, like AsMT1s, confirmed in both populations. The predicted AsCTR proteins showed homology to vacuolar (AsCTR1 and AsCTR4) and plasma membrane (AsCTR2 and AsCTR3) CTRs. Heterologous expression of AsCTR2, AsCTR3 and their translational fusions with green fluorescent protein (GFP) in Cu uptake-deficient S. cerevisiae indicated that both AsCTRs are functional Cu and Ag uptake transporters recombinant genes complemented growth defects and increased Cu and Ag uptake rates in yeasts and the GFP-tagged protein localized to the cell periphery. Site directed mutagenesis revealed the importance of the conserved-among-CTRs M-X3-M motif for the AsCTR2- and AsCTR3-mediated transport of both Cu and Ag. These results provide the first evidence that fungal CTRs can recognize Ag for transport.