Abstract
As we have shown previously, the Cu and Ag concentrations in the sporocarps of Ag-hyperaccumulating Amanita strobiliformis are correlated, and both metals share the same uptake system and are sequestered by the same metallothioneins intracellularly. To further improve our knowledge of the Cu and Ag handling in A. strobiliformis cells, we searched its transcriptome for the P1B-1-ATPases, recognizing Cu+ and Ag+ for transport. We identified transcripts encoding 1097-amino acid (AA) AsCRD1 and 978-AA AsCCC2, which were further subjected to functional studies in metal sensitive Saccharomyces cerevisiae. The expression of AsCRD1 conferred highly increased Cu and Ag tolerance to metal sensitive yeasts in which the functional AsCRD1:GFP (green fluorescent protein) fusion localized exclusively to the tonoplast, indicating that the AsCRD1-mediated Cu and Ag tolerance was a result of vacuolar sequestration of the metals. Increased accumulation of AsCRD1 transcripts observed in A. strobiliformis mycelium upon the treatments with Cu and Ag (8.7- and 4.5-fold in the presence of 5 μM metal, respectively) supported the notion that AsCRD1 can be involved in protection of the A. strobiliformis cells against the toxicity of both metals. Neither Cu nor Ag affected the levels of AsCCC2 transcripts. Heterologous expression of AsCCC2 in mutant yeasts did not contribute to Cu tolerance, but complemented the mutant genotype of the S. cerevisiae ccc2Δ strain. Consistent with the role of the yeast Ccc2 in the trafficking of Cu from cytoplasm to nascent proteins via post-Golgi, the GFP fluorescence in AsCCC2-expressing ccc2Δ yeasts localized among Golgi-like punctate foci within the cells. The AsCRD1- and AsCCC2-associated phenotypes were lost in yeasts expressing mutant transporter variants in which a conserved phosphorylation/dephosphorylation site was altered. Altogether, the data support the roles of AsCRD1 and AsCCC2 as genuine P1B-1-ATPases, and indicate their important functions in the removal of toxic excess of Cu and Ag from the cytoplasm and charging the endomembrane system with Cu, respectively.
Highlights
Studies have revealed that ectomycorrhizal (EM) fungi effectively mobilize heavy metals from soils and minerals (Gadd et al, 2012) and that ectomycorrhizae improve plant fitness in metal polluted environments because metal tolerant mycobionts function as a barrier for the entry of metals into plant tissues (Colpaert et al, 2011; Reddy et al, 2016)
We have documented that the intracellular detoxification of Cu and Ag in A. strobiliformis largely relies upon binding with cysteinyl-rich, cytosolic metallothionein (MT) peptides, AsMT1a, 1b, and 1c (Osobová et al, 2011; Beneš et al, 2016; Hložková et al, 2016); and that two A. strobiliformis transporters of the copper transporter family (CTR; AsCTR2 and AsCTR3) can recognize Cu, and Ag for uptake (Beneš et al, 2016)
These involve putative N-terminal Cu/Agbinding CxxC motifs and two P1B−1 subgroup signature sequences in predicted transmembrane domains (TMD), Nx6YNx4P (x represents any amino acid (AA) residue), and Px6MxxSSx5S, which are in P1B−1-ATPases conserved in TMD7 and TMD8, respectively (Figure 1 and Supplementary Figure S1)
Summary
Studies have revealed that ectomycorrhizal (EM) fungi effectively mobilize heavy metals from soils and minerals (Gadd et al, 2012) and that ectomycorrhizae improve plant fitness in metal polluted environments because metal tolerant mycobionts function as a barrier for the entry of metals into plant tissues (Colpaert et al, 2011; Reddy et al, 2016). It is noteworthy that studies indicate that macrofungi could be considered the most effective Ag accumulators among eukaryotes with two known outstanding EM species, Amanita strobiliformis and Amanita solitaria (Borovicka et al, 2007, 2010). Studies in eukaryotes have revealed that while CTRs transport Cu ions into the cytoplasm, the members of P1B−1 subgroup of P1B-type ATPases ( called heavy metal ATPases, HMA) contribute to the homeostasis and redistribution of essential Cu by exporting the metal ion from the cytoplasm into the subcellular compartments or out of the cell (Nevitt et al, 2012; Bashir et al, 2016). The transporters highly specific for monovalent Cu ions (the dominant intracellular Cu species in eukaryotes; Nevitt et al, 2012) comprise P1B−1-subgroup, while P1B−2, P1B−3, and P1B−4 transport Cd2+/Zn2+/Pb2+, Cu+/Cu2+, and Co2+, respectively
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