Three metallothionein isoforms and sequestration of intracellular silver in the hyperaccumulator Amanita strobiliformis.
Metallothioneins (MTs) are cysteine-rich peptides involved in heavy metal tolerance of many eukaryotes. Here, we examined their involvement in intracellular binding of silver (Ag) in the ectomycorrhizal fungus Amanita strobiliformis. The Ag complexes and their peptide ligands were characterized using chromatography and mass spectrometry. The full-length coding sequences obtained from a cDNA library were used for complementation assays in yeast mutant strains. Abundance of respective transcripts in A. strobiliformis was measured by quantitative real-time reverse-transcribed polymerase chain reaction (qRT-PCR). Ag-speciation analyses showed that intracellular Ag was in wild-grown fruit bodies and cultured extraradical mycelia of A. strobiliformis sequestered by metallothioneins. The determined sequence of the peptide facilitated isolation of three cDNA clones, AsMT1a, AsMT1b and AsMT1c. These encode isomorphic MTs consisting of 34 amino acid residues and sharing 82% identity. In mycelia the expression of AsMT1s is induced by Ag. All AsMT1s expressed in yeasts complemented hypersensitivity of mutants to cadmium (Cd) and copper (Cu) and formed Ag complexes. Only the Ag-AsMT1a complex was detected in the A. strobiliformis fruit body in which AsMT1a was the prevailing transcript. The present study identified the existence of metallothionein isoforms in ectomycorrhizal fungi. We demonstrated that intracellular sequestration of Ag in fruit bodies and mycelia of hyperaccumulating A. strobiliformis is dominated by metallothioneins.
Department of Biochemistry and Microbiology, Institute of Chemical Technology, Prague, Technická 3, Prague, 166 28 Czech Republic.
Michaela
M
Osobová
Department of Biochemistry and Microbiology, Institute of Chemical Technology, Prague, Technická 3, Prague, 166 28 Czech Republic.
Václav
V
Urban
Department of Parasitology, Faculty of Science, Charles University in Prague, Viničná 7, Prague, 128 44 Czech Republic.
Petr L
PL
Jedelský
Nuclear Physics Institute, v.v.i, Academy of Sciences of the Czech Republic, 250 68 Řež, Czech Republic.
Jan
J
Borovička
Institute of Microbiology, v.v.i., Academy of Sciences of the Czech Republic, Vídeňská 1083, CZ-142 20 Prague 4, Czech Republic.
Milan
M
Gryndler
Department of Biochemistry and Microbiology, Institute of Chemical Technology, Prague, Technická 3, Prague, 166 28 Czech Republic.
Tomas
T
Ruml
Department of Biochemistry and Microbiology, Institute of Chemical Technology, Prague, Technická 3, Prague, 166 28 Czech Republic.
Pavel
P
Kotrba
D000545:Amanita / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D002104:Cadmium / Q000378:metabolism; D003300:Copper / Q000378:metabolism; D048690:Fruiting Bodies, Fungal / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D008668:Metallothionein / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D025282:Mycelium / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D017386:Sequence Homology, Amino Acid; D012834:Silver / Q000378:metabolism*
D005656:Fungal Proteins; D020033:Protein Isoforms; D002104:Cadmium; D012834:Silver; D003300:Copper; D008668:Metallothionein
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