title: Functional analysis of two genes coding for distinct cation diffusion facilitators of the ectomycorrhizal Zn-accumulating fungus Russula atropurpurea.
abstract: Russula atropurpurea can accumulate remarkably high concentrations of Zn in its sporocarps. We have previously demonstrated that 40 % of the intracellular Zn in this species is sequestered by MT-like RaZBP peptides. To see what other mechanisms for the handling of the accumulated Zn are available to R. atropurpurea, we searched its transcriptome for cDNAs coding for transporters of the cation diffusion facilitator (CDF) family. The transcriptome search enabled us to identify RaCDF1 and RaCDF2, which were further subjected to functional studies in metal sensitive Saccharomyces cerevisiae. The expression of RaCDF1 and its translational fusion with green fluorescent protein (GFP) protected the yeasts against Zn and Co, but not Cd or Mn, toxicity and led to increased Zn accumulation in the cells. The GFP fluorescence, observed in the RaCDF1::GFP-expressing yeasts on tonoplasts, indicated that the RaCDF1-mediated Zn and Co tolerance was a result of vacuolar sequestration of the metals. The expression of RaCDF2 supported Zn, but not Mn, tolerance in the yeasts and reduced the cellular uptake of Zn, which is congruent with the proposed idea of the Zn-efflux function of RaCDF2, supported by the localization of GFP-derived fluorescence on the plasma membrane of the yeasts expressing functional RaCDF2::GFP. Contrarily, RaCDF2 increased the sensitivity to Co and Cd in the yeasts and significantly promoted Cd uptake, which suggested that it can act as a bidirectional metal transporter. The notion that RaCDF1 and RaCDF2 are genuine CDF transporters in R. atropurputrea was further reinforced by the fact that the RaCDF-associated metal tolerance and uptake phenotypes were lost upon the replacement of histidyl (in RaCDF1) and aspartyl (in RaCDF2), which are highly conserved in the second transmembrane domain and known to be essential for the function of CDF proteins.
authors.affiliation: Department of Biochemistry and Microbiology, University of Chemistry and Technology, Prague, Technická 3, 166 28, Prague, Czech Republic.
authors.forename: Jan
authors.initials: J
authors.lastname: Sácký
authors.affiliation: Department of Biochemistry and Microbiology, University of Chemistry and Technology, Prague, Technická 3, 166 28, Prague, Czech Republic.
authors.forename: Tereza
authors.initials: T
authors.lastname: Leonhardt
authors.affiliation: Department of Biochemistry and Microbiology, University of Chemistry and Technology, Prague, Technická 3, 166 28, Prague, Czech Republic. pavel.kotrba@vscht.cz.
authors.forename: Pavel
authors.initials: P
authors.lastname: Kotrba
mesh_terms: D000222:Adaptation, Physiological; D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000494:pharmacology; D003035:Cobalt / Q000494:pharmacology; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D050296:Microbial Viability; D038821:Mycorrhizae / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000254:growth & development; D015533:Transcriptional Activation; D015032:Zinc / Q000378:metabolism / Q000494:pharmacology
chemical_list: D000935:Antifungal Agents; D005656:Fungal Proteins; D026901:Membrane Transport Proteins; D003035:Cobalt; D015032:Zinc
keywords: Cation diffusion facilitator (CDF) family; Ectomycorrhizal fungi; Metal tolerance; Russula atropurpurea; Zinc transport