Anchoring plant metallothioneins to the inner face of the plasma membrane of Saccharomyces cerevisiae cells leads to heavy metal accumulation

PLoS One. 2017 May 31;12(5):e0178393. doi: 10.1371/journal.pone.0178393. eCollection 2017.

Abstract

In this study we engineered yeast cells armed for heavy metal accumulation by targeting plant metallothioneins to the inner face of the yeast plasma membrane. Metallothioneins (MTs) are cysteine-rich proteins involved in the buffering of excess metal ions, especially Cu(I), Zn(II) or Cd(II). The cDNAs of seven Arabidopsis thaliana MTs (AtMT1a, AtMT1c, AtMT2a, AtMT2b, AtMT3, AtMT4a and AtMT4b) and four Noccaea caerulescens MTs (NcMT1, NcMT2a, NcMT2b and NcMT3) were each translationally fused to the C-terminus of a myristoylation green fluorescent protein variant (myrGFP) and expressed in Saccharomyces cerevisiae cells. The myrGFP cassette introduced a yeast myristoylation sequence which allowed directional targeting to the cytosolic face of the plasma membrane along with direct monitoring of the intracellular localization of the recombinant protein by fluorescence microscopy. The yeast strains expressing plant MTs were investigated against an array of heavy metals in order to identify strains which exhibit the (hyper)accumulation phenotype without developing toxicity symptoms. Among the transgenic strains which could accumulate Cu(II), Zn(II) or Cd(II), but also non-canonical metal ions, such as Co(II), Mn(II) or Ni(II), myrGFP-NcMT3 qualified as the best candidate for bioremediation applications, thanks to the robust growth accompanied by significant accumulative capacity.

MeSH terms

  • Arabidopsis / metabolism*
  • Arabidopsis Proteins / genetics
  • Arabidopsis Proteins / metabolism*
  • Cell Membrane / metabolism*
  • Cloning, Molecular
  • DNA, Complementary / genetics
  • Green Fluorescent Proteins / genetics
  • Metallothionein / genetics
  • Metallothionein / metabolism*
  • Metals, Heavy / metabolism*
  • Reverse Transcriptase Polymerase Chain Reaction
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism*

Substances

  • Arabidopsis Proteins
  • DNA, Complementary
  • Metals, Heavy
  • Green Fluorescent Proteins
  • Metallothionein

Grant support

The research leading to these results has received funding from the Romanian – EEA Research Programme operated by the Ministry of National Education under the EEA Financial Mechanism 2009-2014 and Project Contract No 21 SEE/ 30.06.2014. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.