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

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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.

Figures

  • Table 1. Plasmids used to clone and express myrGFP-MTx.
  • Fig 1. Expression of myrGFP-MTx in yeast cells. A. Expression of myrGFP-MTx was checked by agarose gel electrophoresis on amplicons obtained by Reverse-Transcription PCR made on RNA extracted from BY4741 cells transformed with pGRD-myrGFP::MTx series (Table 1) and grown in SGal-Ura as described in Materials and methods. myrGFP::mt, cDNA fragment containing myrGFP and 67 bp of the MT sequence. ACT1, fragment of actin gene (control). B. Cellular localization of myrGFP-MTx. Transgenic cells expressing myrGFP-MTx were prepared for visualization as described in Materials and methods. Live cell fluorescence revealed the concentration at the plasma membrane of the transgenic myrGFP-MTx studied. Cells expressing cytosolic GFP (up, left) were obtained by transforming yeast with pGREG600 [33], a plasmid harboring GFP under GAL1 control. The experiments were done on at least three independent transformants, with similar results. For each strain, one representative example is shown.
  • Fig 2. Growth of yeast cells expressing myrGFP-MTx. The BY4741 cells transformed with the pGRD-myrGFP::MTx series were shifted to SGal-Ura at density 1 x 106 cells/mL (OD600 = 0.1) for transgene induction, as described in Materials and methods. The growth was determined for each strain spectrophotometrically (OD600) 24 h after the galactose shift. Values are means ± standard deviation of three independent data. Asterisks indicate that the mean of the myrGFP-MTx strain is significantly different from the mean of the myrGFP control under the same conditions, according to one sample t test. *p < 0.05, **p < 0.01.
  • Table 2. Metal accumulation from minimal medium by yeast cells expressing myrGFP-MTx.
  • Fig 3. Cd(II) accumulation by yeast cells expressing myrGFP-MTx. Early log phase growing cells transformed with pGRD-myrGFP::MTx series were shifted to SGal-Ura for transgene induction as described in Materials and methods. Four hours after the galactose shift, CdCl2 was added (0.05 mM final concentration) A. Growth of yeast cells expressing myrGFP-MTx under Cd(II) surplus. The cell growth was determined for each strain spectrophotometrically (OD600) 20 h after adding the metal salt. B. Cd(II) accumulation. Cells were exposed to Cd(II) for 2 hours (30˚C, 200 rpm) before being processed for metal assay by ICP-MS. The accumulated metal was normalized to cell total protein. Values are mean ± standard deviation of three independent data. Asterisks indicate that the mean of the myrGFP-MTx strain was significantly different from the mean of the myrGFP control under the same conditions, according to one sample t test. *p < 0.05, **p < 0.01.
  • Fig 4. Co(II) accumulation by yeast cells expressing myrGFP-MTx. Yeast cells were manipulated as described in Fig 3, except that CoCl2 was added at final concentration 0.5 mM. A. Growth of yeast cells expressing myrGFP-MTx under Co(II) surplus. The cell growth was determined for each strain spectrophotometrically (OD600) 20 h after adding the metal salt. B. Co(II) accumulation. Cells were exposed to Co(II) for 2 hours (30˚C, 200 rpm) before being processed for metal assay by ICP-MS. Accumulated metal was normalized to cell total protein. Values are mean ± standard deviation of three independent data. Asterisks indicate that the mean of the myrGFP-MTx strain was significantly different from the mean of the myrGFP control under the same conditions, according to one sample t test. *p < 0.05, **p < 0.01, ***p < 0.001.
  • Fig 5. Cu(II) accumulation by yeast cells expressing myrGFP-MTx. Yeast cells were manipulated as described in Fig 3, except that CuCl2 was added at a final concentration of 0.5 mM. A. Growth of yeast cells expressing myrGFP-MTx under Cu(II) surplus. The cell growth was determined for each strain spectrophotometrically (OD600) 20 h after adding the metal salt. B. Cu(II) accumulation. Cells were exposed to Cu(II) for 2 hours (30˚C, 200 rpm) before being processed for metal assay by ICP-MS. Accumulated metal was normalized to cell total protein. Values are mean ± standard deviation of three independent data. Asterisks indicate that the mean of the myrGFP-MTx strain was significantly different from the mean of the myrGFP control under the same conditions, according to one sample t test. *p < 0.05, **p < 0.01, ***p < 0.001.C. Cu(II) accumulation from solid medium. Cells were 10-fold serially diluted in a multi-well plate and stamped with a replicator on SGal-Ura plates containing the specified concentrations of Cu(II). Plates were photographed after 6 days’ incubation at 30˚C. One representative plate is shown out of three similar experiments.
  • Fig 6. Mn(II) accumulation by yeast cells expressing myrGFP-MTx. Yeast cells were manipulated as described in Fig 3, except that MnCl2 was added at a final concentration of 1 mM. A. Growth of yeast cells expressing myrGFP-MTx under Mn(II) surplus. The cell growth was determined for each strain spectrophotometrically (OD600) 20 h after adding the metal. B. Mn(II) accumulation. Cells were exposed to Mn(II) for 2 hours (30˚C, 200 rpm) before being processed for metal assay by ICP-MS. Accumulated metal was normalized to cell total protein. Values are mean ± standard deviation of three independent data. Asterisks indicate that the mean of the myrGFP-MT strain is significantly different from the mean of the myrGFP control under the same conditions, according to one sample t test. *p < 0.05, **p < 0.01.

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Ruta, L. L., Lin, Y. F., Kissen, R., Nicolau, I., Neagoe, A. D., Ghenea, S., … Farcasanu, I. C. (2017). Anchoring plant metallothioneins to the inner face of the plasma membrane of Saccharomyces cerevisiae cells leads to heavy metal accumulation. PLoS ONE, 12(5). https://doi.org/10.1371/journal.pone.0178393

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