1. Anchoring plant metallothioneins to the inner face of the plasma membrane of Saccharomyces cerevisiae cells leads to heavy metal accumulation
- Author
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Atle M. Bones, Ileana C. Farcasanu, Simona Ghenea, Ioana Nicolau, Lavinia L. Ruta, Ralph Kissen, Aurora Neagoe, and Ya-Fen Lin
- Subjects
0106 biological sciences ,0301 basic medicine ,Cell Membranes ,Arabidopsis ,lcsh:Medicine ,Yeast and Fungal Models ,Heavy Metals ,Toxicology ,Pathology and Laboratory Medicine ,Biochemistry ,01 natural sciences ,Green fluorescent protein ,Medicine and Health Sciences ,Fluorescence microscope ,Toxins ,Arabidopsis thaliana ,Cloning, Molecular ,lcsh:Science ,Multidisciplinary ,biology ,Reverse Transcriptase Polymerase Chain Reaction ,Plants ,Complementary DNA ,Nucleic acids ,Chemistry ,Experimental Organism Systems ,Physical Sciences ,Cellular Structures and Organelles ,Research Article ,Chemical Elements ,DNA, Complementary ,Forms of DNA ,Arabidopsis Thaliana ,Green Fluorescent Proteins ,Toxic Agents ,Saccharomyces cerevisiae ,Brassica ,Research and Analysis Methods ,Saccharomyces ,03 medical and health sciences ,Model Organisms ,Plant and Algal Models ,Metals, Heavy ,010608 biotechnology ,Genetics ,Molecular Biology Techniques ,Molecular Biology ,Myristoylation ,Arabidopsis Proteins ,lcsh:R ,Cell Membrane ,Organisms ,Fungi ,Biology and Life Sciences ,Membrane Proteins ,Cell Biology ,DNA ,biology.organism_classification ,Yeast ,Cytosol ,030104 developmental biology ,Membrane protein ,lcsh:Q ,Metallothionein ,Cloning - 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. © 2017 Ruta et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
- Published
- 2017