Parallel topology of genetically fused EmrE homodimers

Authors

  • Sonia Steiner-Mordoch,

    1. Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel
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    • These authors contributed equally to this work
  • Misha Soskine,

    1. Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel
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    • These authors contributed equally to this work
  • Dalia Solomon,

    1. Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel
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  • Dvir Rotem,

    1. Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel
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    • Present address: Department of Chemistry, Oxford University, Oxford, UK
  • Ayala Gold,

    1. Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel
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  • Michal Yechieli,

    1. Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel
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  • Yoav Adam,

    1. Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel
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  • Shimon Schuldiner

    Corresponding author
    1. Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel
    • Corresponding author. Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Silberman Bldg 3-345, Jerusalem, Givat Ram 91904, Israel. Tel.: +972 2 658 5992; Fax: +972 2 563 4625; E-mail: Shimon.Schuldiner@huji.ac.il

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Abstract

EmrE is a small H+-coupled multidrug transporter in Escherichia coli. Claims have been made for an antiparallel topology of this homodimeric protein. However, our own biochemical studies performed with detergent-solubilized purified protein support a parallel topology of the protomers. We developed an alternative approach to constrain the relative topology of the protomers within the dimer so that their activity can be assayed also in vivo before biochemical handling. Tandem EmrE was built with two identical monomers genetically fused tail to head (C-terminus of the first to N-terminus of the second monomer) with hydrophilic linkers of varying length. All the constructs conferred resistance to ethidium by actively removing it from the cytoplasm. The purified proteins bound substrate and transported methyl viologen into proteoliposomes by a proton-dependent mechanism. A tandem where one of the essential glutamates was replaced with glutamine transported only monovalent substrates and displayed a modified stoichiometry. The results support a parallel topology of the protomers in the functional dimer. The implications regarding insertion and evolution of membrane proteins are discussed.

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