Parallel topology of genetically fused EmrE homodimers.
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.
Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel.
Sonia
S
Steiner-Mordoch
Misha
M
Soskine
Dalia
D
Solomon
Dvir
D
Rotem
Ayala
A
Gold
Michal
M
Yechieli
Yoav
Y
Adam
Shimon
S
Schuldiner
D000595:Amino Acid Sequence; D017920:Antiporters / Q000737:chemistry* / Q000235:genetics*; D001693:Biological Transport, Active / Q000235:genetics; D003593:Cytoplasm / Q000737:chemistry / Q000235:genetics; D019281:Dimerization; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics*; D004579:Electron Transport / Q000235:genetics; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics*; D004996:Ethidium / Q000737:chemistry / Q000493:pharmacokinetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics*; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis* / Q000737:chemistry / Q000235:genetics*; D013379:Substrate Specificity / Q000235:genetics; D013816:Thermodynamics
D017920:Antiporters; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C075436:EmrE protein, E coli; D004996:Ethidium