Membrane topology of a multidrug efflux transporter, AcrB, in Escherichia coli

J Biochem. 2002 Jan;131(1):145-51. doi: 10.1093/oxfordjournals.jbchem.a003069.

Abstract

AcrA/B in Escherichia coli is a multicomponent system responsible for intrinsic resistance to a wide range of toxic compounds, and probably cooperates with the outer membrane protein TolC. In this study, acrAB genes were cloned from the E. coli W3104 chromosome. To determine the topology of the inner membrane component AcrB, we employed a chemical labeling approach to analyse mutants of AcrB in which a single cysteine residue had been introduced. The cysteine-free AcrB mutant, in which the two intrinsic Cys residues were replaced by Ala, retained full drug resistance. We constructed 33 cysteine mutants in which a single cysteine was introduced into each putative hydrophilic loop region of the cysteine-free AcrB. The binding of [(14)C]N-ethylmaleimide (NEM) to the Cys residue and the competition of NEM binding with the binding of a membrane-impermeant maleimide, 4-acetamide-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS), in intact cells were investigated. The results revealed that the N- and C-terminals are localized on the cytoplasmic surface of the membrane and the two large loops are localized on the periplasmic surface of the membrane. The results supported the 12-membrane-spanning structure of AcrB. Three of the four short periplasmic loop regions were covered by the two large periplasmic loop domains and were not exposed to the water phase until one of the two large periplasmic loops was removed.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Binding, Competitive
  • Carbon Radioisotopes
  • Carrier Proteins*
  • Cloning, Molecular
  • Cysteine / chemistry
  • Cysteine / genetics
  • Cysteine / metabolism
  • Cytoplasm / chemistry
  • Drug Resistance, Multiple / genetics
  • Drug Resistance, Multiple / physiology*
  • Escherichia coli / chemistry*
  • Escherichia coli / metabolism
  • Escherichia coli Proteins*
  • Ethylmaleimide / chemistry
  • Ethylmaleimide / metabolism
  • Membrane Proteins / chemistry*
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism
  • Molecular Sequence Data
  • Multidrug Resistance-Associated Proteins
  • Mutagenesis, Site-Directed
  • Periplasm / chemistry
  • Protein Conformation
  • Radioligand Assay
  • Stilbenes / chemistry
  • Stilbenes / metabolism
  • Sulfonic Acids / chemistry
  • Sulfonic Acids / metabolism

Substances

  • AcrB protein, E coli
  • Bacterial Proteins
  • Carbon Radioisotopes
  • Carrier Proteins
  • Escherichia coli Proteins
  • Membrane Proteins
  • Multidrug Resistance-Associated Proteins
  • Stilbenes
  • Sulfonic Acids
  • 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid
  • Cysteine
  • Ethylmaleimide