Reconstitution of a chloroplast protein import channel

EMBO J. 1997 Dec 15;16(24):7351-60. doi: 10.1093/emboj/16.24.7351.

Abstract

The chloroplastic outer envelope protein OEP75 with a molecular weight of 75 kDa probably forms the central pore of the protein import machinery of the outer chloroplastic membrane. Patch-clamp analysis shows that heterologously expressed, purified and reconstituted OEP75 constitutes a voltage-gated ion channel with a unit conductance of Lambda = 145pS. Activation of the OEP75 channel in vitro is completely dependent on the magnitude and direction of the voltage gradient. Therefore, movements of protein charges of parts of OEP75 in the membrane electric field are required either for pore formation or its opening. In the presence of precursor protein from only one side of the bilayer, strong flickering and partial closing of the channel was observed, indicating a specific interaction of the precursor with OEP75. The comparatively low ionic conductance of OEP75 is compatible with a rather narrow aqueous pore (dporeapproximately equal to 8-9 A). Provided that protein and ion translocation occur through the same pore, this implies that the environment of the polypeptide during the transit is mainly hydrophilic and that protein translocation requires almost complete unfolding of the precursor.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chloroplasts / physiology*
  • Chloroplasts / ultrastructure
  • Cloning, Molecular
  • Electric Conductivity
  • Escherichia coli
  • Intracellular Membranes / physiology
  • Intracellular Membranes / ultrastructure
  • Ion Channels / biosynthesis
  • Ion Channels / physiology*
  • Lipid Bilayers
  • Membrane Potentials
  • Membrane Proteins / biosynthesis
  • Membrane Proteins / chemistry
  • Membrane Proteins / physiology*
  • Models, Chemical
  • Models, Molecular
  • Patch-Clamp Techniques
  • Peas
  • Plant Proteins / physiology
  • Protein Conformation
  • Protein Precursors / biosynthesis
  • Protein Precursors / chemistry
  • Protein Precursors / physiology*
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry

Substances

  • Ion Channels
  • Lipid Bilayers
  • Membrane Proteins
  • OEP75 protein precursor, plant
  • Plant Proteins
  • Protein Precursors
  • Recombinant Proteins