Reconstitution of a chloroplast protein import channel

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 (d(pore) congruent to 8-9 Angstrom), 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.