RECONSTITUTION OF A CHLOROPLAST PROTEIN IMPORT CHANNEL

The chloroplastic outer envelope protein OEP75 with a molecular weight of 75 kDa probably forms the central pore of the protein import machi nery of the outer chloroplastic membrane, Patch-clamp analysis shows t hat heterologously expressed, purified and reconstituted OEP75 constit utes a voltage-gated ion channel with a unit conductance of Lambda = 1 45pS, Activation of the OEP75 channel in vitro is completely dependent on the magnitude and direction of the voltage gradient, Therefore, mo vements of protein charges of parts of OEP75 in the membrane electric field are required either for pore formation or its opening, In the pr esence 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 aqu eous pore (d(pore) congruent to 8-9 Angstrom), Provided that protein a nd ion translocation occur through the same pore, this implies that th e environment of the polypeptide during the transit is mainly hydrophi lic and that protein translocation requires almost complete unfolding of the precursor.