We have used the oxidant CuCl2 to study its effect on precursor protein import into chloroplasts and on the components involved. CuCl2 reversibly oxidizes thiol groups, which in turn, can form disulfide bridges. Concentrations of 40 μM CuCl2 almost completely inhibit precursor protein binding and subsequent translocation into chloroplasts. This inhibitory effect is reversible by a dithiothreitol treatment. Disulfide bridges, which form upon oxidation by CuCl2, are build up intramolecular and intermolecular, if the thiol groups are in close vicinity to each other. CuCl2 can thus be used as a thiol cleavable crosslinker without an additional spacer distance between the two targets. When purified outer envelope membranes were treated with CuCl2, a hetero oligomeric complex is detected, consisting of OEP86, OEP75 and OEP34, indicating the close vicinity and protein-protein interaction between polypeptides in situ, which are involved in protein translocation into chloroplasts.
