COPPER CHLORIDE, AN INHIBITOR OF PROTEIN IMPORT INTO CHLOROPLASTS

We have used the oxidant CuCl2 to study its effect on precursor protei n import into chloroplasts and on the components involved. CuCl2 rever sibly oxidizes thiol groups, which in turn, can form disulfide bridges . Concentrations of 40 mu M CuCl2 almost completely inhibit precursor protein binding and subsequent translocation into chloroplasts. This i nhibitory effect is reversible by a dithiothreitol treatment. Disulfid e bridges, which form upon oxidation by CuCl2, are build up intramolec ular and intermolecular, if the thiol groups are in close vicinity to each other. CuCl2 can thus be used as a thiol cleavable crosslinker wi thout an additional spacer distance between the two targets. When puri fied outer envelope membranes were treated with CuCl2, a hetero oligom eric complex is detected, consisting of OEP86, OEP75 and OEP34, indica ting the close vicinity and protein-protein interaction between polype ptides in situ, which are involved in protein translocation into chlor oplasts.