STABLE ASSOCIATION OF CHLOROPLASTIC PRECURSORS WITH PROTEIN TRANSLOCATION COMPLEXES THAT CONTAIN PROTEINS FROM BOTH ENVELOPE MEMBRANES AND A STROMAL HSP-100 MOLECULAR CHAPERONE

Cytoplasmically synthesized precursors interact with translocation com ponents in both the outer and inner envelope membranes during transpor t into chloroplasts. Using co-immunoprecipitation techniques, with ant ibodies specific to known translocation components, we identified stab le interactions between precursor proteins and their associated membra ne translocation components in detergent-solubilized chloroplastic mem brane fractions. Antibodies specific to the outer envelope translocati on components OEP75 and OEP34, the inner envelope translocation compon ent IEP110 and the stromal Hsp100, ClpC, specifically co-immunoprecipi tated precursor proteins under limiting ATP conditions, a stage we hav e called docking, A portion of these same translocation components was coimmunoprecipitated as a complex, and could also be detected by co-s edimentation through a sucrose density gradient. ClpC was observed onl y in complexes with those precursors utilizing the general import appa ratus, and its interaction with precursor-containing translocation com plexes was destabilized by ATP. Finally, ClpC was co-immunoprecipitate d with a portion of the translocation components of both outer and inn er envelope membranes, even in the absence of added precursors. We dis cuss possible roles for stromal Hsp100 in protein import and mechanism s of precursor binding in chloroplasts.