NEWLY IMPORTED RIESKE IRON-SULFUR PROTEIN ASSOCIATES WITH BOTH CPN60 AND HSP70 IN THE CHLOROPLAST STROMA

The precursor of the Rieske FeS protein, a thylakoid membrane protein, was imparted by isolated pea chloroplasts, and the mature protein was shown to be integrated into the cytochrome bf complex of the thylakoi d membranes. Insertion into the thylakoid membrane was sensitive to th e ionophores nigericin and valinomycin, suggesting a requirement for a proton motive force. A considerable proportion of the imported Rieske protein was detected in the stromal fraction of the chloroplasts, and this increased when membrane insertion was blocked with ionophores. E lectrophoresis of the stromal fraction under nondenaturing conditions resolved two distinct complexes containing the Rieske protein. One of these complexes was identified as an association of the Rieske protein with the chaperonin Cpn60 complex by its electrophoretic mobility, Mg -ATP-dependent dissociation, and immunoprecipitation with anti-Cpn60 a ntibodies. Coimmunoprecipitation of imported Rieske protein with anti- heat shock protein 70 (Hsp70) antibodies indicated that the Rieske pro tein was also associated, in an ATP-dissociable form, with a chloropla st Hsp70 homolog. Immunoprecipitation analysis of an import time cours e detected the highest amounts of the Cpn60-Rieske protein complex ear ly in the time course, whereas highest amounts of the Hsp70-Rieske pro tein complex were formed much later. The disappearance of the Cpn60-Ri eske protein complex correlated with increased amounts of the Rieske p rotein in the thylakoid fraction.