Studies on the relation of the Mr 9000 phosphoprotein to cytochrome b-559 in spinach thylakoid membranes.

Cytochrome b-559 was purified from phosphorylated spinach chloroplast thylakoids after activation of kinase activity in the presence of [gamma-32P]ATP in order to determine whether the 9-kDa phosphoprotein in these membranes arises from phosphorylation of the cytochrome b-559. It was established in this work that the 9-kDa phosphoprotein, like the cytochrome b-559 polypeptide, is a PS II component, and that these two proteins migrate very similarly on denaturing gels. However, the initial 2% Triton-4 M urea membrane extract contains most of the cytochrome b-559 and little 32P. A substantially larger amount of stable 32P-labeled 9-kDa phosphoprotein fraction is found in the material that is insoluble in the 2% Triton-4 M urea. Furthermore, the ratio of 32P:heme in cytochrome b-559 purified in the presence of protease inhibitors from phosphorylated membranes was on the order of 1% of that expected if cytochrome b-559 were the sole source of the radiolabel seen in the 9-kDa band. The differential extraction properties of the 32P-labeled 9-kDa phosphoprotein and cytochrome b-559, and the stoichiometry of 32P:heme in the purified cytochrome appear to exclude the cytochrome as a candidate for the 9-kDa phosphoprotein.