Kinetic Resolution of the Incorporation of the D1 Protein into Photosystem II and Localization of Assembly Intermediates in Thylakoid Membranes of Spinach Chloroplasts

Abstract

The chloroplast-encoded D1 protein of photosystem II (PSII) has a much higher turnover rate than the other subunits of the PSII complex as a consequence of photodamage and subsequent repair of its reaction center. The replacement of the D1 protein in existing PSII complexes was followed in two in vitro translation systems consisting of isolated chloroplasts or isolated thylakoid membranes with attached ribosomes. By application of pulse-chase translation experiments, we followed translation elongation, release of proteins from the ribosomes, and subsequent incorporation of newly synthesized products into PSII (sub)complexes. The time course of incorporation of newly synthesized proteins into the different PSII (sub)complexes was analyzed by sucrose density gradient centrifugation. Immediately after termination of translation, the D1 protein was found both unassembled in the membrane as well as already incorporated into PSII reaction center complexes, possibly due to a cotranslational association of the D1 protein with other PSII reaction center components. Later steps in the reassembly of PSII were clearly post-translational and sequential. Different rate-limiting steps in the assembly process were found to be related to the depletion of nuclear encoded and stromal components as well as the lateral migration of subcomplexes within the heterogeneous thylakoid membrane. The slow processing of precursor D1 in the thylakoid translation system revealed that processing was not required for the assembly of the D1 protein into a PSII (sub)complex and that processing of the unassembled precursor could take place. The limited incorporation into PSII subcomplexes of three other PSII core proteins (D2 protein, CP43, and CP47) was clearly post-translational in both translation systems. Radiolabeled assembly intermediates smaller than the PSII core complex were found to be located in the stroma-exposed thylakoid membranes, the site of protein synthesis. Larger PSII assembly intermediates were almost exclusively located in the appressed regions of the membranes.