KINETIC RESOLUTION OF THE INCORPORATION OF THE D1 PROTEIN INTO PHOTOSYSTEM-II AND LOCALIZATION OF ASSEMBLY INTERMEDIATES IN THYLAKOID MEMBRANES OF SPINACH-CHLOROPLASTS

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 cent er. The replacement of the D1 protein in existing PSII complexes was f ollowed in two in vitro translation systems consisting of isolated chl oroplasts or isolated thylakoid membranes with attached ribosomes. By application of pulse-chase translation experiments, we followed transl ation elongation, release of proteins from the ribosomes, and subseque nt incorporation of newly synthesized products into PSII (sub)complexe s. The time course of incorporation of newly synthesized proteins into the different PSII (sub)complexes was analyzed by sucrose density gra dient centrifugation. Immediately after termination of translation, th e D1 protein was found both unassembled in the membrane as well as alr eady incorporated into PSII reaction center complexes, possibly due to a cotranslational association of the D1 protein with other PSII react ion center components, Later steps in the reassembly of PSII were clea rly post-translational and sequential. Different rate-limiting steps i n the assembly process were found to be related to the depletion of nu clear 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 reveale d that processing was not required for the assembly of the D1 protein into a PSII (sub)complex and that processing of the unassembled precur sor could take place, The limited incorporation into PSII subcomplexes of three other PSII core proteins (D2 protein, CP43, and CP47) was cl early post-translational in both translation systems. Radiolabeled ass embly intermediates smaller than the PSII core complex were found to b e located in the stroma-exposed thylakoid membranes, the site of prote in synthesis. Larger PSII assembly intermediates were almost exclusive ly located in the appressed regions of the membranes.