The D1 reaction center protein of the membrane-bound photosystem II complex (PSII) has a much higher turnover rate than the other PSII proteins. Thus, the D1 protein has to be replaced while the other PSII components are not newly synthesized. In this study, this D1 protein replacement into PSII complexes was followed in two in vitro translation systems: isolated chloroplasts and a homologous run-off translation system consisting primarily of isolated thylakoids with attached ribosomes. The incorporation of newly synthesized radiolabeled products into different (sub)complexes was analyzed by sucrose density gradient centrifugation of n-dodecyl -D-maltoside-solubilized thylakoid membranes. This analysis allowed us to follow the release of the nascent polypeptide chains from the ribosomes and identification of at least four assembly steps of the PSII complex, as shown below.

(i) Both in isolated chloroplasts and in thylakoids, newly synthesized D1 protein is predominantly incorporated into existing PSII subcomplexes, indicating that synthesis and import of nuclear-encoded factors is not needed for D1 protein replacement.

(ii) In chloroplasts, D1 protein incorporation into PSII core complexes is more efficient than during translation in isolated thylakoids. In the thylakoid translation system, a large percentage of radiolabeled D1 protein is found in smaller PSII subcomplexes, like PSII reaction center particles, and as unassembled protein in the membrane. This indicates that stromal factors are required in the replacement process of the D1 protein.

(iii) Both in isolated chloroplasts and in thylakoids, the other PSII core proteins D2, CP43, and CP47 are also synthesized and released from the membrane-bound ribosomes, but incorporation into PSII complexes occurs to a much smaller extent than the D1 protein. Instead they accumulate predominantly as unassembled proteins in the thylakoid membrane.

(iv) In chloroplasts, synthesis of the D1 protein seems to be adjusted according to the possibilities of incorporation into PSII complexes, while synthesis of the D2 protein, CP43, and CP47 is less regulated and their accumulation as unassembled protein in the membrane is abundant.

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