IN-VITRO SYNTHESIS AND ASSEMBLY OF PHOTOSYSTEM-II CORE PROTEINS - THED1 PROTEIN CAN BE INCORPORATED INTO PHOTOSYSTEM-II IN ISOLATED-CHLOROPLASTS AND THYLAKOIDS

The D1 reaction center protein of the membrane-bound photosystem II co mplex (PSII) has a much higher turnover rate than the other PSII prote ins, Thus, the D1 protein has to be replaced while the other PSII comp onents are not newly synthesized. In this study, this D1 protein repla cement into PSII complexes was followed in two in vitro translation sy stems: isolated chloroplasts and a homologous run-off translation syst em consisting primarily of isolated thylakoids with attached ribosomes . The incorporation of newly synthesized radiolabeled products into di fferent (sub)complexes was analyzed by sucrose density gradient centri fugation of n-dodecyl beta-D-maltoside-solubilized thylakoid membranes , This analysis allowed us to follow the release of the nascent polype ptide chains from the ribosomes and identification of at least four as sembly steps of the PSII complex, as shown below. (i) Both in isolated chloroplasts and in thylakoids, newly synthesized D1 protein is predo minantly 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 in to PSII core complexes is more efficient than during translation in is olated thylakoids, In the thylakoid translation system, a large percen tage of radiolabeled D1 protein is found in smaller PSII: subcomplexes , like PSII reaction center particles, and as unassembled protein in t he membrane. This indicates that stromal factors are required in the r eplacement process of the D1 protein. (iii) Both in isolated chloropla sts 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 unassemb led proteins in the thylakoid membrane. (iv) In chloroplasts, synthesi s of the D1 protein seems to be adjusted according to the possibilitie s of incorporation into PSII complexes, while synthesis of the D2 prot ein, CP43, and CP47 is less regulated and their accumulation as unasse mbled protein in the membrane is abundant.