The D1 reaction center protein of the membranebound 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. Photosystem II (PSII)1 is a multiprotein complex of more than 25 different proteins in the thylakoid membrane and catalyzes the light-driven reduction of plastoquinone by electrons derived from water (see Refs. 1 and 2 for review). The heart of the PSII complex can be isolated as the PSII reaction center particle and is composed of a heterodimer of two homologous proteins, the D1 and D2 protein, the two cytochrome b 559 subunits (4 and 9 kDa), and at least one small protein (4.8 kDa, psbI gene product) of unknown function (see Ref. 3). Recently also a nuclear-encoded, small (6.1 kDa) protein has been suggested to be part of the PSII reaction center (4). Based on immunological data (5) and structural similarities between the bacterial reaction center and the reaction center of higher plants (6), the D1 and D2 proteins are predicted to contain five memb...

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It has been shown for the first time that several photosystem-I1 thylakoid proteins and the main chlorophyllulh light-harvesting complex can be phosphorylated with inorganic pyrophosphate as phosphate donor. With pyrophosphate, as with ATP, the protein-kinase reaction is dependent on light or a strong reducing agent. The reaction which can be demonstrated in well-washed spinach thylakoids is dependent on electron transport and is controlled by the redox state of the plastoquinone pool. It is suggested that the pyrophosphate-dependent thylakoid protein phosphorylation is mediated by the same kinase which is responsible for the ATP-dependent protein phosphorylation. This pyrophosphate-dependent kinase activity may be derived from an evolutionary precursor from which ATP-dependent protein phosphorylation also developed.Both the light-harvesting chlorophyll-u/b protein complex (LHC 11) and several photosystem-I1 (PS 11) thylakoid proteins are known to be phosphorylated by an ATP-dependent protein kinase [l -31. This kinase is controlled by the redox state of the plastoquinone pool [4]. More recent results also emphasize a role for the cytochrome-bf'complex in the activation of the kinase [5 -71. The phosphorylated proteins are dephosphorylated by a membrane-bound phosphatase that does not require activation [2]. This reversible protein phosphorylation leads to rearrangements in the thylakoid membrane through the lateral migration of a phosphorylated LHC I1 subpopulation from the appressed into the non-appressed thylakoid membrane regions [8 -101. It has been suggested that LHC I1 protein phosphorylation and the accompanying changes in organization of the LHC I1 antenna regulate the excitation energy distribution between PS I and PS I1 [ll], protect PS I1 against photoinhibition [12] and regulate protein turnover [13].