Abstract
Photosystem II (PSII) is a large membrane protein complex that catalyzes oxidation of water to molecular oxygen. During its normal function, PSII is damaged and frequently turned over. The maturation of the D1 protein, a key component in PSII, is a critical step in PSII biogenesis. The precursor form of D1 (pD1) contains a C-terminal extension, which is removed by the protease CtpA to yield PSII complexes with oxygen evolution activity. To determine the temporal position of D1 processing in the PSII assembly pathway, PSII complexes containing only pD1 were isolated from a CtpA-deficient strain of the cyanobacterium Synechocystis 6803. Although membranes from the mutant cell had nearly 50% manganese, no manganese was detected in isolated DeltactpAHT3 PSII, indicating a severely decreased manganese affinity. However, chlorophyll fluorescence decay kinetics after a single saturating flash suggested that the donor Y(Z) was accessible to exogenous Mn(2+) ions. Furthermore, the extrinsic proteins PsbO, PsbU, and PsbV were not present in PSII isolated from this mutant. However, PsbO and PsbV were present in mutant membranes, but the amount of PsbV protein was consistently less in the mutant membranes compared with the control membranes. We conclude that D1 processing precedes manganese binding and assembly of the extrinsic proteins into PSII. Interestingly, the Psb27 protein was found to be more abundant in DeltactpAHT3 PSII than in HT3 PSII, suggesting a possible role of Psb27 as an assembly factor during PSII biogenesis.
Highlights
Phytins, plastoquinones, manganese, non-heme iron, calcium, and chloride atoms as well as two heme groups (2, 3)
The precursor form of D1 (pD1) protein is co-translationally inserted into the thylakoid membrane, it associates with other Photosystem II (PSII) membrane components, and is cleaved after Ala-344 by CtpA, a C-terminal processing protease, to yield the mature D1 protein (12, 13)
D1 Processing and Manganese Ion Binding—It has been previously established that D1 processing mutants do not evolve oxygen, because these mutants cannot completely assemble the tetra-nuclear manganese cluster, the catalytic core of the oxygen-evolving complex (9, 12, 13, 16, 25)
Summary
Bacterial Strains and Culture Conditions—Synechocystis 6803 cultures were grown at 30 °C under 30 mol of photons mϪ2 sϪ1 of white light in TES-buffered BG-11 medium (20) with air bubbling. Samples were subjected to continuous actinic light (300 mol of photons mϪ2 sϪ1) at 30% intensity for 10 s and fluorescence (F) was monitored by 33-s measuring flashes every 40 ms. Cells were harvested and washed with 20 mM HEPES, pH 7.8, 5 mM EDTA buffer according to Ref. 22, resuspended in HCMS buffer (50 mM HEPES-NaOH, 5 mM CaCl2, 10 mM MgCl2, and 1 M sucrose, pH 7.8) to a concentration of 500 g of Chl/ml, and broken with 0.17-mm glass beads. Protein Detection—PSII samples (7 g of Chl/lane) were fractionated using the SDS-PAGE system described in Ref. 2 and visualized with Coomassie Blue staining (Sigma). The resulting cell lysate (ϳ300 g of Chl/ml) was treated with 0.04% (w/v) of the detergent -dodecyl maltoside (Amresco) and immediately centrifuged at 60,000 ϫ g for 40 min to pellet the membranes. The ImageJ program was used to quantify the relative amounts of different proteins in the samples
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