The mechanism of D1 protein degradation was investigated during photoinhibitory illumination of isolated photosystem II core preparations. The studies revealed that a proteolytic activity resides within the photosystem II core complex. A relationship between the inhibition of D1 protein degradation and the binding of the highly specific serine protease inhibitor diisopropyl fluorophosphate to isolated complexes of photosystem II was observed, evidence that this protease is of the serine type. Using radiolabeled inhibitor, it was shown that the binding site, representing the active serine of the catalytic site, is located on a 43-kDa polypeptide, probably the chlorophyll a protein CP43. The protease is apparently active in darkness, with the initiation of breakdown being dependent on high light-induced substrate activation. The proteolysis, which has an optimum at pH 7.5, gives rise to primary degradation fragments of 23 and 16 kDa. In addition, D1 protein fragments of 14, 13, and 10 kDa were identified. Experiments with phosphate-labeled D1 protein and sequence-specific antisera showed that the 23- and 16-kDa fragments originate from the N- and C-termini, respectively, suggesting a primary cleavage of the D1 protein at the outer thylakoid surface in the region between transmembrane helices D and E.

The chloroplast compartment enclosed by the thylakoid membrane, the "lumen," is poorly characterized. The major aims of this work were to design a procedure for the isolation of the thylakoid lumen which could be generally used to characterize lumenal proteins. The preparation was a stepwise procedure in which thylakoid membranes were isolated from intact chloroplasts. Loosely associated thylakoid surface proteins were removed, and following Yeda press fragmentation the lumenal content was recovered in the supernatant following centrifugation. The purity and yield of lumenal proteins were determined using appropriate marker proteins specific for the different chloroplast compartments. Quantitative immunoblot analyses showed that the recovery of soluble lumenal proteins was 60 -65% (as judged by the presence of plastocyanin), whereas contamination with stromal enzymes was less than 1% (ribulose-bisphosphate carboxylase) and negligible for thylakoid integral membrane proteins (D1 protein). Approximately 25 polypeptides were recovered in the lumenal fraction, of which several were identified for the first time. Enzymatic measurements and/or amino-terminal sequencing revealed the presence of proteolytic activities, violaxanthin de-epoxidase, polyphenol oxidase, peroxidase, as well as a novel prolyl cis/trans-isomerase.The chloroplast is the photosynthetic organelle of green algae and higher plants. The chloroplast architecture comprises an envelope membrane, which encloses the soluble stroma as well as the highly specialized thylakoid membrane. The stromal compartment contains mainly the components of the Calvin cycle, which are required for the fixation of carbon dioxide. The thylakoids, on the other hand, carry out the light reactions of photosynthesis leading to the production of NADPH and ATP. The thylakoid membrane has a characteristic flat shape and is differentiated into appressed grana stacks and single non-appressed stroma-exposed lamellae. The inner surface of the thylakoid membrane encloses a narrow, continuous compartment, the lumen (1, 2). Electron microscopy studies of spinach thylakoids have suggested that the lumen is a densely packed space (3). No isolation method has so far been available for obtaining a high yield of pure thylakoid lumen. Thus, the present knowledge of the lumen from a compositional and functional point of view is fragmentary and is gathered from several independent approaches, addressing only single aspects of this compartment.By developing a technique for obtaining inside-out thylakoids, the investigation of the membrane surface of the lumenal side became possible (4). This work contributed to the discovery of the extrinsic proteins PsbO, PsbP, and PsbQ (5, 6) that bind to the lumenal side of photosystem II and are thought to stabilize the water oxidizing complex (7,8). More recent studies have shown that these subunits of photosystem II occur also as soluble lumenal proteins (9). This pool of unassembled PsbO, PsbQ, and PsbP was resistant to proteolytic degradation and was capa...

The repair of photoinhibitory damage to photosystem II involves the rapid degradation and turnover of the D1 reaction center subunit. Additional protein subunits which show a limited degradation at high light intensities are the complementary reaction center subunit, D2, and the two chlorophyll a binding proteins, CP 47 and CP 43. In this work, we provide the first evidence for light-induced degradation of a nuclear-encoded subunit of photosystem II, the recently discovered PsbW protein. This 6.1 kDa protein is predicted to have a single membrane span and was found to be closely associated with the photosystem II reaction center. The degradation of the PsbW protein was demonstrated by photoinhibitory experiments, both in vitro, using thylakoid membranes and photosystem II core particles, and in vivo using leaf discs. The PsbW protein showed almost the same rate and extent of degradation as the D1 protein, and its degradation was more pronounced compared to the D2 and CP 43 proteins. The degradation of the PsbW protein was shown to share many mechanistic similarities with the more well characterized D1 protein degradation, such as oxygen dependence, sensitivity to serine protease inhibitors, and high light triggering while the actual degradation could readily occur in total darkness. The degradation of the PsbW protein was impaired by protein phosphorylation, although this protein was not itself phosphorylated. This impairment was correlated to the phosphorylation of the D1 protein which has been shown to block its degradation during photoinhibitory conditions. It is concluded that the PsbW protein is not degraded as a direct consequence of primary photodamage but due to a general destabilization of the photosystem II complex under conditions were the D1 protein becomes degraded in the absence of a sufficient repair system. The results are discussed in terms of a requirement for coordination between degradation and protein synthesis/integration during the repair process of photodamaged photosystem II reaction centers.

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The biochemical isolation of pure and active proteins or chlorophyll protein complexes has been crucial for elucidating the mechanism of photosynthetic energy conversion. Most of the proteins involved in this process are embedded in the photosynthetic membrane. The isolation of such hydrophobic integral membrane proteins is not trivial, and involves the use of detergents often combined with various time-consuming isolation procedures. We have applied the new procedure of perfusion chromatography for the rapid isolation of photosynthetic membrane proteins. Perfusion chromatography combines a highly reactive surface per bed volume with extremely high elution flow rates. We present an overview of this chromatographic method and show the rapid isolation of reaction centres from plant Photosystems I and II and photosynthetic purple bacteria, as well as the fractionation of the chlorophyll a/b-binding proteins of Photosystem I (LHC I). The isolation times have been drastically reduced compared to earlier approaches. The pronounced reduction in time for separation of photosynthetic complexes is convenient and permits purification of proteins in a more native state, including the maintainance of ligands and the possibility to isolate proteins trapped in intermediate metabolic or structural states.

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