The photosystem II reaction center D1 protein is known to turn over frequently. This protein is prone to irreversible damage caused by reactive oxygen species that are formed in the light; the damaged, nonfunctional D1 protein is degraded and replaced by a new copy. However, the proteases responsible for D1 protein degradation remain unknown. In this study, we investigate the possible role of the FtsH protease, an ATP-dependent zinc metalloprotease, during this process. The primary light-induced cleavage product of the D1 protein, a 23-kD fragment, was found to be degraded in isolated thylakoids in the dark during a process dependent on ATP hydrolysis and divalent metal ions, suggesting the involvement of FtsH. Purified FtsH degraded the 23-kD D1 fragment present in isolated photosystem II core complexes, as well as that in thylakoid membranes depleted of endogenous FtsH. In this study, we definitively identify the chloroplast protease acting on the D1 protein during its light-induced turnover. Unlike previously identified membrane-bound substrates for FtsH in bacteria and mitochondria, the 23-kD D1 fragment represents a novel class of FtsH substratefunctionally assembled proteins that have undergone irreversible photooxidative damage and cleavage. INTRODUCTIONProteins that are rendered nonfunctional due to interactions with reactive oxygen species or free radicals undergo proteolysis and are replaced by newly synthesized copies. This is particularly significant in the chloroplast thylakoid membrane. Here, enzymes operate in a highly oxidizing environment and therefore are susceptible to impairment of structure and function. Within the thylakoid membrane, photosystem II (PSII) is the component most sensitive to oxidative damage. This sensitivity is partially due to its function in water splitting, a reaction that requires an oxidizing potential of 1.1 V, but it is also due to the intrinsic formation by PSII of toxic oxygen species (Andersson and Barber, 1994). PSII is a large multisubunit protein complex integral to the thylakoid membrane (Andersson and Barber, 1994). Its reaction center contains the homologous D1 and D2 proteins, PsbI, PsbW (in which Psb stands for PSII, and I and W denote specific subunits), and cytochrome b 559 . The D1/D2 heterodimer binds all of the chlorophylls, quinones, and metal ligands necessary to perform primary PSII photochemistry and electron transport. The structure of the PSII reaction center recently has been determined at a resolution of 8 Å (Rhee et al., 1998), and the structure of the dimeric PSII core complex has been set at a resolution of ‫ف‬ 9 Å (Hankamer et al., 1999).Under conditions of high light intensity, electron transport within the complex is arrested, and consequently, the photosynthetic process is inactivated. This phenomenon is known as photoinhibition (Barber and Andersson, 1992;Prasil et al., 1992). The process is thought to occur via overreduction of the acceptor side of PSII, chlorophyll triplet formation, and production of toxic singlet oxygen (Vass et a...

In an attempt to identify and characterize chloroplast proteases, we performed an immunological analysis of chloroplasts using an antibody against Escherichia coli FtsH protease, which is an ATP-dependent metalloprotease bound to the cytoplasmic membrane. A cross-reacting protein of 78 kDa was found in the thylakoid membrane of spinach, but not in the soluble stromal fraction. Alkali and high salt washes, as well as trypsin treatment of thylakoid membranes, suggest that the chloroplastic FtsH protein is integral to the membrane, with its hydrophilic portion exposed to the stroma. The protein is not bound to any photosynthetic complex and is exclusively located in the stromally exposed regions of the thylakoid membrane. Its expression is dependent on light, as it is present in green pea seedlings, but absent from etiolated ones. An Arabidopsis cDNA was isolated, and the deduced amino acid sequence demonstrated high similarity to the E. coli FtsH protein, especially in the central region of the protein, containing the ATP-and zinc-binding sites. The product of this clone was capable of import into isolated pea chloroplasts, where it was processed to its mature form and targeted to the thylakoid membrane. The trans-bilayer orientation and lateral location of the FtsH protein in the thylakoid membrane suggest its involvement in the degradation of both soluble stromal proteins and newly inserted or turning-over thylakoid proteins.Numerous examples of protein degradation in chloroplasts have been accumulated over the years. These include degradation of unassembled proteins, apoproteins lacking their prosthetic groups or pigments, photo-or otherwise-damaged proteins, and developmentally or environmentally regulated proteins (see Ref. 1, and references therein). In particular, emphasis has been placed on the degradation of the D1 protein in connection with photoinhibitory damage of the PSII 1 reaction center (2, 3). However, despite extensive documentation of these processes, little is known about the identity of the proteases involved. Only recently, the existence of Clp protease, a homologue of a well characterized bacterial ATP-dependent serine protease (4, 5), has been demonstrated in chloroplasts of higher plants (1, 6 -8). The occurrence of Clp subunits in plants subjected to a variety of environmental conditions suggests its function in chloroplasts as a housekeeping protease.The existence of a protein related to a bacterial protease in the chloroplast, together with other prokaryotic characteristics of this organelle, led us to hypothesize that additional chloroplast proteases may resemble prokaryotic ones. This notion was recently substantiated by the finding that the chloroplast genomes of red and brown algae (9, 10) contain genes homologous to another bacterial protease, FtsH. FtsH and related proteins are found in different bacteria and yeast mitochondria; it was demonstrated to be a 74-kDa (11), ATP-dependent metalloprotease, involved in the response of Escherichia coli to heat shock (12, 13), degradation o...

Light-dependent regulation of a growing number of chloroplast enzymatic activities has been found to occur through the reversible reduction of intra-or intermolecular disulphides by thioredoxins. In cyanobacteria, despite their similarity to chloroplasts, no proteins have hitherto been shown to interact with thioredoxins, and the role of the cyanobacterial ferredoxin͞thioredoxin system has remained obscure. By using an immobilized cysteine 35-to-serine site-directed mutant of the Synechocystis sp. PCC 6803 thioredoxin TrxA as bait, we screened the Synechocystis cytosolic and peripheral membrane protein complements for proteins interacting with TrxA. The covalent bond between the isolated target proteins and mutated TrxA was confirmed by nonreducing͞reducing twodimensional SDS͞PAGE. Thus, we have identified 18 cytosolic proteins and 8 membrane-associated proteins as candidate thioredoxin substrates. Twenty of these proteins have not previously been associated with thioredoxin-mediated regulation. Phosphoglucomutase, one of the previously uncharacterized thioredoxinlinked enzymes, has not earlier been considered a target for metabolic control through disulphide reduction. In this article, we show that phosphoglucomutase is inhibited under oxidizing conditions and activated by DTT and reduced wild-type TrxA in vitro. The results imply that thioredoxin-mediated redox regulation is as extensive in cyanobacteria as in chloroplasts but that the subjects of regulation are largely different. Dithiol͞disulphide exchange catalyzed by thioredoxin (Trx) forms the molecular basis for light-dependent regulation of many enzymes in the chloroplasts of higher plants and algae (1-3). Ferredoxin receives reducing equivalents from the photosynthetic electron transport in the light and reduces Trx m and f by means of ferredoxin-Trx reductase. The Trxs, in turn, convert disulphides to dithiols in their respective target enzymes, thereby modulating their activities. The earliest discovered targets for Trx-mediated regulation belong to the Calvin cycle of CO 2 assimilation (1). Since the initial discoveries, several more chloroplast enzymes have been recognized as substrates for Trx (3). A breakthrough in the investigation of chloroplast redox regulation came with the introduction of a new method to isolate Trx target proteins (4-6). The method involves mutation of the buried redox-active cysteine of Trx, which favors the formation of stable mixed disulphides with its target proteins. This experimental approach confirmed the interaction between Trx and its known targets and more than doubled the number of potential Trx-regulated proteins (6).Cyanobacteria are oxygenic photosynthetic prokaryotes that probably share a common ancestor with the chloroplast. The complete sequence of the cyanobacterium Synechocystis sp. PCC 6803 genome (Cyanobase, www.kazusa.or.jp͞cyano͞cyano. html) reveals that this organism, hereafter referred to as Synechocystis, contains ferredoxin-Trx reductase and at least four different Trxs. Nevertheless, attempts to ...