The Thylakoid FtsH Protease Plays a Role in the Light-Induced Turnover of the Photosystem II D1 Protein

Abstract

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...

Proteomic analysis of the response to high-salinity stress in Physcomitrella patens

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