Torill Hundal

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

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 substrate— functionally assembled proteins that have undergone irreversible photooxidative damage and cleavage.

In vitro studies on light-induced inhibition of Photosystem II and D1-protein degradation at low temperatures

Abstract In order to get information on the molecular background behind the aggrevated photodamage to photosynthesis at low temperatures and to investigate the general mechanism of D1-protein degradation, isolated spinach thylakoids were subjected to photoinhibitory treatment at various temperatures. The results reveal that: (i) the Photosystem II electron transport per se is less sensitive to high light at low temperatures in contrast to the overall photosynthetic process; (ii) the degradation of D1-protein is severely retarded below 7°C; (iii) inhibition of Photosystem II electron transport and D1-protein degradation are separate events the two reactions could be completely separated in time; (iv) D1-protein is degraded by enzymatic proteolysis and not by a direct photocleavage reaction; (v) degradation of the D1-protein readily proceeds in the dark but its triggering for the proteolytic attack requires light; (vi) strong illumination at low temperature does not induce any lateral rearrangement in the location of Photosystem II; and (vii) D1-protein fragments can be identified in vitro and be used to verify the specificity of D1-protein degradation under various experimental conditions.

Changes in the organization of Photosystem II following light-induced D1-protein degradation

Abstract The composition and organization of photosystem II was studied in thylakoid membranes and subfractions which had been subjected to photoinhibitory light conditions. The results show that D1-protein degradation can occur in vitro leading to a 50–60% loss of the protein. Apart from the D2-protein, which shows a limited decline, there was no loss of any other photosystem II proteins. The D1-protein degradation induced several changes in the organization of the photosystem II complex. Using inside-out thylakoid vesicles we demonstrate that concomitant with the D1-protein degradation there is a release of the extrinsic 33, 23 and 16 kDa proteins from the inner thylakoid surface into the lumenal space. In addition, there is a release of four manganese ions per D1-protein degraded. The correlation between the D1-protein degradation and the release of manganese is also seen in inside-out thylakoid vesicles that have been CaCl2-washed to remove the three extrinsic proteins prior to photoinhibitory illumination. Subfractionation of thylakoids subsequent to photoinhibitory treatment suggests a migration of certain photosystem II subunits from the appressed to the non-appressed thylakoid regions following D1-protein degradation. The photosystem II subunits showed an individual migration behaviour, suggesting a disassembly of the photosystem II core. Our data suggest that repair of photodamaged photosystem II involves, apart from reinsertion of new D1-proteins, reassembly of the photosystem II complex including lateral movement of proteins between the two thylakoid regions and religation of the manganese cluster.

Photosystem II reaction centres stay intact during low temperature photoinhibition

Photoinhibition of photosynthesis was studied in intact barley leaves at 5 and 20°C, to reveal if Photosystem II becomes predisposed to photoinhibition at low temperature by 1) creation of excessive excitation of Photosystem II or, 2) inhibition of the repair process of Photosystem II. The light and temperature dependence of the reduction state of QA was measured by modulated fluorescence. Photon flux densities giving 60% of QA in a reduced state at steady-state photosynthesis (300 μmol m−2s−1 at 5°C and 1200 μmol m−2s−1 at 20°C) resulted in a depression of the photochemical efficiency of Photosystem II (Fv/Fm) at both 5 and 20°C. Inhibition of Fv/Fm occurred with initially similar kinetics at the two temperatures. After 6h, Fv/Fm was inhibited by 30% and had reached steady-state at 20°C. However, at 5°C, Fv/Fm continued to decrease and after 10h, Fv/Fm was depressed to 55% of control. The light response of the reduction state of QA did not change during photoinhibition at 20°C, whereas after photoinhibition at 5°C, the proportion of closed reaction centres at a given photon flux density was 10–20% lower than before photoinhibition.Changes in the D1-content were measured by immunoblotting and by the atrazine binding capacity during photoinhibition at high and low temperatures, with and without the addition of chloramphenicol to block chloroplast encoded protein synthesis. At 20°C, there was a close correlation between the amount of D1-protein and the photochemical efficiency of photosystem II, both in the presence or in the absence of an active repair cycle. At 5°C, an accumulation of inactive reaction centres occurred, since the photochemical efficiency of Photosystem II was much more depressed than the loss of D1-protein. Furthermore, at 5°C the repair cycle was largely inhibited as concluded from the finding that blockage of chloroplast encoded protein synthesis did not enhance the susceptibility to photoinhibition at 5°C.It is concluded that, the kinetics of the initial decrease of Fv/Fm was determined by the reduction state of the primary electron acceptor QA, at both temperatures. However, the further suppression of Fv/Fm at 5°C after several hours of photoinhibition implies that the inhibited repair cycle started to have an effect in determining the photochemical efficiency of Photosystem II.

Amino acid sequence of the oligomycin sensitivity-conferring protein (OSCP) of beef-heart mitochondria and its homology with the δ-subunit of the F1-ATPase of Escherichia coli

The complete amino acid sequence of the oligomycin sensitivity‐conferring protein (OSCP) of beef‐heart mitochondria is reported. The protein contains 190 amino acids and has a molecular mass of 20 967. Its structure is characterized by a concentration of charged amino acids in the two terminal segments (N 1–77 and C 128–190) of the protein, whereas its central region is more hydrophobic. The earlier reported homology of the protein with the δ‐subunit of E. coli F1, based on the terminal amino acid sequences of OSCP, is further substantiated.

Restoration of light induced photosystem II inhibition without de novo protein synthesis

Illumination of isolated spinach thylakoid membranes under anaerobic conditions gave rise to severe inhibition of photosystem II electron transport but did not result in D1‐protein degradation. When these photoinhibited thylakoids were incubated in total darkness the photosystem II activity could be fully restored in vitro in a process that required 1–2 h for completion.

Lack of ability of trypsin-treated mitochondrial F1-ATPase to bind the oligomycin-sensitivity conferring protein (OSCP).

Soluble beef‐heart mitochondrial F1‐ATPase modified in its α‐subunit by mild trypsin treatment (α′‐F1) can no longer bind oligomycin‐sensitivity conferring protein (OSCP) but is still capable of binding to F1‐depleted submitochondrial particles, giving rise to a maximally oligomycin‐sensitive ATPase, provided the particles contain their native complement of OSCP. When OSCP is removed from the particles, α′‐F1 can still bind to the particles, but added OSCP induces only a low degree of oligomycin sensitivity. The possible role of OSCP in the functional coupling of the catalytic (F1) and H+‐translocating (Fo) moieties of mitochondrial ATPase is discussed. The results suggest a functional similarity between the OSCP component of mitochondrial ATPase and the δ‐subunit of E. coli ATPase, which is in accordance with the structural homology recently found to exist between the two polypeptides.

GTP enhances the degradation of the photosystem II D1 protein irrespective of its conformational heterogeneity at the Q(B) site

The light exposure history and/or binding of different herbicides at the QB site may induce heterogeneity of photosystem II acceptor side conformation that affects D1 protein degradation under photoinhibitory conditions. GTP was recently found to stimulate the D1 protein degradation of photoinactivated photosystem II (Spetea, C., Hundal, T., Lohmann, F., and Andersson, B. (1999) Proc. Natl. Acad. Sci. U. S. A.96, 6547–6552). Here we report that GTP enhances the cleavage of the D1 protein D-E loop following exposure of thylakoid membranes to either high light, low light, or repetitive single turnover flashes but not to trypsin. GTP does not stimulate D1 protein degradation in the presence of herbicides known to affect the accessibility of the cleavage site to proteolysis. However, GTP stimulates degradation that can be induced even in darkness in some photosystem II conformers following binding of the PNO8 herbicide (Nakajima, Y., Yoshida, S., Inoue, Y., Yoneyama, K., and Ono, T. (1995) Biochim. Biophys. Acta 1230, 38–44). Both the PNO8- and the light-induced primary cleavage of the D1 protein occur in the grana membrane domains. The subsequent migration of photosytem II containing the D1 protein fragments to the stroma domains for secondary proteolysis is light-activated. We conclude that the GTP effect is not confined to a specific photoinactivation pathway nor to the conformational state of the photosytem II acceptor side. Consequently, GTP does not interact with the site of D1 protein cleavage but rather enhances the activity of the endogenous proteolytic system.

Oligomycin sensitivity-conferring protein (OSCP) of beef heart mitochondria Internal sequence homology and structural relationship with other proteins

Structural analysis of oligomycin sensitivity‐conferring protein (OSCP) revealed repeating sequences (residues 1‐89, 105‐190) suggesting an evolution of the protein by gene duplication. In addition to the reported homology with the δ‐subunit of Escherichia coli F1ATPase, OSCP also shows a certain homology with the b‐subunit of E. coli F0 and the ADP/ATP carrier of mitochondria.

The oligomycin sensitivity conferring protein (OSCP) of beef heart mitochondria: Studies of its binding to F1 and its function

The binding of “oligomycin sensitivity conferring protein” (OSCP) to soluble beef-heart mitochondrial ATPase (F1) has been investigated. OSCP forms a stable complex with F1, and the F1 · OSCP complex is capable of restoring oligomycin- and DCCD-sensitive ATPase activity to F1- and OSCP-depleted submitochondrial particles. The F1 · OSCP complex retains 50% of its ATPase activity upon cold exposure while free F1 is inactivated by 90% or more. Both free F1 and the F1 · OSCP complex release upon cold exposure a part—probably 1 out of 3—of their β subunits; whether α subunits are also lost is uncertain. The cold-treated F1 · OSCP complex is still capable of restoring oligomycin- and DCCD-sensitive ATPase activity to F1- and OSCP-depleted particles. OSCP also protects F1 against modification of its α subunit by mild trypsin treatment. This finding together with the earlier demonstration that trypsin-modified F1 cannot bind OSCP indicates that OSCP binds to the α subunit of F1 and that F1 contains three binding sites for OSCP. The results are discussed in relation to the possible role of OSCP in the interaction of F1 with the membrane sector of the mitochondrial ATPase system.