Marika Lindahl

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.

Identification, Characterization, and Molecular Cloning of a Homologue of the Bacterial FtsH Protease in Chloroplasts of Higher Plants

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.

Thioredoxin-linked processes in cyanobacteria are as numerous as in chloroplasts, but targets are different

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 ferredoxin/thioredoxin 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 nonreducing/reducing two-dimensional SDS/PAGE. 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 thioredoxin-linked 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.

Disulphide proteomes and interactions with thioredoxin on the track towards understanding redox regulation in chloroplasts and cyanobacteria.

Light-dependent disulphide/dithiol exchange catalysed by thioredoxin is a classical example of redox regulation of chloroplast enzymes. Recent proteome studies have mapped thioredoxin target proteins in all chloroplast compartments ranging from the envelope to the thylakoid lumen. Progress in the methodologies has made it possible to identify which cysteine residues interact with thioredoxin and to tackle membrane-bound thioredoxin targets. To date, more than hundred targets of thioredoxin and glutaredoxin have been found in plastids from Arabidopsis, spinach, poplar and Chlamydomonas reinhardtii. Thioredoxin-mediated redox control appears to be a feature of the central pathways for assimilation and storage of carbon, sulphur and nitrogen, as well as for translation and protein folding. Cyanobacteria are oxygenic photosynthetic prokaryotes, which presumably share a common ancestor with higher plant plastids. As in chloroplasts, cyanobacterial thioredoxins receive electrons from the photosynthetic electron transport, and thioredoxin-targeted proteins are therefore highly interesting in the context of acclimation of these organisms to their environment. Studies of the unicellular model cyanobacterium Synechocystis sp. PCC 6803 revealed 77 thioredoxin target proteins. Notably, the functions of all these thioredoxin targets highlight essentially the same processes as those described in chloroplasts suggesting that thioredoxin-mediated redox signalling is equally significant in oxygenic photosynthetic prokaryotes and eukaryotes.

Thioredoxin targets of the plant chloroplast lumen and their implications for plastid function

The light‐dependent regulation of stromal enzymes by thioredoxin (Trx)‐catalysed disulphide/dithiol exchange is known as a classical mechanism for control of chloroplast metabolism. Recent proteome studies show that Trx targets are present not only in the stroma but in all chloroplast compartments, from the envelope to the thylakoid lumen. Trx‐mediated redox control appears to be a common feature of important pathways, such as the Calvin cycle, starch synthesis and tetrapyrrole biosynthesis. However, the extent of thiol‐dependent redox regulation in the thylakoid lumen has not been previously systematically explored. In this study, we addressed Trx‐linked redox control in the chloroplast lumen of Arabidopsis thaliana. Using complementary proteomics approaches, we identified 19 Trx target proteins, thus covering more than 40% of the currently known lumenal chloroplast proteome. We show that the redox state of thiols is decisive for degradation of the extrinsic PsbO1 and PsbO2 subunits of photosystem II. Moreover, disulphide reduction inhibits activity of the xanthophyll cycle enzyme violaxanthin de‐epoxidase, which participates in thermal dissipation of excess absorbed light. Our results indicate that redox‐controlled reactions in the chloroplast lumen play essential roles in the function of photosystem II and the regulation of adaptation to light intensity.

Overoxidation of 2-Cys Peroxiredoxin in Prokaryotes CYANOBACTERIAL 2-CYS PEROXIREDOXINS SENSITIVE TO OXIDATIVE STRESS

In eukaryotic organisms, hydrogen peroxide has a dual effect; it is potentially toxic for the cell but also has an important signaling activity. According to the previously proposed floodgate hypothesis, the signaling activity of hydrogen peroxide in eukaryotes requires a transient increase in its concentration, which is due to the inactivation by overoxidation of 2-Cys peroxiredoxin (2-Cys Prx). Sensitivity to overoxidation depends on the structural GGLG and YF motifs present in eukaryotic 2-Cys Prxs and is believed to be absent from prokaryotic enzymes, thus representing a paradoxical gain of function exclusive to eukaryotic organisms. Here we show that 2-Cys Prxs from several prokaryotic organisms, including cyanobacteria, contain the GG(L/V/I)G and YF motifs characteristic of sensitive enzymes. In search of the existence of overoxidation-sensitive 2-Cys Prxs in prokaryotes, we have analyzed the sensitivity to overoxidation of 2-Cys Prxs from two cyanobacterial strains, Anabaena sp. PCC7120 and Synechocystis sp. PCC6803. In vitro analysis of wild type and mutant variants of the Anabaena 2-Cys Prx showed that this enzyme is overoxidized at the peroxidatic cysteine residue, thus constituting an exception among prokaryotes. Moreover, the 2-Cys Prx from Anabaena is readily and reversibly overoxidized in vivo in response to high light and hydrogen peroxide, showing higher sensitivity to overoxidation than the Synechocystis enzyme. These cyanobacterial strains have different strategies to cope with hydrogen peroxide. While Synechocystis has low content of less sensitive 2-Cys Prx and high catalase activity, Anabaena contains abundant and sensitive 2-Cys Prx, but low catalase activity, which is remarkably similar to the chloroplast system.

The diversity and complexity of the cyanobacterial thioredoxin systems

Cyanobacteria perform oxygenic photosynthesis, which makes them unique among the prokaryotes, and this feature together with their abundance and worldwide distribution renders them a central ecological role. Cyanobacteria and chloroplasts of plants and algae are believed to share a common ancestor and the modern chloroplast would thus be the remnant of an endosymbiosis between a eukaryotic cell and an ancestral oxygenic photosynthetic prokaryote. Chloroplast metabolic processes are coordinated with those of the other cellular compartments and are strictly controlled by means of regulatory systems that commonly involve redox reactions. Disulphide/dithiol exchange catalysed by thioredoxin is a fundamental example of such regulation and represents the molecular mechanism for light-dependent redox control of an ever-increasing number of chloroplast enzymatic activities. In contrast to chloroplast thioredoxins, the functions of the cyanobacterial thioredoxins have long remained elusive, despite their common origin. The sequenced genomes of several cyanobacterial species together with novel experimental approaches involving proteomics have provided new tools for re-examining the roles of the thioredoxin systems in these organisms. Thus, each cyanobacterial genome encodes between one and eight thioredoxins and all components necessary for the reduction of thioredoxins. Screening for thioredoxin target proteins in cyanobacteria indicates that assimilation and storage of nutrients, as well as some central metabolic pathways, are regulated by mechanisms involving disulphide/dithiol exchange, which could be catalysed by thioredoxins or related thiol-containing proteins.

A Comprehensive Analysis of the Peroxiredoxin Reduction System in the Cyanobacterium Synechocystis sp. Strain PCC 6803 Reveals that All Five Peroxiredoxins Are Thioredoxin Dependent

Cyanobacteria perform oxygenic photosynthesis, which gives rise to the continuous production of reactive oxygen species, such as superoxide anion radicals and hydrogen peroxide, particularly under unfavorable growth conditions. Peroxiredoxins, which are present in both chloroplasts and cyanobacteria, constitute a class of thiol-dependent peroxidases capable of reducing hydrogen peroxide as well as alkyl hydroperoxides. Chloroplast peroxiredoxins have been studied extensively and have been found to use a variety of endogenous electron donors, such as thioredoxins, glutaredoxins, or cyclophilin, to sustain their activities. To date, however, the endogenous reduction systems for cyanobacterial peroxiredoxins have not been systematically studied. We have expressed and purified all five Synechocystis sp. strain PCC 6803 peroxiredoxins, which belong to the classes 1-Cys Prx, 2-Cys Prx, type II Prx (PrxII), and Prx Q, and we have examined their capacities to interact with and receive electrons from the m-, x-, and y-type thioredoxins from the same organism, which are called TrxA, TrxB, and TrxQ, respectively. Assays for peroxidase activity demonstrated that all five enzymes could use thioredoxins as electron donors, whereas glutathione and Synechocystis sp. strain PCC 6803 glutaredoxins were inefficient. The highest catalytic efficiency was obtained for the couple consisting of PrxII and TrxQ thioredoxin. Studies of transcript levels for the peroxiredoxins and thioredoxins under different stress conditions highlighted the similarity between the PrxII and TrxQ thioredoxin expression patterns.

Expression of ELIPs and PS II-S protein in spinach during acclimative reduction of the Photosystem II antenna in response to increased light intensities

The PS II-S protein and the so-called early light-inducible proteins (ELIPs) are homologous to the chlorophyll a/b-binding (Cab) gene products functioning in light-harvesting. The functional significance of these two CAB homologues is not known although they have been considered to bind pigments and in the case of the PS II–S protein this has been experimentally supported. The role of these two proteins does not appear to be light-harvesting but instead they are suggested to play a role as quenchers of free chlorophyll molecules during biogenesis and/or degradation of pigment-binding proteins. Such a role would be essential to eliminate the toxic and damaging effects that can be induced by free chlorophyll in the light. To this end the expression and characteristics of the ELIPs and the PS II–S protein were investigated in spinach leaves acclimating from low to high light intensities. Under these conditions there is a reduction in the antenna size of Photosystem II due to proteolytic digestion of its major chlorophyll a/b-binding protein (LHC II). During this acclimative proteolysis, up to one third of LHC II can be degraded and consequently substantial amounts of chlorophyll molecules will lose their binding sites. Our results reveal that there is a close correlation between ELIP accumulation and the onset of the LHC II degradation as low light-grown spinach leaves are subjected to increased light intensities. In contrast, there was no change in the relative level of the PS II–S protein during the acclimation process. It is concluded that the role for the ELIPs may be related to binding of liberated chlorophyll molecules and quenching of the toxic effects during LHC II degradation. In addition it was shown that in spinach four different ELIP species can be expressed and that they show different accumulation patterns in response to increased light intensities.

A comparative analysis of the NADPH thioredoxin reductase C -2-Cys peroxiredoxin system from plants and cyanobacteria

Redox regulation based on disulfide-dithiol conversion catalyzed by thioredoxins is an important component of chloroplast function. The reducing power is provided by ferredoxin reduced by the photosynthetic electron transport chain. In addition, chloroplasts are equipped with a peculiar NADPH-dependent thioredoxin reductase, termed NTRC, with a joint thioredoxin domain at the carboxyl terminus. Because NADPH can be produced by the oxidative pentose phosphate pathway during the night, NTRC is important to maintain the chloroplast redox homeostasis under light limitation. NTRC is exclusive for photosynthetic organisms such as plants, algae, and some, but not all, cyanobacteria. Phylogenetic analysis suggests that chloroplast NTRC originated from an ancestral cyanobacterial enzyme. While the biochemical properties of plant NTRC are well documented, little is known about the cyanobacterial enzyme. With the aim of comparing cyanobacterial and plant NTRCs, we have expressed the full-length enzyme from the cyanobacterium Anabaena species PCC 7120 as well as site-directed mutant variants and truncated polypeptides containing the NTR or the thioredoxin domains of the protein. Immunological and kinetic analysis showed a high similarity between NTRCs from plants and cyanobacteria. Both enzymes efficiently reduced 2-Cys peroxiredoxins from plants and from Anabaena but not from the cyanobacterium Synechocystis. Arabidopsis (Arabidopsis thaliana) NTRC knockout plants were transformed with the Anabaena NTRC gene. Despite a lower content of NTRC than in wild-type plants, the transgenic plants showed significant recovery of growth and pigmentation. Therefore, the Anabaena enzyme fulfills functions of the plant enzyme in vivo, further emphasizing the similarity between cyanobacterial and plant NTRCs.