D1 protein of photosystem II: The light sensor in chloroplasts

Light, controls the “blueprint” for chloroplast development, but at high intensities is toxic to the chloroplast. Excessive light intensities inhibit primarily photosystem II electron transport. This results in generation of toxic singlet oxygen due to impairment of electron transport on the acceptor side between pheophytin and Q_B -the secondary electron acceptor. High light stress also impairs electron transport on the donor side of photosystem II generating highly oxidizing species Z⁺ and P680⁺. A conformationsl change in the photosystem II reaction centre protein Dl affecting its Q_B-binding site is involved in turning the damaged protein into a substrate for proteolysis. The evidence indicates that the degradation of D1 is an enzymatic process and the protease that degrades D1 protein has been shown to be a serine protease Although there is evidence to indicate that the chlorophyll a-protein complex CP43 acts as a serine-type protease degrading Dl, the observed degradation of Dl protein in photosystem II reaction centre particlesin vitro argues against the involvement of CP43 in Dl degradation. Besides the degradation during high light stress of Dl, and to a lesser extent D2-the other reaction centre protein, CP43 and CP29 have also been shown to undergo degradation. In an oxygenic environment, Dl is cleaved from its N-and C-termini and the disassembly of the photosystem II complex involves simultaneous release of manganese and three extrinsic proteins involved in oxygen evolution. It is known that protein with PEST sequences are subject to degradation; D1 protein contains a PEST sequence adjacent to the site of cleavage on the outer side of thylakoid membrane between helices IV and V. The molecular processes of “triggering” of Dl for proteolytic degradation are not clearly understood. The changes in structural organization of photosystem II due to generation of oxy-radicals and other highly oxidizing species have also not been resolved. Whether CP43 or a component of the photosystem II reaction centre itself (Dl. D2 or cy1 b559 subunits), which may be responsible for degradation of Dl, is also subject to light modification to become an active protease, is also not known. The identity of proteases degrading Dl, LHCII and CP43 and C29 remains to be established     

Relationship between photoinhibition of photosynthesis, D1 protein turnover and chloroplast structure: effects of protein synthesis inhibitors

Irradiation of Spinach oleracea intact leaf tissue and of mesophyll protoplasts of Valerianella locusta at 20° C with strong light resulted in severe (40–80%) inhibition of photosynthesis, measured as photosystem II electron transport activity in isolated thylakoids or as fluorescence parameter F_V/F_M on intact leaf disks. No net degradation of the D1 protein of photosystem II was seen under these conditions. However, in the presence of streptomycin, an inhibitor of chloroplast protein synthesis, net D1 degradation (up to about 80%) did occur with a half-time of 4–6h, and photoinhibition was enhanced. Thylakoid ultrastructure remained stable during photoinhibition, even when substantial degradation of D1 took place in the presence of streptomycin. When leaf disks were irradiated at 2°C, streptomycin did not influence the degree of photoinhibition, and net Dl degradation did not occur. These results suggest that in excess (photoinhibitory) light at 20°C, turnover (coordinated degradation and synthesis) of D1 diminished the degree of photoinhibition. The observed photoinhibition is thought to be due to the accumulation of inactive photosystem II reaction centres still containing D1. In the presence of streptomycin, the Dl protein was degraded (probably in the previously inactivated centres), but restoration of active centres via D1 synthesis was blocked, leading to more severe photoinhibition. Low temperature (2°C), by restricting both degradation and resynthesis of D1, favoured the accumulation of inactive centres. Streptomycin and chloramphenicol (another inhibitor of chloroplast protein synthesis) were tested for side-effects on photosynthesis. Strong inhibitory effects of chloramphenicol, but much less severe effects of streptomycin were observed.     

Photodamaged D1 protein is degraded in Arabidopsis mutants lacking the Deg2 protease

In plants exposed to high irradiances of visible light, the D1 protein in the reaction center of photosystem II is oxidatively damaged and rapidly degraded. Earlier work in our laboratory showed that the serine protease Deg2 performs the primary cleavage of photodamaged D1 protein in vitro. Here, we demonstrate that the rate of D1 protein degradation under light stress conditions in Arabidopsis mutants lacking the Deg2 protease is similar to those in wild-type plants. Therefore, we propose that several redundant D1 protein degradation pathways might exist in vivo.     

Photosynthetic Properties of Photosystem Ⅱ in Arabidopsis thaliana Ipa1 Mutant

In a previous study, we characterized a high chlorophyll fluorescence Ipal mutant of Arabidopsis thallana, in which approximately 20% photosystem （PS） Ⅱ protein is accumulated. In the present study, analysis of fluorescence decay kinetics and thermoluminescence profiles demonstrated that the electron transfer reaction on either the donor or acceptor side of PSII remained largely unaffected in the Ipa1 mutant. In the mutant, maximal photochemical efficiency （Fv/Fm, where Fm is the maximum fluorescence yield and Fv is variable fluorescence） decreased with increasing light intensity and remained almost unchanged in wildtype plants under different light conditions. The Fv/Fm values also increased when mutant plants were transferred from standard growth light to low light conditions. Analysis of PSll protein accumulation further confirmed that the amount of PSll reaction center protein is correlated with changes in Fv/Fm in Ipal plants. Thus, the assembled PSll in the mutant was functional and also showed increased photosensitivity compared with wild-type plants.     

Photosynthetic Properties of Photosystem II in Arabidopsis thaliana lpa1 Mutant

In a previous study, we characterized a high chlorophyll fluorescence lpa1 mutant of Arabidopsis thaliana, in which approximately 20% photosystem (PS) II protein is accumulated. In the present study, analysis of fluorescence decay kinetics and thermoluminescence profiles demonstrated that the electron transfer reaction on either the donor or acceptor side of PSII remained largely unaffected in the lpa1 mutant. In the mutant, maximal photochemical efficiency (Fv/Fm, where Fm is the maximum fluorescence yield and Fv is variable fluorescence) decreased with increasing light intensity and remained almost unchanged in wild-type plants under different light conditions. The Fv/Fm values also increased when mutant plants were transferred from standard growth light to low light conditions. Analysis of PSII protein accumulation further confirmed that the amount of PSII reaction center protein is correlated with changes in Fv/Fm in lpa1 plants. Thus, the assembled PSII in the mutant was functional and also showed increased photosensitivity compared with wild-type plants.(Author for correspondence. Tel: +86 (0)10 6283 6256; Fax: +86 (0)10 8259 9384; E-mail: zhanglixin@ibcas.ac.cn)     

Recovery from photoinhibition: effect of light and inhibition of protein synthesis of 32-kD chloroplast protein

Recovery from photoinhibition of photosynthesis in intact Lemna gibba was studied in presence of the protein synthesis inhibitors chloramphenicol and cycloheximide. Exposure to an irradiance of 1000 mol m^-2s^-1 in N₂ for 90 min induced 80% photoinhibition. The plants recovered photosynthesis when transfered to normal irradiances (210 mol m^-2s^-1) and air. Chloramphenicol added to the medium was taken up by the plant and reduced photosynthesis slightly. Recovery from photoinhibition was more inhibited than photosynthesis. Cycloheximide was also taken up by the plants and reduced synthesis of light harvesting chlorophyll protein: however, neither photosynthesis nor recovery were much affected. Synthesis of 32-kD chloroplast protein during recovery was inhibited by chloramphenicol, but not by cycloheximide. Synthesis of 32-kD protein was enhanced by 20–210 mol m^-2s^-1 light. The results support the hypothesis that synthesis of 32-kD protein is important for recovery of photosynthesis after photoinhibition.     

The Arabidopsis PsbO2 protein regulates dephosphorylation and turnover of the photosystem II reaction centre D1 protein

The extrinsic photosystem II (PSII) protein of 33 kDa (PsbO), which stabilizes the water-oxidizing complex, is represented in Arabidopsis thaliana (Arabidopsis) by two isoforms. Two T-DNA insertion mutant lines deficient in either the PsbO1 or the PsbO2 protein were retarded in growth in comparison with the wild type, while differing from each other phenotypically. Both PsbO proteins were able to support the oxygen evolution activity of PSII, although PsbO2 was less efficient than PsbO1 under photoinhibitory conditions. Prolonged high light stress led to reduced growth and fitness of the mutant lacking PsbO2 as compared with the wild type and the mutant lacking PsbO1. During a short period of treatment of detached leaves or isolated thylakoids at high light levels, inactivation of PSII electron transport in the PsbO2-deficient mutant was slowed down, and the subsequent degradation of the D1 protein was totally inhibited. The steady-state levels of in vivo phosphorylation of the PSII reaction centre proteins D1 and D2 were specifically reduced in the mutant containing only PsbO2, in comparison with the mutant containing only PsbO1 or with wild-type plants. Phosphorylation of PSII proteins in vitro proceeded similarly in thylakoid membranes from both mutants and wild-type plants. However, dephosphorylation of the D1 protein occurred much faster in the thylakoids containing only PsbO2. We conclude that the function of PsbO1 in Arabidopsis is mostly in support of PSII activity, whereas the interaction of PsbO2 with PSII regulates the turnover of the D1 protein, increasing its accessibility to the phosphatases and proteases involved in its degradation.     

Proteasome-dependent turnover of protein disulfide isomerase in oxidatively stressed cells.

Generalized increases in protein oxidation and protein degradation in response to mild oxidative stress have been widely reported, but only a few individual proteins have actually been shown to undergo selective, oxidation-induced proteolysis. Our goal was to find such proteins in Clone 9 liver cells exposed to hydrogen peroxide. Using metabolic radiolabeling of intracellular proteins with [35S]cysteine/methionine, and analysis by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), we found at least three labeled proteins ("A," "B," and "C") whose levels were decreased significantly more than the generalized protein loss after mild oxidative stress. "Protein C" was excised from 2-D PAGE and subjected to N-terminal amino acid microsequencing. "Protein C" was identified as Protein Disulfide Isomerase or PDI (E.C. 5.3.4.1), and this identity was reconfirmed by Western blotting with a C-terminal anti-PDI monoclonal antibody. A combination of quantitative radiometry and Western blotting in 2-D PAGE revealed that PDI was selectively degraded and then new PDI was synthesized, following H2O2 exposure. PDI degradation was blocked by inhibitors of the proteasome, and by cell treatment with proteasome C2 subunit antisense oligonucleotides, indicating that the proteasome was largely responsible for oxidation-induced PDI degradation.     

D1 protein degradation during photoinhibition of intact leaves a modification of the D1 protein precedes degradation

Illumination of intact pumpkin leaves with high light led to severe photoinhibition of photosystem II with no net degradation of the D1 protein. Instead, however, a modified form of D1 protein with slightly slower electrophoretic mobility was induced with corresponding loss in the original form of the D1 protein. When the leaves were illuminated in the presence of chloramphenicol the modified form was degraded, which led to a decrease in the total amount of the D1 protein. Subfractionation of the thylakoid membranes further supported the conclusion that the novel form of the D1 protein was not a precursor but a high-light modified form that was subsequently degraded.     

Isolation and characterization of an Arabidopsis thaliana C-8,7 sterol isomerase: functional and structural similarities to mammalian C-8,7 sterol isomerase/emopamil-binding protein

The yeast C-8,7 sterol isomerase contains a polyvalent high-affinity drug binding site similar to mammalian sigma receptors. Exogenously supplied sigma ligands inhibit sterol biosynthesis in yeast, demonstrating a pharmacological relationship between sigma ligand-binding and C-8,7 sterol isomerase activity. We report the isolation of an Arabidopsis thaliana C-8,7 sterol isomerase by functional complementation of the corresponding sterol mutant in yeast and its characterization by exposure to sigma ligands. The yeast erg2 mutant, which lacks the C-8,7 sterol isomerase gene and activity, was transformed with an Arabidopsis cDNA yeast expression library. Transformed colonies were selected for restoration of C-8,7 sterol isomerase activity (i.e. wild-type ergosterol production) by enhanced resistance to the antibiotic cycloheximide. Sterols produced in complemented lines were characterized by gas chromatography-mass spectroscopy (GC-MS). The full-length A. thaliana cDNA (pA.t.SI1) that complemented the erg2 mutation contains an open reading frame encoding a 21 kDa protein that shares 68% similarity and 35% amino acid identity to the recently isolated mouse C-8,7 sterol isomerase. The sigma ligands, haloperidol, ifenprodil and verapamil inhibited the production of ergosterol in wild-type Saccharomyces cerevisiae and in the erg2 mutant complemented with pA.t.SI1. Structural and biochemical similarities between the A. thaliana C-8,7 sterol isomerase and the mammalian emopamil-binding protein (EBP) are discussed.     

Construction of a chloroplast protein interaction network and functional mining of photosynthetic proteins in Arabidopsis thaliana

Chloroplast is a typical plant cell organelle where photosynthesis takes place. In this study, a total of 1 808 chloroplast core proteins in Arabidopsis thaliana were reliably identified by combining the results of previously published studies and our own predictions. We then constructed a chloroplast protein interaction network primarily based on these core protein interactions. The network had 22 925 protein interaction pairs which involved 2 214 proteins. A total of 160 previously uncharacterized proteins were annotated in this network. The subunits of the photosynthetic complexes were modularized, and the functional relationships among photosystem Ⅰ （PSI）, photosystem Ⅱ （PSII）, light harvesting complex of photosystem Ⅰ （LHC Ⅰ） and light harvesting complex of photosystem Ⅰ （LHC Ⅱ） could be deduced from the predicted protein interactions in this network. We further confirmed an interaction between an unknown protein AT1G52220 and a photosynthetic subunit PSI-D2 by yeast two-hybrid analysis. Our chloroplast protein interaction network should be useful for functional mining of photosynthetic proteins and investigation of chloroplast-related functions at the systems biology level in Arabidopsis.     

Reduction of protein disulfide isomerase results in open conformations and stimulates dynamic exchange between structural ensembles

Human protein disulfide isomerase (PDI) is an essential redox-regulated enzyme required for oxidative protein folding. It comprises four thioredoxin domains, two catalytically active (a, a’) and two inactive (b, b’), organized to form a flexible abb’a’ U-shape. Snapshots of unbound oxidized and reduced PDI have been obtained by X-ray crystallography. Yet, how PDI’s structure changes in response to the redox environment and inhibitor binding remains controversial. Here, we used multiparameter confocal single-molecule FRET to track the movements of the two catalytic domains with high temporal resolution. We found that at equilibrium, PDI visits three structurally distinct conformational ensembles, two “open” (O₁ and O₂) and one “closed” (C). We show that the redox environment dictates the time spent in each ensemble and the rate at which they exchange. While oxidized PDI samples O₁, O₂, and C more evenly and in a slower fashion, reduced PDI predominantly populates O₁ and O₂ and exchanges between them more rapidly, on the submillisecond timescale. These findings were not expected based on crystallographic data. Using mutational analyses, we further demonstrate that the R300-W396 cation-π interaction and active site cysteines dictate, in unexpected ways, how the catalytic domains relocate. Finally, we show that irreversible inhibitors targeting the active sites of reduced PDI did not abolish these protein dynamics but rather shifted the equilibrium toward the closed ensemble. This work introduces a new structural framework that challenges current views of PDI dynamics, helps rationalize its multifaceted role in biology, and should be considered when designing PDI-targeted therapeutics.     

Yeast Mpd1p reveals the structural diversity of the protein disulfide isomerase family

Oxidoreductases belonging to the protein disulfide isomerase (PDI) family promote proper disulfide bond formation in substrate proteins in the endoplasmic reticulum. In plants and metazoans, new family members continue to be identified and assigned to various functional niches. PDI-like proteins typically contain tandem thioredoxin-fold domains. The limited information available suggested that the relative orientations of these domains may be quite uniform across the family, and structural models based on this assumption are appearing. However, the X-ray crystal structure of the yeast PDI family protein Mpd1p, described here, demonstrates the radically different domain orientations and surface properties achievable with multiple copies of the thioredoxin fold. A comparison of Mpd1p with yeast Pdi1p expands our perspective on the contexts in which redox-active motifs are presented in the PDI family.     

Light-dependent phosphorylation of D1 reaction centre protein of photosystem II: hypothesis for the functional role in vivo

Reversible phosphorylation of the D1 reaction centre protein of photosystem II (PSII) occurs in thylakoid membranes of higher plants. The significance of D1 protein phosphorylation in the function of PSII is not yet clear. This paper summarizes the data implying that phosphorylation of D1 protein in higher plants is involved in the regulation of the repair cycle of photoinhibited PSII centres. Photoinhibition of PSII, D1 protein phosphorylation and degradation have been studied in vivo in higher plant leaves acclimated to different growth irradiances. It is shown that photoinhibitory illumination induces maximal phosphorylation of the D1 protein. Under these conditions D1 turnover is also saturated. We postulate that phosphorylation retards the degradation of damaged D1 protein under conditions where rapid replacement by a new D1 copy is not possible. This would protect PSII from total disassembly and degradation of all PSII subunits. We conclude that the phosphorylation of D1 protein and the regulation of D1 protein degradation may have evolved together. Furthermore, these characteristics seem to be related to the highly organized structure of higher-plant type thylakoid membranes, since the capability to phosphorylate D1 protein is restricted to seed plants.     

A novel allele of the Arabidopsis thaliana MACPF protein CAD1 results in deregulated immune signaling

Immune recognition in plants is governed by two major classes of receptors: pattern recognition receptors (PRRs) and nucleotide-binding leucine-rich repeat receptors (NLRs). Located at the cell surface, PRRs bind extracellular ligands originating from microbes (indicative of “non-self”) or damaged plant cells (indicative of “infected-self”), and trigger signaling cascades to protect against infection. Located intracellularly, NLRs sense pathogen-induced physiological changes and trigger localized cell death and systemic resistance. Immune responses are under tight regulation in order to maintain homeostasis and promote plant health. In a forward-genetic screen to identify regulators of PRR-mediated immune signaling, we identified a novel allele of the membrane-attack complex and perforin (MACPF)-motif containing protein CONSTITUTIVE ACTIVE DEFENSE 1 (CAD1) resulting from a missense mutation in a conserved N-terminal cysteine. We show that cad1-5 mutants display deregulated immune signaling and symptoms of autoimmunity dependent on the lipase-like protein ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), suggesting that CAD1 integrity is monitored by the plant immune system. We further demonstrate that CAD1 localizes to both the cytosol and plasma membrane using confocal microscopy and subcellular fractionation. Our results offer new insights into immune homeostasis and provide tools to further decipher the intriguing role of MACPF proteins in plants.     

Enhancement of protein secretion in Pichia pastoris by overexpression of protein disulfide isomerase

A potential vaccine candidate, Necator americanus secretory protein (Na-ASP1), against hookworm infections, has been expressed in Pichia pastoris. Na-ASP1, a 45 kDa protein containing 20 cysteines, was directed outside the cell by fusing the protein to the preprosequence of the alpha-mating factor of Saccharomyces cerevisiae. Most of the protein produced by single copy clones was secreted outside the cell. However, increasing gene copy number of Na-ASP1 protein in P. pastoris saturated secretory capacity and therefore, decreased the amount of secreted protein in clones harboring multiple copies of Na-ASP1 gene. Overexpression of the endoplasmic reticulum (ER) resident, homologous chaperone protein, protein disulfide isomerase (PDI) was able to increase the secretion of (Na-ASP1) protein in high copy clones. The effect of PDI levels on secretion of Na-ASP1 protein was examined in clones with varying copy number of PDI gene. Increase in secreted Na-ASP1 secretion is correlated well with the PDI copy number. Increasing levels of PDI also increased overall Na-ASP1 protein production in all the clones. Nevertheless, there was still accumulation of intracellular Na-ASP1 protein in P. pastoris clones over-expressing Na-ASP1 and PDI proteins.     

S‐nitrosylated protein disulfide isomerase contributes to mutant SOD1 aggregates in amyotrophic lateral sclerosis

A major hallmark of mutant superoxide dismutase (SOD1)‐linked familial amyotrophic lateral sclerosis is SOD1‐immunopositive inclusions found within motor neurons. The mechanism by which SOD1 becomes aggregated, however, remains unclear. In this study, we aimed to investigate the role of nitrosative stress and S‐nitrosylation of protein disulfide isomerase (PDI) in the formation of SOD1 aggregates. Our data show that with disease progression inducible nitric oxide synthase (iNOS) was up‐regulated, which generated high levels of nitric oxide (NO) and subsequently induced S‐nitrosylation of PDI in the spinal cord of mutant SOD1 transgenic mice. This was further confirmed by in vitro observation that treating SH‐SY5Y cells with NO donor S‐nitrosocysteine triggered a dose‐dependent formation of S‐nitrosylated PDI. When mutant SOD1 was over‐expressed in SH‐SY5Y cells, the iNOS expression was up‐regulated, and NO generation was consequently increased. Furthermore, both S‐nitrosylation of PDI and the formation of mutant SOD1 aggregates were detected in the cells expressing mutant SOD1^G93A. Blocking NO generation with the NOS inhibitor N‐nitro‐l ‐arginine attenuated the S‐nitrosylation of PDI and inhibited the formation of mutant SOD1 aggregates. We conclude that NO‐mediated S‐nitrosylation of PDI is a contributing factor to the accumulation of mutant SOD1 aggregates in amyotrophic lateral sclerosis.