CO2 limitation induces specific redox-dependent protein phosphorylation in Chlamydomonas reinhardtii

Acclimation of the green alga Chlamydomonas reinhardtii to limiting environmental CO2 induced specific protein phosphorylation at the surface of photosynthetic thylakoid membranes. Four phosphopeptides were identified and sequenced by nanospray quadrupole TOF MS from the cells acclimating to limiting CO2. One phosphopeptide originated from a protein that has not been annotated. We found that this unknown expressed protein (UEP) was encoded in the genome of C. reinhardtii. Three other phosphorylated peptides belonged to Lci5 protein encoded by the low-CO2-inducible gene 5 (lci5). The phosphorylation sites were mapped in the tandem repeats of Lci5 ensuring phosphorylation of four serine and three threonine residues in the protein. Immunoblotting with Lci5-specific antibodies revealed that Lci5 was localized in chloroplast and confined to the stromal side of the thylakoid membranes. Phosphorylation of Lci5 and UEP occurred strictly at limiting CO2; it required reduction of electron carriers in the thylakoid membrane, but was not induced by light. Both proteins were phosphorylated in the low-CO2-exposed algal mutant deficient in the light-activated protein kinase Stt7. Phosphorylation of previously unknown basic proteins UEP and Lci5 by specific redox-dependent protein kinase(s) in the photosynthetic membranes reveals the early response of green algae to limitation in the environmental inorganic carbon.     

TIC62 redox-regulated translocon composition and dynamics

The preprotein translocon at the inner envelope of chloroplasts (Tic complex) facilitates the import of nuclear-encoded preproteins into the organelle. Seven distinct subunits have been identified so far. For each of those, specific functions have been proposed based on structural prediction or experimental evidence. Three of those subunits possess modules that could act as redox-active regulatory components in the import process. To date, however, the mode of redox regulation of the import process remains enigmatic. To investigate how the chloroplast redox state influences translocon behavior and composition, we studied the Tic component and the putative redox sensor Tic62 in more detail. The experimental results provide evidence that Tic62 can act as a bona fide dehydrogenase in vitro, and that it changes its localization in the chloroplast dependent on the NADP+/NADPH ratio in the stroma. Moreover, the redox state influences the interactions of Tic62 with the translocon and the flavoenzyme ferredoxin-NADP+ oxidoreductase. Additionally, we give initial experimental insights into the Tic62 structure using circular dichroism measurements and demonstrate that the protein consists of two structurally different domains. Our results indicate that Tic62 possesses redox-dependent properties that would allow it to fulfill a role as redox sensor protein in the chloroplast.     

Effect of precursor protein phosphorylation on import into isolated chloroplasts from Chlamydomonas.

In higher plants, chloroplast-destined precursor proteins are thought to be phosphorylated. Mediated by a specific 14-3-3 protein, these phosphorylated proteins bind to the chloroplast surface and are subsequently imported into the chloroplast. We demonstrate that also in the green alga Chlamydomonas reinhardtii the precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase becomes phosphorylated by a plant protein kinase and that the phosphorylation site is located in the transit peptide. The phosphorylation status of the precursor protein regulates its import into chloroplasts especially at an early step during this process. The possible physiological function is discussed.     

In vitro reconstitution of monogalactosyldiacylglycerol (MGDG) synthase regulation by thioredoxin

Monogalactosyldiacylglycerol (MGDG), a major membrane lipid of chloroplasts, is synthesized by MGDG synthase (MGD) localized in chloroplast envelope membranes. We investigated whether MGD activity is regulated in a redox-dependent manner using recombinant cucumber MGD overexpressed in Escherichia coli. We found that MGD activity is reversibly regulated by reduction and oxidation in vitro and that an intramolecular disulfide bond(s) is involved in MGD activation. Because thioredoxin efficiently reduced disulfide bonds to enhance MGD activity in vitro, MGD is potentially an envelope-bound thioredoxin target protein in higher plants.     

Chloroplast transit peptides: structure, function and evolution

It is thought that two to three thousand different proteins are targeted to the chloroplast, and the 'transit peptides' that act as chloroplast targeting sequences are probably the largest class of targeting sequences in plants. At a primary structural level, transit peptide sequences are highly divergent in length, composition and organization. An emerging concept suggests that transit peptides contain multiple domains that provide either distinct or overlapping functions. These functions include direct interaction with envelope lipids, chloroplast receptors and the stromal processing peptidase. The genomic organization of transit peptides suggests that these domains might have originated from distinct exons, which were shuffled and streamlined throughout evolution to yield a modern, multifunctional transit peptide. Although still poorly characterized, this evolutionary process could yield transit peptides with different domain organizations. The plasticity of transit peptide design is consistent with the diverse biological functions of chloroplast proteins.     

L-Buthionine-(S,R)-sulfoximine, an irreversible inhibitor of gamma-glutamylcysteine synthetase, augments LPS-mediated pro-inflammatory cytokine biosynthesis: evidence for the implication of an IkappaB-alpha/NF-kappaB insensitive pathway.

The pro-inflammatory cytokines, including tumor necrosis factor (TNF)-alpha and interleukin (IL)-6, contribute to the exacerbation of pathophysiological conditions in the lung. The regulation of cytokines involves the reduction-oxidation (redox)-sensitive nuclear factor-kappaB (NF-kappaB), the activation of which is mediated through an upstream kinase that regulates the phosphorylation and subsequent degradation of inhibitory-kappaB (IkappaB)-alpha, the major cytosolic inhibitor of NF-kappaB. It was hypothesized that lipopolysaccharide (LPS)-induced biosynthesis of TNF-alpha and IL-6 in vitro is tightly regulated by redox equilibrium. Furthermore, the likely involvement of the IkappaB-alpha/NF-kappaB signalling transduction pathway in mediating redox-dependent regulation of LPS-induced cytokine biosynthesis was revealed. Using alveolar epithelial cells, the role of L-buthionine-(S,R)-sulfoximine (BSO), a specific and irreversible inhibitor of gamma-glutamylcysteine synthetase (gamma-GCS), the rate-limiting enzyme in glutathione (GSH - an antioxidant thiol) biosynthesis, in regulating LPS-mediated TNF-alpha and IL-6 production and the IkappaB-alpha/NF-kappaB signalling pathway was investigated. Pre-treatment with BSO, prior to exposure to LPS augmented, in a dose-dependent manner, LPS-induced TNF-alpha and IL-6 biosynthesis, an effect associated with the induction of intracellular accumulation of reactive oxygen species (ROS). Interestingly, BSO blocked the phosphorylation of IkappaB-alpha, reduced its degradation, thereby allowing its cytosolic accumulation, and subsequently inhibited the activation of NF-kappaB. These results indicate that there are ROS and redox-mediated effects regulating pro-inflammatory cytokines, and that the IkappaB-alpha/NF-kappaB pathway is redox-sensitive and differentially involved in mediating redox-dependent regulation of cytokine signaling.     

Light-harvesting complex II protein CP29 binds to photosystem I of Chlamydomonas reinhardtii under State 2 conditions

The State 1 to State 2 transition in the photosynthetic membranes of plants and green algae involves the functional coupling of phosphorylated light-harvesting complexes of photosystem II (LHCII) to photosystem I (PSI). We present evidence suggesting that in Chlamydomonas reinhardtii this coupling may be aided by a hyper-phosphorylated form of the LHCII-like CP29 protein (Lhcbm4). MS analysis of CP29 showed that Thr6, Thr16 and Thr32, and Ser102 are phosphorylated in State 2, whereas in State 1-exposed cells only phosphorylation of Thr6 and Thr32 could be detected. The LHCI-PSI supercomplex isolated from the alga in State 2 was found to contain strongly associated CP29 in phosphorylated form. Electron microscopy suggests that the binding site for this highly phosphorylated CP29 is close to the PsaH protein. It is therefore postulated that redox-dependent multiple phosphorylation of CP29 in green algae is an integral part of the State transition process in which the structural changes of CP29, induced by reversible phosphorylation, determine the affinity of LHCII for either of the two photosystems.     

Glutaredoxin modulates platelet-derived growth factor-dependent cell signaling by regulating the redox status of low molecular weight protein-tyrosine phosphatase

Glutaredoxin (GRX) is a glutathione-disulfide oxidoreductase involved in various cellular functions, including the redox-dependent regulation of certain integral proteins. Here we demonstrated that overexpression of GRX suppressed the proliferation of myocardiac H9c2 cells treated with platelet-derived growth factor (PDGF)-BB. After stimulation with PDGF-BB, the phosphorylation of PDGF receptor (PDGFR) beta was suppressed in GRX gene-transfected cells, compared with controls. Conversely, the phosphorylation was enhanced by depletion of GRX by RNA interference. In this study we focused on the role of low molecular weight protein-tyrosine phosphatase (LMW-PTP) in the dephosphorylation of PDGFRbeta via a redox-dependent mechanism. We found that depletion of LMW-PTP using RNA interference enhanced the PDGF-BB-induced phosphorylation of PDGFRbeta, indicating that LMW-PTP works for PDGFRbeta. The enhancement of the phosphorylation of PDGFRbeta was well correlated with inactivation of LMW-PTP by cellular peroxide generated in the cells stimulated with PDGF-BB. In vitro, with hydrogen peroxide treatment, LMW-PTP showed decreased activity with the concomitant formation of dithiothreitol-reducible oligomers. GRX protected LMW-PTP from hydrogen peroxide-induced oxidation and inactivation in concert with glutathione, NADPH, and glutathione disulfide reductase. This strongly suggests that retention of activity of LMW-PTP by enhanced GRX expression suppresses the proliferation of cells treated with PDGF-BB via enhanced dephosphorylation of PDGFRbeta. Thus, GRX plays an important role in PDGF-BB-dependent cell proliferation by regulating the redox state of LMW-PTP.     

Changes in Phosphorylation Status of Wheat Plastid Polypeptides as Influenced by Light, Calcium and Cyclic AMP

The polypeptides of etioplast and chloroplast fractions, purified on Percoll discontinuous gradient, were phosphorylated in vitro using (γ-³²P)ATP, resolved by SDS-PAGE and autoradiographed. In general, about 15-18 phosphopolypeptides in the range of 14-150 kD were distinctly visible in autoradiograms of both organelle fractions with varying degree of radiolabel incorporation. Although short-term irradiation with red or far-red light did not have any significant effect on phosphorylation status of etioplast polypeptides, in vivo irradiation with 1 h white light, followed by in vitro phosphorylation, decreased phosphorylation of a 116 kD polypeptide and increased the phosphorylation of polypeptides of 38 kD and a doublet around 20 kD. Strikingly, the phosphorylation status of 116 kD etioplast polypeptide was adversely affected by Ca²⁺ as well, and this phosphopolypeptlde was not distinctly visible in the autoradiogram of the chloroplast fraction proteins. However, in vitro phosphorylation of 98, 57 and 50 kD polypeptides of both etioplast and chloroplast fractions was found to be Ca²⁺ dependent. Unlike Ca²⁺, 3′,5′-cyclic AMP down-regulated the phosphorylation of several polypeptides of both etioplasts and chloroplasts, including 98 and 50 kD, and up-regulated the phosphorylation of 32 and 57 kD polypeptides. The significance of these observations on changes in phosphoprotein profile of etioplasts and chloroplasts, as influenced by light, Ca²⁺ and cyclic nucleotides, has been discussed.     

Cross-talk between calcium signalling and protein phosphorylation at the thylakoid

The role of protein phosphorylation for adjusting chloroplast functions to changing environmental needs is well established, whereas calcium signalling in the chloroplast is only recently becoming appreciated. The work presented here explores the potential cross-talk between calcium signalling and protein phosphorylation in chloroplasts and provides the first evidence for targets of calcium-dependent protein phosphorylation at the thylakoid membrane. Thylakoid proteins were screened for calcium-dependent phosphorylation by 2D gel electrophoresis combined with phospho-specific labelling and PsaN, CAS, and VAR1, among other proteins, were identified repeatedly by mass spectrometry. Subsequently their calcium-dependent phosphorylation was confirmed in kinase assays using the purified proteins and chloroplast extracts. This is the first report on the protein targets of calcium-dependent phosphorylation of thylakoid proteins and provides ground for further studies in this direction.     

Mitochondria: Biological roles in platelet physiology and pathology

Mitochondria are key regulators of cellular energy and redox metabolism, also playing a central role in cell signaling and death pathways. A number of processes occur within mitochondria, including redox-dependent ATP synthesis by oxidative phosphorylation and reactive oxygen species production. Mitochondrial permeability transition is a reversible process that may lead to cell death and is regulated by calcium and reactive oxygen species. Functional mitochondria are present in platelets, and evidence has demonstrated the direct involvement of these organelles in cellular ATP production, redox balance, as well as in platelet activation and apoptosis. Here, we review aspects of platelet physiology in which mitochondria are involved, as well as assess their function as new tools for studying a number of human diseases.     

Nitric oxide donor-mediated inhibition of phosphorylation shows that light-mediated degradation of photosystem II D1 protein and phosphorylation are not tightly linked

An outcome of the photochemistry during oxygenic photosynthesis is the rapid turn over of the D1 protein in the light compared to the other proteins of the photosystem II (PS II) reaction center. D1 is a major factor of PS II instability and its replacement a primary event of the PS II repair cycle. D1 also undergoes redox-dependent phosphorylation prior to its degradation. Although it has been suggested that phosphorylation modulates D1 metabolism, reversible D1 phosphorylation was reported not to be essential for PS II repair in Arabidopsis. Thus, the involvement of phosphorylation in D1 degradation is controversial. We show here that nitric oxide donors inhibit in vivo phosphorylation of the D1 protein in Spirodela without inhibiting degradation of the protein. Thus, D1 phosphorylation is not tightly linked to D1 degradation in the intact plant.     

Extracellular cysteine/cystine redox regulates the p44/p42 MAPK pathway by metalloproteinase-dependent epidermal growth factor receptor signaling

Previous research shows that stimulation of proliferation of colon carcinoma (Caco-2) cells by a more reduced extracellular cysteine/cystine (Cys/CySS) redox state occurs with no apparent effect on intracellular glutathione and that this stimulation is lost on addition of epidermal growth factor. The purpose of the present study was to determine whether a more reduced extracellular Cys/CySS redox state activates the mitogenic p44/p42 mitogen-activated protein kinase (MAPK) pathway and whether this is signaled through the epidermal growth factor receptor (EGFR). Caco-2 cells were exposed to a range of physiological extracellular redox conditions from -150 to 0 mV. In the absence of added growth factors, the most reduced (-150 mV) redox state induced an 80% increase in EGFR phosphorylation, and this was followed by a marked increase in phosphorylation of p44/p42 MAPK. Inhibitors of EGFR (AG1478) and p44/p42 MAPK (U0126) phosphorylation blocked redox-dependent p44/p42 phosphorylation, indicating that signaling occurred by EGFR. These effects were inhibited by pretreatment with a nonpermeant alkylating agent, showing that signaling involved thiols accessible to the extracellular space. The EGFR ligand TGF-alpha was increased in culture medium at more reduced redox states. Redox-dependent phosphorylation of EGFR was completely prevented by a metalloproteinase inhibitor (GM6001), and an antibody to TGF-alpha partially inhibited the phosphorylation of p44/p42 MAPK by redox. Thus the data show that a redox-dependent activation of metalloproteinase can stimulate the mitogenic p44/p42 MAPK pathway by a TGF-alpha-dependent mechanism. Because Cys availability and Cys/CySS redox are dependent on nutrition, disease, and environmental exposures, the results suggest that cell proliferation could be influenced physiologically by Cys-dependent redox effects on growth factor signaling pathways.     

Chloroplast DNA insertions into the nuclear genome of rice: the genes,sites and ages of insertion involved

Rice (Oryza sativa) is one of three predominant grain crops, and its nuclear and organelle genomes have been sequenced. Following genome analysis revealed many exchanges of DNA sequences between the nuclear and organelle genomes. In this study, a total of 45 chloroplast DNA insertions more than 2 kb in length were detected in rice nuclear genome. A homologous recombination mechanism is expected for those chloroplast insertions with high similarity between their flanking sequences. Only five chloroplast insertions with high sequence similarity between two flanking sequences from an insertion were found in the 45 insertions, suggesting that rice might follow the non-homologous end-joining (NHEJ) repair of double-stranded breaks mechanism, which is suggested to be common to all eukaryotes. Our studies indicate that the most chloroplast insertions occurred at a nuclear region characterized by a sharp change of repetitive sequence density. One potential explanation is that regions such as this might be susceptible target sites or “hotspots” of DNA damage. Our results also suggest that the insertion of retrotransposon elements or non-chloroplast DNA into chloroplast DNA insertions may contribute significantly to their fragmentation process. Moreover, based on chloroplast insertions in nuclear genomes of two subspecies (indica and japonica) of cultivated rice, our results strongly suggest that they diverged during 0.06–0.22 million years ago. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effect of ATP on the Content of Inactive PSII Complexes in Chlorella

A prolonged (20 h) dark incubation of Chlorella pyrenoidosa algae at 37°C resulted in a twofold rise of the slowly rising phase (10–15 min), sF _v, in the kinetics of variable chlorophyll fluorescence, F _v (F _v = F _m – F ₀) in diuron-treated cells. This effect suggests the accumulation of inactive photosystem II (PSII) complexes with low efficiency of primary quinone acceptor of electron of PSII (Q_A) reduction. The presence of methylamine (MA), a thylakoid membrane uncoupler, or N, N-dicyclohexylcarbodiimide, an inhibitor of ATPase, precluded the accumulation of inactive PSII complexes. When salicylhydroxamate promoted the reduction of the plastoquinone (PQ) pool, exogenous ATP accelerated the accumulation of inactive complexes. Dark PQ oxidation in the presence of nonmetabolized glucose analog, 2-deoxy-D-glucose, lowered the content of inactive PSII complexes, and NaF, an inhibitor of chloroplast phosphatases, retarded this process. These data are considered as evidence for a mechanism regulating the content of inactive PSII complexes in the process of redox-dependent phosphorylation of D1- and/or D2-proteins of PSII.     

The phosphorylation state of chloroplast transit peptides regulates preprotein import

Import of nuclear encoded proteins into chloroplast is an essential and well-regulated mechanism. The cytosolic kinases STY8, STY17 and STY46 have been shown to phosphorylate chloroplast preprotein transit peptides advantaging the binding of a 14-3-3 dimer. Analyses of sty8 sty17 sty46 mutant plants revealed a role for the kinases in chloroplast differentiation, possibly due to lack of transit peptide phosphorylation. Moreover we could show that not only phosphorylation but also transit peptide dephosphorylation appears to be required for the fine regulation of the back-transport of nuclear encoded proteins to the chloroplast.     

A gateway to chloroplasts - protein translocation and beyond

The biogenesis of chloroplasts requires the coordinated interplay with the nucleus and the cytoplasm. The majority of chloroplast proteins are encoded by the nuclear genome and must be faithfully and efficiently delivered to the organelle upon completion of translation in the cytosol. This high-fidelity targeting is accomplished by specific chloroplast targeting signal peptides. Several cytoplasmic factors recognise, modify, and bind this targeting sequence, and deliver the preproteins to the chloroplast translocation machinery. The multisubunit translocation complex at the outer envelope contains receptor proteins, a translocation channel, and accessory subunits. Complete import into the stroma utilizes both outer and inner envelope translocons and molecular chaperones in the intermembrane space and in the stroma. The entire import process appears to be regulated by phosphorylation, nucleotide binding, and hydrolysis. Recent evidence indicates that several subunits of the chloroplast import machinery may have evolved from cyanobacterial ancestors.