Type II metacaspase mediates light-dependent programmed cell death in Chlamydomonas reinhardtii.

Among the crucial processes that preside over the destiny of cells from any type of organism are those involving their self-destruction. This process is well characterized and conceptually logical to understand in multicellular organisms; however, the levels of knowledge and comprehension of its existence are still quite enigmatic in unicellular organisms. We use Chlamydomonas (Chlamydomonas reinhardtii) to lay the foundation for understanding the mechanisms of programmed cell death (PCD) in a unicellular photosynthetic organism. In this paper, we show that while PCD induces the death of a proportion of cells, it allows the survival of the remaining population. A quantitative proteomic analysis aiming at unveiling the proteome of PCD in Chlamydomonas allowed us to identify key proteins that led to the discovery of essential mechanisms. We show that in Chlamydomonas, PCD relies on the light dependence of a photosynthetic organism to generate reactive oxygen species and induce cell death. Finally, we obtained and characterized mutants for the 2 metacaspase genes in Chlamydomonas and showed that a type II metacaspase is essential for PCD execution.

New RoxS sRNA Targets Identified in Bacillus subtilis by Pulsed SILAC.

Non-coding RNAs (sRNA) play a key role in controlling gene expression in bacteria, typically by base-pairing with ribosome binding sites to block translation. The modification of ribosome traffic along the mRNA generally affects its stability. However, a few cases have been described in bacteria where sRNAs can affect translation without a major impact on mRNA stability. To identify new sRNA targets in Bacillus subtilis potentially belonging to this class of mRNAs, we used pulsed-SILAC (stable isotope labeling by amino acids in cell culture) to label newly synthesized proteins after short expression of the RoxS sRNA, the best characterized sRNA in this bacterium. RoxS sRNA was previously shown to interfere with the expression of genes involved in central metabolism, permitting control of the NAD+/NADH ratio in B. subtilis. In this study, we confirmed most of the known targets of RoxS, showing the efficiency of the method. We further expanded the number of mRNA targets encoding enzymes of the TCA cycle and identified new targets. One of these is YcsA, a tartrate dehydrogenase that uses NAD+ as co-factor, in excellent agreement with the proposed role of RoxS in management of NAD+/NADH ratio in Firmicutes. IMPORTANCE Non-coding RNAs (sRNA) play an important role in bacterial adaptation and virulence. The identification of the most complete set of targets for these regulatory RNAs is key to fully identifying the perimeter of its function(s). Most sRNAs modify both the translation (directly) and mRNA stability (indirectly) of their targets. However, sRNAs can also influence the translation efficiency of the target primarily, with little or no impact on mRNA stability. The characterization of these targets is challenging. We describe here the application of the pulsed SILAC method to identify such targets and obtain the most complete list of targets for a defined sRNA.

Structural snapshots of nitrosoglutathione binding and reactivity underlying S-nitrosylation of photosynthetic GAPDH.

S-nitrosylation is a redox post-translational modification widely recognized to play an important role in cellular signaling as it can modulate protein function and conformation. At the physiological level, nitrosoglutathione (GSNO) is considered the major physiological NO-releasing compound due to its ability to transfer the NO moiety to protein thiols but the structural determinants regulating its redox specificity are not fully elucidated. In this study, we employed photosynthetic glyceraldehyde-3-phosphate dehydrogenase from Chlamydomonas reinhardtii (CrGAPA) to investigate the molecular mechanisms underlying GSNO-dependent thiol oxidation. We first observed that GSNO causes reversible enzyme inhibition by inducing S-nitrosylation. While the cofactor NADP+ partially protects the enzyme from GSNO-mediated S-nitrosylation, protein inhibition is not observed in the presence of the substrate 1,3-bisphosphoglycerate, indicating that the S-nitrosylation of the catalytic Cys149 is responsible for CrGAPA inactivation. The crystal structures of CrGAPA in complex with NADP+ and NAD+ reveal a general structural similarity with other photosynthetic GAPDH. Starting from the 3D structure, we carried out molecular dynamics simulations to identify the protein residues involved in GSNO binding. The reaction mechanism of GSNO with CrGAPA Cys149 was investigated by quantum mechanical/molecular mechanical calculations, which permitted to disclose the relative contribution of protein residues in modulating the activation barrier of the trans-nitrosylation reaction. Based on our findings, we provide functional and structural insights into the response of CrGAPA to GSNO-dependent regulation, possibly expanding the mechanistic features to other protein cysteines susceptible to be oxidatively modified by GSNO.

How abiotic stress-induced socialization leads to the formation of massive aggregates in Chlamydomonas.

Multicellular organisms implement a set of reactions involving signaling and cooperation between different types of cells. Unicellular organisms, on the other hand, activate defense systems that involve collective behaviors between individual organisms. In the unicellular model alga Chlamydomonas (Chlamydomonas reinhardtii), the existence and the function of collective behaviors mechanisms in response to stress remain mostly at the level of the formation of small structures called palmelloids. Here, we report the characterization of a mechanism of abiotic stress response that Chlamydomonas can trigger to form massive multicellular structures. We showed that these aggregates constitute an effective bulwark within which the cells are efficiently protected from the toxic environment. We generated a family of mutants that aggregate spontaneously, the socializer (saz) mutants, of which saz1 is described here in detail. We took advantage of the saz mutants to implement a large-scale multiomics approach that allowed us to show that aggregation is not the result of passive agglutination, but rather genetic reprogramming and substantial modification of the secretome. The reverse genetic analysis we conducted allowed us to identify positive and negative regulators of aggregation and to make hypotheses on how this process is controlled in Chlamydomonas.

The C-Terminal Acidic Tail Modulates the Anticancer Properties of HMGB1.

Energy metabolism reprogramming was recently listed as a hallmark of cancer. In this process, the switch from pyruvate kinase isoenzyme type M1 to pyruvate kinase isoenzyme type M2 (PKM2) is believed to play a crucial role. Interestingly, the activity of the active form of PKM2 can efficiently be inhibited by the high-mobility group box 1 (HMGB1) protein, leading to a rapid blockage of glucose-dependent aerobic respiration and cancer cell death. HMGB1 is a member of the HMG protein family. It contains two DNA-binding HMG-box domains and an acidic C-terminal tail capable of positively or negatively modulating its biological properties. In this work, we report that the deletion of the C-terminal tail of HMGB1 increases its activity towards a large panel of cancer cells without affecting the viability of normal immortalized fibroblasts. Moreover, in silico analysis suggests that the truncated form of HMGB1 retains the capacity of the full-length protein to interact with PKM2. However, based on the capacity of the cells to circumvent oxidative phosphorylation inhibition, we were able to identify either a cytotoxic or cytostatic effect of the proteins. Together, our study provides new insights in the characterization of the anticancer activity of HMGB1.

Crystal structure of chloroplastic thioredoxin z defines a type-specific target recognition.

Thioredoxins (TRXs) are ubiquitous disulfide oxidoreductases structured according to a highly conserved fold. TRXs are involved in a myriad of different processes through a common chemical mechanism. Plant TRXs evolved into seven types with diverse subcellular localization and distinct protein target selectivity. Five TRX types coexist in the chloroplast, with yet scarcely described specificities. We solved the crystal structure of a chloroplastic z-type TRX, revealing a conserved TRX fold with an original electrostatic surface potential surrounding the redox site. This recognition surface is distinct from all other known TRX types from plant and non-plant sources and is exclusively conserved in plant z-type TRXs. We show that this electronegative surface endows thioredoxin z (TRXz) with a capacity to activate the photosynthetic Calvin-Benson cycle enzyme phosphoribulokinase. The distinct electronegative surface of TRXz thereby extends the repertoire of TRX-target recognitions.

Structural and functional insights into nitrosoglutathione reductase from Chlamydomonas reinhardtii.

Protein S-nitrosylation plays a fundamental role in cell signaling and nitrosoglutathione (GSNO) is considered as the main nitrosylating signaling molecule. Enzymatic systems controlling GSNO homeostasis are thus crucial to indirectly control the formation of protein S-nitrosothiols. GSNO reductase (GSNOR) is the key enzyme controlling GSNO levels by catalyzing its degradation in the presence of NADH. Here, we found that protein extracts from the microalga Chlamydomonas reinhardtii catabolize GSNO via two enzymatic systems having specific reliance on NADPH or NADH and different biochemical features. Scoring the Chlamydomonas genome for orthologs of known plant GSNORs, we found two genes encoding for putative and almost identical GSNOR isoenzymes. One of the two, here named CrGSNOR1, was heterologously expressed and purified. Its kinetic properties were determined and the three-dimensional structures of the apo-, NAD+- and NAD+/GSNO-forms were solved. These analyses revealed that CrGSNOR1 has a strict specificity towards GSNO and NADH, and a conserved folding with respect to other plant GSNORs. The catalytic zinc ion, however, showed an unexpected variability of the coordination environment. Furthermore, we evaluated the catalytic response of CrGSNOR1 to thermal denaturation, thiol-modifying agents and oxidative modifications as well as the reactivity and position of accessible cysteines. Despite being a cysteine-rich protein, CrGSNOR1 contains only two solvent-exposed/reactive cysteines. Oxidizing and nitrosylating treatments have null or limited effects on CrGSNOR1 activity and folding, highlighting a certain resistance of the algal enzyme to redox modifications. The molecular mechanisms and structural features underlying the response to thiol-based modifications are discussed.

Redox Modification of the Iron-Sulfur Glutaredoxin GRXS17 Activates Holdase Activity and Protects Plants from Heat Stress.

Heat stress induces misfolding and aggregation of proteins unless they are guarded by chaperone systems. Here, we examined the function of the glutaredoxin GRXS17, a member of thiol reductase families in the model plant Arabidopsis (Arabidopsis thaliana). GRXS17 is a nucleocytosolic monothiol glutaredoxin consisting of an N-terminal thioredoxin domain and three CGFS active-site motif-containing GRX domains that coordinate three iron-sulfur (Fe-S) clusters in a glutathione-dependent manner. As an Fe-S cluster-charged holoenzyme, GRXS17 is likely involved in the maturation of cytosolic and nuclear Fe-S proteins. In addition to its role in cluster biogenesis, GRXS17 presented both foldase and redox-dependent holdase activities. Oxidative stress in combination with heat stress induced loss of its Fe-S clusters followed by subsequent formation of disulfide bonds between conserved active-site cysteines in the corresponding thioredoxin domains. This oxidation led to a shift of GRXS17 to a high-molecular-weight complex and thus activated its holdase activity in vitro. Moreover, GRXS17 was specifically involved in plant tolerance to moderate high temperature and protected root meristematic cells from heat-induced cell death. Finally, GRXS17 interacted with a different set of proteins upon heat stress, possibly protecting them from heat injuries. Therefore, we propose that the Fe-S cluster enzyme GRXS17 is an essential guard that protects proteins against moderate heat stress, likely through a redox-dependent chaperone activity. We reveal the mechanism of an Fe-S cluster-dependent activity shift that converts the holoenzyme GRXS17 into a holdase, thereby preventing damage caused by heat stress.

Secondary Metabolites from the Culture of the Marine-derived Fungus Paradendryphiella salina PC 362H and Evaluation of the Anticancer Activity of Its Metabolite Hyalodendrin.

High-throughput screening assays have been designed to identify compounds capable of inhibiting phenotypes involved in cancer aggressiveness. However, most studies used commercially available chemical libraries. This prompted us to explore natural products isolated from marine-derived fungi as a new source of molecules. In this study, we established a chemical library from 99 strains corresponding to 45 molecular operational taxonomic units and evaluated their anticancer activity against the MCF7 epithelial cancer cell line and its invasive stem cell-like MCF7-Sh-WISP2 counterpart. We identified the marine fungal Paradendryphiella salina PC 362H strain, isolated from the brown alga Pelvetia caniculata (PC), as one of the most promising fungi which produce active compounds. Further chemical and biological characterizations of the culture of the Paradendryphiella salina PC 362H strain identified (-)-hyalodendrin as the active secondary metabolite responsible for the cytotoxic activity of the crude extract. The antitumor activity of (-)-hyalodendrin was not only limited to the MCF7 cell lines, but also prominent on cancer cells with invasive phenotypes including colorectal cancer cells resistant to chemotherapy. Further investigations showed that treatment of MCF7-Sh-WISP2 cells with (-)-hyalodendrin induced changes in the phosphorylation status of p53 and altered expression of HSP60, HSP70 and PRAS40 proteins. Altogether, our study reveals that this uninvestigated marine fungal crude extract possesses a strong therapeutic potential against tumor cells with aggressive phenotypes and confirms that members of the epidithiodioxopiperazines are interesting fungal toxins with anticancer activities.

Glutathionylation primes soluble glyceraldehyde-3-phosphate dehydrogenase for late collapse into insoluble aggregates.

Protein aggregation is a complex physiological process, primarily determined by stress-related factors revealing the hidden aggregation propensity of proteins that otherwise are fully soluble. Here we report a mechanism by which glycolytic glyceraldehyde-3-phosphate dehydrogenase of Arabidopsis thaliana (AtGAPC1) is primed to form insoluble aggregates by the glutathionylation of its catalytic cysteine (Cys149). Following a lag phase, glutathionylated AtGAPC1 initiates a self-aggregation process resulting in the formation of branched chains of globular particles made of partially misfolded and totally inactive proteins. GSH molecules within AtGAPC1 active sites are suggested to provide the initial destabilizing signal. The following removal of glutathione by the formation of an intramolecular disulfide bond between Cys149 and Cys153 reinforces the aggregation process. Physiological reductases, thioredoxins and glutaredoxins, could not dissolve AtGAPC1 aggregates but could efficiently contrast their growth. Besides acting as a protective mechanism against overoxidation, S-glutathionylation of AtGAPC1 triggers an unexpected aggregation pathway with completely different and still unexplored physiological implications.

Detection of IgG directed against a recombinant form of Epstein-Barr virus BALF0/1 protein in patients with nasopharyngeal carcinoma.

BALF0/1 is a putative Epstein-Barr virus (EBV) protein that has been described as a modulator of apoptosis. So far, the lack of specific immunological reagents impaired the detection of native BALF0/1 in EBV-infected cells. This study describes the expression and purification of a truncated form of BALF0/1 (tBALF0) using a heterologous bacterial expression system. tBALF0 was further used as an antigen in an indirect Enzyme-linked Immunosorbent Assay (ELISA) that unraveled the presence of low titer IgGs to BALF0/1 during primary (10.0%) and past (13.3%) EBV infection. Conversely high-titer IgGs to BALF0/1 were detected in 33.3% of nasopharyngeal carcinoma (NPC) patients suggesting that BALF0/1 and/or humoral response against it may contribute to NPC pathogenesis.

Arabidopsis and Chlamydomonas phosphoribulokinase crystal structures complete the redox structural proteome of the Calvin-Benson cycle.

In land plants and algae, the Calvin-Benson (CB) cycle takes place in the chloroplast, a specialized organelle in which photosynthesis occurs. Thioredoxins (TRXs) are small ubiquitous proteins, known to harmonize the two stages of photosynthesis through a thiol-based mechanism. Among the 11 enzymes of the CB cycle, the TRX target phosphoribulokinase (PRK) has yet to be characterized at the atomic scale. To accomplish this goal, we determined the crystal structures of PRK from two model species: the green alga Chlamydomonas reinhardtii (CrPRK) and the land plant Arabidopsis thaliana (AtPRK). PRK is an elongated homodimer characterized by a large central ß-sheet of 18 strands, extending between two catalytic sites positioned at its edges. The electrostatic surface potential of the catalytic cavity has both a positive region suitable for binding the phosphate groups of substrates and an exposed negative region to attract positively charged TRX-f. In the catalytic cavity, the regulatory cysteines are 13 Å apart and connected by a flexible region exclusive to photosynthetic eukaryotes-the clamp loop-which is believed to be essential for oxidation-induced structural rearrangements. Structural comparisons with prokaryotic and evolutionarily older PRKs revealed that both AtPRK and CrPRK have a strongly reduced dimer interface and an increased number of random-coiled regions, suggesting that a general loss in structural rigidity correlates with gains in TRX sensitivity during the molecular evolution of PRKs in eukaryotes.

Structural and Biochemical Insights into the Reactivity of Thioredoxin h1 from Chlamydomonas reinhardtii.

Thioredoxins (TRXs) are major protein disulfide reductases of the cell. Their redox activity relies on a conserved Trp-Cys-(Gly/Pro)-Pro-Cys active site bearing two cysteine (Cys) residues that can be found either as free thiols (reduced TRXs) or linked together by a disulfide bond (oxidized TRXs) during the catalytic cycle. Their reactivity is crucial for TRX activity, and depends on the active site microenvironment. Here, we solved and compared the 3D structure of reduced and oxidized TRX h1 from Chlamydomonas reinhardtii (CrTRXh1). The three-dimensional structure was also determined for mutants of each active site Cys. Structural alignments of CrTRXh1 with other structurally solved plant TRXs showed a common spatial fold, despite the low sequence identity. Structural analyses of CrTRXh1 revealed that the protein adopts an identical conformation independently from its redox state. Treatment with iodoacetamide (IAM), a Cys alkylating agent, resulted in a rapid and pH-dependent inactivation of CrTRXh1. Starting from fully reduced CrTRXh1, we determined the acid dissociation constant (pKa) of each active site Cys by Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry analyses coupled to differential IAM-based alkylation. Based on the diversity of catalytic Cys deprotonation states, the mechanisms and structural features underlying disulfide redox activity are discussed.

Redox Homeostasis in Photosynthetic Organisms: Novel and Established Thiol-Based Molecular Mechanisms.

Significance: Redox homeostasis consists of an intricate network of reactions in which reactive molecular species, redox modifications, and redox proteins act in concert to allow both physiological responses and adaptation to stress conditions. Recent Advances: This review highlights established and novel thiol-based regulatory pathways underlying the functional facets and significance of redox biology in photosynthetic organisms. In the last decades, the field of redox regulation has largely expanded and this work is aimed at giving the right credit to the importance of thiol-based regulatory and signaling mechanisms in plants. Critical Issues: This cannot be all-encompassing, but is intended to provide a comprehensive overview on the structural/molecular mechanisms governing the most relevant thiol switching modifications with emphasis on the large genetic and functional diversity of redox controllers (i.e., redoxins). We also summarize the different proteomic-based approaches aimed at investigating the dynamics of redox modifications and the recent evidence that extends the possibility to monitor the cellular redox state in vivo. The physiological relevance of redox transitions is discussed based on reverse genetic studies confirming the importance of redox homeostasis in plant growth, development, and stress responses. Future Directions: In conclusion, we can firmly assume that redox biology has acquired an established significance that virtually infiltrates all aspects of plant physiology.

Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii.

Microalgae are regarded as promising organisms to develop innovative concepts based on their photosynthetic capacity that offers more sustainable production than heterotrophic hosts. However, to realize their potential as green cell factories, a major challenge is to make microalgae easier to engineer. A promising approach for rapid and predictable genetic manipulation is to use standardized synthetic biology tools and workflows. To this end we have developed a Modular Cloning toolkit for the green microalga Chlamydomonas reinhardtii. It is based on Golden Gate cloning with standard syntax, and comprises 119 openly distributed genetic parts, most of which have been functionally validated in several strains. It contains promoters, UTRs, terminators, tags, reporters, antibiotic resistance genes, and introns cloned in various positions to allow maximum modularity. The toolkit enables rapid building of engineered cells for both fundamental research and algal biotechnology. This work will make Chlamydomonas the next chassis for sustainable synthetic biology.

MinOmics, an Integrative and Immersive Tool for Multi-Omics Analysis.

Proteomic and transcriptomic technologies resulted in massive biological datasets, their interpretation requiring sophisticated computational strategies. Efficient and intuitive real-time analysis remains challenging. We use proteomic data on 1417 proteins of the green microalga Chlamydomonas reinhardtii to investigate physicochemical parameters governing selectivity of three cysteine-based redox post translational modifications (PTM): glutathionylation (SSG), nitrosylation (SNO) and disulphide bonds (SS) reduced by thioredoxins. We aim to understand underlying molecular mechanisms and structural determinants through integration of redox proteome data from gene- to structural level. Our interactive visual analytics approach on an 8.3 m2 display wall of 25 MPixel resolution features stereoscopic three dimensions (3D) representation performed by UnityMol WebGL. Virtual reality headsets complement the range of usage configurations for fully immersive tasks. Our experiments confirm that fast access to a rich cross-linked database is necessary for immersive analysis of structural data. We emphasize the possibility to display complex data structures and relationships in 3D, intrinsic to molecular structure visualization, but less common for omics-network analysis. Our setup is powered by MinOmics, an integrated analysis pipeline and visualization framework dedicated to multi-omics analysis. MinOmics integrates data from various sources into a materialized physical repository. We evaluate its performance, a design criterion for the framework.

Pyrenoid functions revealed by proteomics in Chlamydomonas reinhardtii.

Organelles are intracellular compartments which are themselves compartmentalized. Biogenic and metabolic processes are localized to specialized domains or microcompartments to enhance their efficiency and suppress deleterious side reactions. An example of intra-organellar compartmentalization is the pyrenoid in the chloroplasts of algae and hornworts. This microcompartment enhances the photosynthetic CO2-fixing activity of the Calvin-Benson cycle enzyme Rubisco, suppresses an energetically wasteful oxygenase activity of Rubisco, and mitigates limiting CO2 availability in aquatic environments. Hence, the pyrenoid is functionally analogous to the carboxysomes in cyanobacteria. However, a comprehensive analysis of pyrenoid functions based on its protein composition is lacking. Here we report a proteomic characterization of the pyrenoid in the green alga Chlamydomonas reinhardtii. Pyrenoid-enriched fractions were analyzed by quantitative mass spectrometry. Contaminant proteins were identified by parallel analyses of pyrenoid-deficient mutants. This pyrenoid proteome contains 190 proteins, many of which function in processes that are known or proposed to occur in pyrenoids: e.g. the carbon concentrating mechanism, starch metabolism or RNA metabolism and translation. Using radioisotope pulse labeling experiments, we show that pyrenoid-associated ribosomes could be engaged in the localized synthesis of the large subunit of Rubisco. New pyrenoid functions are supported by proteins in tetrapyrrole and chlorophyll synthesis, carotenoid metabolism or amino acid metabolism. Hence, our results support the long-standing hypothesis that the pyrenoid is a hub for metabolism. The 81 proteins of unknown function reveal candidates for new participants in these processes. Our results provide biochemical evidence of pyrenoid functions and a resource for future research on pyrenoids and their use to enhance agricultural plant productivity. Data are available via ProteomeXchange with identifier PXD004509.

The Deep Thioredoxome in Chlamydomonas reinhardtii: New Insights into Redox Regulation.

Thiol-based redox post-translational modifications have emerged as important mechanisms of signaling and regulation in all organisms, and thioredoxin plays a key role by controlling the thiol-disulfide status of target proteins. Recent redox proteomic studies revealed hundreds of proteins regulated by glutathionylation and nitrosylation in the unicellular green alga Chlamydomonas reinhardtii, while much less is known about the thioredoxin interactome in this organism. By combining qualitative and quantitative proteomic analyses, we have comprehensively investigated the Chlamydomonas thioredoxome and 1188 targets have been identified. They participate in a wide range of metabolic pathways and cellular processes. This study broadens not only the redox regulation to new enzymes involved in well-known thioredoxin-regulated metabolic pathways but also sheds light on cellular processes for which data supporting redox regulation are scarce (aromatic amino acid biosynthesis, nuclear transport, etc). Moreover, we characterized 1052 thioredoxin-dependent regulatory sites and showed that these data constitute a valuable resource for future functional studies in Chlamydomonas. By comparing this thioredoxome with proteomic data for glutathionylation and nitrosylation at the protein and cysteine levels, this work confirms the existence of a complex redox regulation network in Chlamydomonas and provides evidence of a tremendous selectivity of redox post-translational modifications for specific cysteine residues.

Modulation of the specific glutathionylation of mitochondrial proteins in the yeast Saccharomyces cerevisiae under basal and stress conditions.

The potential biological consequences of oxidative stress and changes in glutathione levels include the oxidation of susceptible protein thiols and reversible covalent binding of glutathione to the -SH groups of proteins by S-glutathionylation. Mitochondria are central to the response to oxidative stress and redox signaling. It is therefore crucial to explore the adaptive response to changes in thiol-dependent redox status in these organelles. We optimized the purification protocol of glutathionylated proteins in the yeast Saccharomyces cerevisiae and present a detailed proteomic analysis of the targets of protein glutathionylation in cells undergoing constitutive metabolism and after exposure to various stress conditions. This work establishes the physiological importance of the glutathionylation process in S. cerevisiae under basal conditions and provides evidence for an atypical and unexpected cellular distribution of the process between the cytosol and mitochondria. In addition, our data indicate that each oxidative condition (diamide, GSSG, H2O2, or the presence of iron) elicits an adaptive metabolic response affecting specific mitochondrial metabolic pathways, mainly involved in the energetic maintenance of the cells. The correlation of protein modifications with intracellular glutathione levels suggests that protein deglutathionylation may play a role in protecting mitochondria from oxidative stress. This work provides further insights into the diversity of proteins undergoing glutathionylation and the role of this post-translational modification as a regulatory process in the adaptive response of the cell.

A Light Switch Based on Protein S-Nitrosylation Fine-Tunes Photosynthetic Light Harvesting in Chlamydomonas.

Photosynthetic eukaryotes are challenged by a fluctuating light supply, demanding for a modulated expression of nucleus-encoded light-harvesting proteins associated with photosystem II (LHCII) to adjust light-harvesting capacity to the prevailing light conditions. Here, we provide clear evidence for a regulatory circuit that controls cytosolic LHCII translation in response to light quantity changes. In the green unicellular alga Chlamydomonas reinhardtii, the cytosolic RNA-binding protein NAB1 represses translation of certain LHCII isoform mRNAs. Specific nitrosylation of Cys-226 decreases NAB1 activity and could be demonstrated in vitro and in vivo. The less active, nitrosylated form of NAB1 is found in cells acclimated to limiting light supply, which permits accumulation of light-harvesting proteins and efficient light capture. In contrast, elevated light supply causes its denitrosylation, thereby activating the repression of light-harvesting protein synthesis, which is needed to control excitation pressure at photosystem II. Denitrosylation of recombinant NAB1 is efficiently performed by the cytosolic thioredoxin system in vitro. To our knowledge, NAB1 is the first example of stimulus-induced denitrosylation in the context of photosynthetic acclimation. By identifying this novel redox cross-talk pathway between chloroplast and cytosol, we add a new key element required for drawing a precise blue print of the regulatory network of light harvesting.