Pseudomonas aeruginosa MipA-MipB envelope proteins act as new sensors of polymyxins.

Due to the rising incidence of antibiotic-resistant infections, the last-line antibiotics, polymyxins, have resurged in the clinics in parallel with new bacterial strategies of escape. The Gram-negative opportunistic pathogen Pseudomonas aeruginosa develops resistance to colistin/polymyxin B by distinct molecular mechanisms, mostly through modification of the lipid A component of the LPS by proteins encoded within the arnBCDATEF-ugd (arn) operon. In this work, we characterized a polymyxin-induced operon named mipBA, present in P. aeruginosa strains devoid of the arn operon. We showed that mipBA is activated by the ParR/ParS two-component regulatory system in response to polymyxins. Structural modeling revealed that MipA folds as an outer-membrane ß-barrel, harboring an internal negatively charged channel, able to host a polymyxin molecule, while the lipoprotein MipB adopts a ß-lactamase fold with two additional C-terminal domains. Experimental work confirmed that MipA and MipB localize to the bacterial envelope, and they co-purify in vitro. Nano differential scanning fluorimetry showed that polymyxins stabilized MipA in a specific and dose-dependent manner. Mass spectrometry-based quantitative proteomics on P. aeruginosa membranes demonstrated that ∆mipBA synthesized fourfold less MexXY-OprA proteins in response to polymyxin B compared to the wild-type strain. The decrease was a direct consequence of impaired transcriptional activation of the mex operon operated by ParR/ParS. We propose MipA/MipB to act as membrane (co)sensors working in concert to activate ParS histidine kinase and help the bacterium to cope with polymyxin-mediated envelope stress through synthesis of the efflux pump, MexXY-OprA.IMPORTANCEDue to the emergence of multidrug-resistant isolates, antibiotic options may be limited to polymyxins to eradicate Gram-negative infections. Pseudomonas aeruginosa, a leading opportunistic pathogen, has the ability to develop resistance to these cationic lipopeptides by modifying its lipopolysaccharide through proteins encoded within the arn operon. Herein, we describe a sub-group of P. aeruginosa strains lacking the arn operon yet exhibiting adaptability to polymyxins. Exposition to sub-lethal polymyxin concentrations induced the expression and production of two envelope-associated proteins. Among those, MipA, an outer-membrane barrel, is able to specifically bind polymyxins with an affinity in the 10-µM range. Using membrane proteomics and phenotypic assays, we showed that MipA and MipB participate in the adaptive response to polymyxins via ParR/ParS regulatory signaling. We propose a new model wherein the MipA-MipB module functions as a novel polymyxin sensing mechanism.

Proteomics unveil a central role for peroxisomes in butyrate assimilation of the heterotrophic Chlorophyte alga Polytomella sp.

Volatile fatty acids found in effluents of the dark fermentation of biowastes can be used for mixotrophic growth of microalgae, improving productivity and reducing the cost of the feedstock. Microalgae can use the acetate in the effluents very well, but butyrate is poorly assimilated and can inhibit growth above 1 gC.L-1. The non-photosynthetic chlorophyte alga Polytomella sp. SAG 198.80 was found to be able to assimilate butyrate fast. To decipher the metabolic pathways implicated in butyrate assimilation, quantitative proteomics study was developed comparing Polytomella sp. cells grown on acetate and butyrate at 1 gC.L-1. After statistical analysis, a total of 1772 proteins were retained, of which 119 proteins were found to be overaccumulated on butyrate vs. only 46 on acetate, indicating that butyrate assimilation necessitates additional metabolic steps. The data show that butyrate assimilation occurs in the peroxisome via the ß-oxidation pathway to produce acetyl-CoA and further tri/dicarboxylic acids in the glyoxylate cycle. Concomitantly, reactive oxygen species defense enzymes as well as the branched amino acid degradation pathway were strongly induced. Although no clear dedicated butyrate transport mechanism could be inferred, several membrane transporters induced on butyrate are identified as potential condidates. Metabolic responses correspond globally to the increased needs for central cofactors NAD, ATP and CoA, especially in the peroxisome and the cytosol.

Quantitative proteomic analyses reveal the impact of nitrogen starvation on the proteome of the model diatom Phaeodactylum tricornutum.

Diatoms are one of the largest groups in phytoplankton biodiversity. Understanding their response to nitrogen variations, present from micromolar to near-zero levels in oceans and fresh waters, is essential to comprehend their ecological success. Nitrogen starvation is used in biotechnological processes, to trigger the remodeling of carbon metabolism in the direction of fatty acids and triacylglycerol synthesis. We evaluated whole proteome changes in Phaeodactylum tricornutum after 7 days of cultivation with 5.5-mM nitrate (+N) or without any nitrogen source (-N). On a total of 3768 proteins detected in biological replicates, our analysis pointed to 384 differentially abundant proteins (DAP). Analysis of proteins of lower abundance in -N revealed an arrest of amino acid and protein syntheses, a remodeling of nitrogen metabolism, and a decrease of the proteasome abundance suggesting a decline in unselective whole-proteome decay. Analysis of proteins of higher abundance revealed the setting up of a general nitrogen scavenging system dependent on deaminases. The increase of a plastid palmitoyl-ACP desaturase appeared as a hallmark of carbon metabolism rewiring in the direction of fatty acid and triacylglycerol synthesis. This dataset is also valuable to select gene candidates for improved biotechnological properties.

Proteomics Evidence of a Systemic Response to Desiccation in the Resurrection Plant Haberlea rhodopensis.

Global warming and drought stress are expected to have a negative impact on agricultural productivity. Desiccation-tolerant species, which are able to tolerate the almost complete desiccation of their vegetative tissues, are appropriate models to study extreme drought tolerance and identify novel approaches to improve the resistance of crops to drought stress. In the present study, to better understand what makes resurrection plants extremely tolerant to drought, we performed transmission electron microscopy and integrative large-scale proteomics, including organellar and phosphorylation proteomics, and combined these investigations with previously published transcriptomic and metabolomics data from the resurrection plant Haberlea rhodopensis. The results revealed new evidence about organelle and cell preservation, posttranscriptional and posttranslational regulation, photosynthesis, primary metabolism, autophagy, and cell death in response to desiccation in H. rhodopensis. Different protective intrinsically disordered proteins, such as late embryogenesis abundant (LEA) proteins, thaumatin-like proteins (TLPs), and heat shock proteins (HSPs), were detected. We also found a constitutively abundant dehydrin in H. rhodopensis whose phosphorylation levels increased under stress in the chloroplast fraction. This integrative multi-omics analysis revealed a systemic response to desiccation in H. rhodopensis and certain targets for further genomic and evolutionary studies on DT mechanisms and genetic engineering towards the improvement of drought tolerance in crops.

The hexosamine pathway and coat complex II promote malignant adaptation to nutrient scarcity.

The glucose-requiring hexosamine biosynthetic pathway (HBP), which produces UDP-N-acetylglucosamine for glycosylation reactions, promotes lung adenocarcinoma (LUAD) progression. However, lung tumor cells often reside in low-nutrient microenvironments, and whether the HBP is involved in the adaptation of LUAD to nutrient stress is unknown. Here, we show that the HBP and the coat complex II (COPII) play a key role in cell survival during glucose shortage. HBP up-regulation withstood low glucose-induced production of proteins bearing truncated N-glycans, in the endoplasmic reticulum. This function for the HBP, alongside COPII up-regulation, rescued cell surface expression of a subset of glycoproteins. Those included the epidermal growth factor receptor (EGFR), allowing an EGFR-dependent cell survival under low glucose in anchorage-independent growth. Accordingly, high expression of the HBP rate-limiting enzyme GFAT1 was associated with wild-type EGFR activation in LUAD patient samples. Notably, HBP and COPII up-regulation distinguished LUAD from the lung squamous-cell carcinoma subtype, thus uncovering adaptive mechanisms of LUAD to their harsh microenvironment.

Argonaute Autoantibodies as Biomarkers in Autoimmune Neurologic Diseases.

OBJECTIVE: To identify and characterize autoantibodies (Abs) as novel biomarkers for an autoimmune context in patients with central and peripheral neurologic diseases. METHODS: Two distinct approaches (immunoprecipitation/mass spectrometry-based proteomics and protein microarrays) and patients' sera and CSF were used. The specificity of the identified target was confirmed by cell-based assay (CBA) in 856 control samples. RESULTS: Using the 2 methods as well as sera and CSF of patients with central and peripheral neurologic involvement, we identified Abs against the family of Argonaute proteins (mainly AGO1 and AGO2), which were already reported in systemic autoimmunity. AGO-Abs were mostly of immunoglobulin G 1 subclass and conformation dependent. Using CBA, AGO-Abs were detected in 21 patients with a high suspicion of autoimmune neurologic diseases (71.4% were women; median age 57 years) and only in 4/856 (0.5%) controls analyzed by CBA (1 diagnosed with small-cell lung cancer and the other 3 with Sjögren syndrome). Among the 21 neurologic patients identified, the main clinical presentations were sensory neuronopathy (8/21, 38.1%) and limbic encephalitis (6/21, 28.6%). Fourteen patients (66.7%) had autoimmune comorbidities and/or co-occurring Abs, whereas AGO-Abs were the only autoimmune biomarker for the remaining 7/21 (33.3%). Thirteen (61.9%) patients were treated with immunotherapy; 8/13 (61.5%) improved, and 3/13 (23.1%) remained stable, suggesting an efficacy of these treatments. CONCLUSIONS: AGO-Abs might be potential biomarkers of autoimmunity in patients with central and peripheral nonparaneoplastic neurologic diseases. In 7 patients, AGO-Abs were the only biomarkers; thus, their identification may be useful to suspect the autoimmune character of the neurologic disorder. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that AGO-Abs are more frequent in patients with autoimmune neurologic diseases than controls.

Mixotrophic growth of the extremophile Galdieria sulphuraria reveals the flexibility of its carbon assimilation metabolism.

Galdieria sulphuraria is a cosmopolitan microalga found in volcanic hot springs and calderas. It grows at low pH in photoautotrophic (use of light as a source of energy) or heterotrophic (respiration as a source of energy) conditions, using an unusually broad range of organic carbon sources. Previous data suggested that G. sulphuraria cannot grow mixotrophically (simultaneously exploiting light and organic carbon as energy sources), its photosynthetic machinery being repressed by organic carbon. Here, we show that G. sulphuraria SAG21.92 thrives in photoautotrophy, heterotrophy and mixotrophy. By comparing growth, biomass production, photosynthetic and respiratory performances in these three trophic modes, we show that addition of organic carbon to cultures (mixotrophy) relieves inorganic carbon limitation of photosynthesis thanks to increased CO2 supply through respiration. This synergistic effect is lost when inorganic carbon limitation is artificially overcome by saturating photosynthesis with added external CO2 . Proteomic and metabolic profiling corroborates this conclusion suggesting that mixotrophy is an opportunistic mechanism to increase intracellular CO2 concentration under physiological conditions, boosting photosynthesis by enhancing the carboxylation activity of Ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) and decreasing photorespiration. We discuss possible implications of these findings for the ecological success of Galdieria in extreme environments and for biotechnological applications.

Plastidial and cytosolic thiol reductases participate in the control of stomatal functioning.

Stomatal movements via the control of gas exchanges determine plant growth in relation to environmental stimuli through a complex signalling network involving reactive oxygen species that lead to post-translational modifications of Cys and Met residues, and alter protein activity and/or conformation. Thiol-reductases (TRs), which include thioredoxins, glutaredoxins (GRXs) and peroxiredoxins (PRXs), participate in signalling pathways through the control of Cys redox status in client proteins. Their involvement in stomatal functioning remains poorly characterized. By performing a mass spectrometry-based proteomic analysis, we show that numerous thiol reductases, like PRXs, are highly abundant in guard cells. When investigating various Arabidopsis mutants impaired in the expression of TR genes, no change in stomatal density and index was noticed. In optimal growth conditions, a line deficient in cytosolic NADPH-thioredoxin reductases displayed higher stomatal conductance and lower leaf temperature evaluated by thermal infrared imaging. In contrast, lines deficient in plastidial 2-CysPRXs or type-II GRXs exhibited compared to WT reduced conductance and warmer leaves in optimal conditions, and enhanced stomatal closure in epidermal peels treated with abscisic acid or hydrogen peroxide. Altogether, these data strongly support the contribution of thiol redox switches within the signalling network regulating guard cell movements and stomatal functioning.

Phylogenetic and functional diversity of aldehyde-alcohol dehydrogenases in microalgae.

KEY MESSAGE: The study shows the biochemical and enzymatic divergence between the two aldehyde-alcohol dehydrogenases of the alga Polytomella sp., shedding light on novel aspects of the enzyme evolution amid unicellular eukaryotes. Aldehyde-alcohol dehydrogenases (ADHEs) are large metalloenzymes that typically perform the two-step reduction of acetyl-CoA into ethanol. These enzymes consist of an N-terminal acetylating aldehyde dehydrogenase domain (ALDH) and a C-terminal alcohol dehydrogenase (ADH) domain. ADHEs are present in various bacterial phyla as well as in some unicellular eukaryotes. Here we focus on ADHEs in microalgae, a diverse and polyphyletic group of plastid-bearing unicellular eukaryotes. Genome survey shows the uneven distribution of the ADHE gene among free-living algae, and the presence of two distinct genes in various species. We show that the non-photosynthetic Chlorophyte alga Polytomella sp. SAG 198.80 harbors two genes for ADHE-like enzymes with divergent C-terminal ADH domains. Immunoblots indicate that both ADHEs accumulate in Polytomella cells growing aerobically on acetate or ethanol. ADHE1 of ~ 105-kDa is found in particulate fractions, whereas ADHE2 of ~ 95-kDa is mostly soluble. The study of the recombinant enzymes revealed that ADHE1 has both the ALDH and ADH activities, while ADHE2 has only the ALDH activity. Phylogeny shows that the divergence occurred close to the root of the Polytomella genus within a clade formed by the majority of the Chlorophyte ADHE sequences, next to the cyanobacterial clade. The potential diversification of function in Polytomella spp. unveiled here likely took place after the loss of photosynthesis. Overall, our study provides a glimpse at the complex evolutionary history of the ADHE in microalgae which includes (i) acquisition via different gene donors, (ii) gene duplication and (iii) independent evolution of one of the two enzymatic domains.

Hybrid cluster proteins in a photosynthetic microalga.

Hybrid cluster proteins (HCPs) are metalloproteins characterized by the presence of an iron-sulfur-oxygen cluster. These proteins occur in all three domains of life. In eukaryotes, HCPs have so far been found only in a few anaerobic parasites and photosynthetic microalgae. With respect to all species harboring an HCP, the green microalga Chlamydomonas reinhardtii stands out by the presence of four HCP genes. The study of the gene and protein structures as well as the phylogenetic analyses strongly support a model in which the HCP family in the alga has emerged from a single gene of alpha proteobacterial origin and then expanded by several rounds of duplications. The spectra and redox properties of HCP1 and HCP3, produced heterologously in Escherichia coli, were analyzed by electron paramagnetic resonance spectroscopy on redox-titrated samples. Both proteins contain a [4Fe-4S]-cluster as well as a [4Fe-2O-2S]-hybrid cluster with paramagnetic properties related to those of HCPs from Desulfovibrio species. Immunoblotting experiments combined with mass spectrometry-based proteomics showed that both nitrate and darkness contribute to the strong upregulation of the HCP levels in C. reinhardtii growing under oxic conditions. The link to the nitrate metabolism is discussed in the light of recent data on the potential role of HCP in S-nitrosylation in bacteria.

Unraveling Hidden Components of the Chloroplast Envelope Proteome: Opportunities and Limits of Better MS Sensitivity.

The chloroplast is a major plant cell organelle that fulfills essential metabolic and biosynthetic functions. Located at the interface between the chloroplast and other cell compartments, the chloroplast envelope system is a strategic barrier controlling the exchange of ions, metabolites and proteins, thus regulating essential metabolic functions (synthesis of hormones precursors, amino acids, pigments, sugars, vitamins, lipids, nucleotides etc.) of the plant cell. However, unraveling the contents of the chloroplast envelope proteome remains a difficult challenge; many proteins constituting this functional double membrane system remain to be identified. Indeed, the envelope contains only 1% of the chloroplast proteins (i.e. 0.4% of the whole cell proteome). In other words, most envelope proteins are so rare at the cell, chloroplast, or even envelope level, that they remained undetectable using targeted MS studies. Cross-contamination of chloroplast subcompartments by each other and by other cell compartments during cell fractionation, impedes accurate localization of many envelope proteins. The aim of the present study was to take advantage of technologically improved MS sensitivity to better define the proteome of the chloroplast envelope (differentiate genuine envelope proteins from contaminants). This MS-based analysis relied on an enrichment factor that was calculated for each protein identified in purified envelope fractions as compared with the value obtained for the same protein in crude cell extracts. Using this approach, a total of 1269 proteins were detected in purified envelope fractions, of which, 462 could be assigned an envelope localization by combining MS-based spectral count analyses with manual annotation using data from the literature and prediction tools. Many of such proteins being previously unknown envelope components, these data constitute a new resource of significant value to the broader plant science community aiming to define principles and molecular mechanisms controlling fundamental aspects of plastid biogenesis and functions.

A Soluble Metabolon Synthesizes the Isoprenoid Lipid Ubiquinone.

Ubiquinone (UQ) is a polyprenylated lipid that is conserved from bacteria to humans and is crucial to cellular respiration. How the cell orchestrates the efficient synthesis of UQ, which involves the modification of extremely hydrophobic substrates by multiple sequential enzymes, remains an unresolved issue. Here, we demonstrate that seven Ubi proteins form the Ubi complex, a stable metabolon that catalyzes the last six reactions of the UQ biosynthetic pathway in Escherichia coli. The SCP2 domain of UbiJ forms an extended hydrophobic cavity that binds UQ intermediates inside the 1-MDa Ubi complex. We purify the Ubi complex from cytoplasmic extracts and demonstrate that UQ biosynthesis occurs in this fraction, challenging the current thinking of a membrane-associated biosynthetic process. Collectively, our results document a rare case of stable metabolon and highlight how the supramolecular organization of soluble enzymes allows the modification of hydrophobic substrates in a hydrophilic environment.

TRIM9 and TRIM67 Are New Targets in Paraneoplastic Cerebellar Degeneration.

To describe autoantibodies (Abs) against tripartite motif-containing (TRIM) protein 9 and 67 in two patients with paraneoplastic cerebellar degeneration (PCD) associated with lung adenocarcinoma. Abs were characterized using immunohistochemistry, Western blotting, cultures of murine cortical, and hippocampal neurons, immunoprecipitation, mass spectrometry, knockout mice for Trim9 and 67, and cell-based assay. Control samples included sera from 63 patients with small cell lung cancer without any paraneoplastic neurological syndrome, 36 patients with lung adenocarcinoma and PNS, CSF from 100 patients with autoimmune encephalitis, and CSF from 165 patients with neurodegenerative diseases. We found Abs targeting TRIM9 and TRIM67 at high concentration in the serum and the cerebrospinal fluid (CSF) of a 78-year-old woman and a 65-year-old man. Both developed subacute severe cerebellar ataxia. Brain magnetic resonance imaging found no abnormality and no cerebellar atrophy. Both had CSF inflammation with mild pleiocytosis and a few oligoclonal bands. We identified a pulmonary adenocarcinoma, confirming the paraneoplastic neurological syndrome in both patients. They received immunomodulatory and cancer treatments without improvement of cerebellar ataxia, even though both were in remission of their cancer (for more than 10 years in one patient). Anti-TRIM9 and anti-TRIM67 Abs were specific to these two patients. All control serum and CSF samples tested were negative for anti-TRIM9 and 67. Anti-TRIM9 and anti-TRIM67 Abs appeared to be specific biomarkers of PCD and should be added to the panel of antigens tested when this is suspected.

Short- and long-term efficacy of electroconvulsive stimulation in animal models of depression: The essential role of neuronal survival.

BACKGROUND: Severe and medication-resistant psychiatric diseases, such as major depressive disorder, bipolar disorder or schizophrenia, can be effectively and rapidly treated by electroconvulsive therapy (ECT). Despite extensive long-standing clinical use, the neurobiological mechanisms underlying the curative action of ECT remain incompletely understood. OBJECTIVE: Unravel biological basis of electroconvulsive stimulation (ECS) efficacy, the animal equivalent of ECT. METHODS: Using MAP6 KO mouse, a genetic model that constitutively exhibits features relevant to some aspects of depression; we analyzed the behavioral and biological consequences of ECS treatment alone (10 stimulations over a 2-week period) and associated with a continuation protocol (2 stimulations per week for 5 weeks). RESULTS: ECS treatment had a beneficial effect on constitutive behavioral defects. We showed that behavioral improvement is associated with a strong increase in the survival and integration of neurons born before ECS treatment. Retroviral infection revealed the larger number of integrated neurons to exhibit increased dendritic complexity and spine density, as well as remodeled synapses. Furthermore, our results show that ECS triggers a cortical increase in synaptogenesis. A sustained newborn neuron survival rate, induced by ECS treatment, is associated with the behavioral improvement, but relapse occurred 40 days after completing the ECS treatment. However, a 5-week continuation protocol following the initial ECS treatment led to persistent improvement of behavior correlated with sustained rate survival of newborn neurons. CONCLUSION: Altogether, these results reveal that increased synaptic connectivity and extended neuronal survival are key to the short and long-term efficacy of ECS.

An algal photoenzyme converts fatty acids to hydrocarbons.

Although many organisms capture or respond to sunlight, few enzymes are known to be driven by light. Among these are DNA photolyases and the photosynthetic reaction centers. Here, we show that the microalga Chlorella variabilis NC64A harbors a photoenzyme that acts in lipid metabolism. This enzyme belongs to an algae-specific clade of the glucose-methanol-choline oxidoreductase family and catalyzes the decarboxylation of free fatty acids to n-alkanes or -alkenes in response to blue light. Crystal structure of the protein reveals a fatty acid-binding site in a hydrophobic tunnel leading to the light-capturing flavin adenine dinucleotide (FAD) cofactor. The decarboxylation is initiated through electron abstraction from the fatty acid by the photoexcited FAD with a quantum yield >80%. This photoenzyme, which we name fatty acid photodecarboxylase, may be useful in light-driven, bio-based production of hydrocarbons.

Characteristics in limbic encephalitis with anti-adenylate kinase 5 autoantibodies.

OBJECTIVE: To report 10 patients with limbic encephalitis (LE) and adenylate kinase 5 autoantibodies (AK5-Abs). METHODS: We conducted a retrospective study in a cohort of 50 patients with LE with uncharacterized autoantibodies and identified a specific target using immunohistochemistry, Western blotting, immunoprecipitation, mass spectrometry, and cell-based assay. RESULTS: AK5 (a known autoantigen of LE) was identified as the target of antibodies in the CSFs and sera of 10 patients with LE (median age 64 years; range 57-80), which was characterized by subacute anterograde amnesia without seizure and sometimes preceded by a prodromal phase of asthenia or mood disturbances. Anterograde amnesia can be isolated, but some patients also complained of prosopagnosia, paroxysmal anxiety, or abnormal behavior. No associated cancer was observed. All 10 patients had bilateral hippocampal hypersignal on a brain MRI. CSF analysis generally showed a mild pleiocytosis with elevated immunoglobulin G index and oligoclonal bands, as well as high levels of tau protein with normal concentration of Aß42 and phospho-tau, suggesting a process of neuronal death. Except for one patient, clinical response to immunotherapy was unfavorable, with persistence of severe anterograde amnesia. Two patients evolved to severe cognitive decline. Hippocampal atrophy was observed on control brain MRI. Using in vitro tests on hippocampal neurons, we did not identify clues suggesting a direct pathogenic role of AK5-Abs. CONCLUSIONS: AK5-Abs should be systematically considered in aged patients with subacute anterograde amnesia. Recognition of this disorder is important to develop new treatment strategies to prevent irreversible limbic damage.

Indolizine-Based Scaffolds as Efficient and Versatile Tools: Application to the Synthesis of Biotin-Tagged Antiangiogenic Drugs.

We describe the design and optimization of polyfunctional scaffolds based on a fluorescent indolizine core derivatized with various orthogonal groups (amines, esters, oximes, alkynes, etc.). To show one application as tools in biology, the scaffold was used to prepare drug-biotin conjugates that were then immobilized onto avidin-agarose for affinity chromatography. More specifically, the antiangiogenic drug COB223, whose mechanism of action remained unclear, was chosen as a proof-of-concept drug. The drug-selective discrimination of proteins observed after elution of the cell lysates through the affinity columns, functionalized either with the biologically active COB223 or a structurally related inactive analogue (COB236), is a clear indication that the presence of the indolizine core does not limit drug-protein interaction and confirms the usefulness of the indolizine scaffold. Furthermore, the separation of COB223-interacting proteins from human placental extracts unveiled unanticipated protein targets belonging to the family of regulatory RNA-binding proteins, which opens the way to new hypotheses on the mode of action of this antiangiogenic drug.

Deletion of FtsH11 protease has impact on chloroplast structure and function in Arabidopsis thaliana when grown under continuous light.

The membrane-integrated metalloprotease FtsH11 of Arabidopsis thaliana is proposed to be dual-targeted to mitochondria and chloroplasts. A bleached phenotype was observed in ftsh11 grown at long days or continuous light, pointing to disturbances in the chloroplast. Within the chloroplast, FtsH11 was found to be located exclusively in the envelope. Two chloroplast-located proteins of unknown function (Tic22-like protein and YGGT-A) showed significantly higher abundance in envelope membranes and intact chloroplasts of ftsh11 and therefore qualify as potential substrates for the FtsH11 protease. No proteomic changes were observed in the mitochondria of 6-week-old ftsh11 compared with wild type, and FtsH11 was not immunodetected in these organelles. The abundance of plastidic proteins, especially of photosynthetic proteins, was altered even during standard growth conditions in total leaves of ftsh11. At continuous light, the amount of photosystem I decreased relative to photosystem II, accompanied by a drastic change of the chloroplast morphology and a drop of non-photochemical quenching. FtsH11 is crucial for chloroplast structure and function during growth in prolonged photoperiod.

Saturating Light Induces Sustained Accumulation of Oil in Plastidal Lipid Droplets in Chlamydomonas reinhardtii.

Enriching algal biomass in energy density is an important goal in algal biotechnology. Nitrogen (N) starvation is considered the most potent trigger of oil accumulation in microalgae and has been thoroughly investigated. However, N starvation causes the slow down and eventually the arrest of biomass growth. In this study, we show that exposing a Chlamydomonas reinhardtii culture to saturating light (SL) under a nonlimiting CO2 concentration in turbidostatic photobioreactors induces a sustained accumulation of lipid droplets (LDs) without compromising growth, which results in much higher oil productivity than N starvation. We also show that the polar membrane lipid fraction of SL-induced LDs is rich in plastidial lipids (approximately 70%), in contrast to N starvation-induced LDs, which contain approximately 60% lipids of endoplasmic reticulum origin. Proteomic analysis of LDs isolated from SL-exposed cells identified more than 200 proteins, including known proteins of lipid metabolism, as well as 74 proteins uniquely present in SL-induced LDs. LDs induced by SL and N depletion thus differ in protein and lipid contents. Taken together, lipidomic and proteomic data thus show that a large part of the sustained oil accumulation occurring under SL is likely due to the formation of plastidial LDs. We discuss our data in relation to the different metabolic routes used by microalgae to accumulate oil reserves depending on cultivation conditions. Finally, we propose a model in which oil accumulation is governed by an imbalance between photosynthesis and growth, which can be achieved by impairing growth or by boosting photosynthetic carbon fixation, with the latter resulting in higher oil productivity.

AtMic60 Is Involved in Plant Mitochondria Lipid Trafficking and Is Part of a Large Complex.

The mitochondrion is an organelle originating from an endosymbiotic event and playing a role in several fundamental processes such as energy production, metabolite syntheses, and programmed cell death. This organelle is delineated by two membranes whose synthesis requires an extensive exchange of phospholipids with other cellular organelles such as endoplasmic reticulum (ER) and vacuolar membranes in yeast. These transfers of phospholipids are thought to occur by a non-vesicular pathway at contact sites between two closely apposed membranes. In plants, little is known about the biogenesis of mitochondrial membranes. Contact sites between ER and mitochondria are suspected to play a similar role in phospholipid trafficking as in yeast, but this has never been demonstrated. In contrast, it has been shown that plastids are able to transfer lipids to mitochondria during phosphate starvation. However, the proteins involved in such transfer are still unknown. Here, we identified in Arabidopsis thaliana a large lipid-enriched complex called the mitochondrial transmembrane lipoprotein (MTL) complex. The MTL complex contains proteins located in the two mitochondrial membranes and conserved in all eukaryotic cells, such as the TOM complex and AtMic60, a component of the MICOS complex. We demonstrate that AtMic60 contributes to the export of phosphatidylethanolamine from mitochondria and the import of galactoglycerolipids from plastids during phosphate starvation. Furthermore, AtMic60 promotes lipid desorption from membranes, likely as an initial step for lipid transfer, and binds to Tom40, suggesting that AtMic60 could regulate the tethering between the inner and outer membranes of mitochondria.