Michel Rossignol

Nitrate Efflux at the Root Plasma Membrane: Identification of an Arabidopsis Excretion Transporter

Root NO3− efflux to the outer medium is a component of NO3− net uptake and can even overcome influx upon various stresses. Its role and molecular basis are unknown. Following a functional biochemical approach, NAXT1 (for NITRATE EXCRETION TRANSPORTER1) was identified by mass spectrometry in the plasma membrane (PM) of Arabidopsis thaliana suspension cells, a localization confirmed using a NAXT1–Green Fluorescent Protein fusion protein. NAXT1 belongs to a subclass of seven NAXT members from the large NITRATE TRANSPORTER1/PEPTIDE TRANSPORTER family and is mainly expressed in the cortex of mature roots. The passive NO3− transport activity (Km = 5 mM) in isolated root PM, electrically coupled to the ATP-dependant H+-pumping activity, is inhibited by anti-NAXT antibodies. In standard culture conditions, NO3− contents were altered in plants expressing NAXT-interfering RNAs but not in naxt1 mutant plants. Upon acid load, unidirectional root NO3− efflux markedly increased in wild-type plants, leading to a prolonged NO3− excretion regime concomitant with a decrease in root NO3− content. In vivo and in vitro mutant phenotypes revealed that this response is mediated by NAXT1, whose expression is upregulated at the posttranscriptional level. Strong medium acidification generated a similar response. In vitro, the passive efflux of NO3− (but not of Cl−) was strongly impaired in naxt1 mutant PM. This identification of NO3− efflux transporters at the PM of plant cells opens the way to molecular studies of the physiological role of NO3− efflux in stressed or unstressed plants.

Arabidopsis thaliana High-Affinity Phosphate Transporters Exhibit Multiple Levels of Posttranslational Regulation

In Arabidopsis, the PHOSPHATE TRANSPORTER1 (PHT1) family encodes the high affinity phosphate transporters. This analysis revealed multiple steps of regulation in various cell compartments modulating the level of PHT1 proteins present in the plasma membrane in response to the level of inorganic phosphate. In Arabidopsis thaliana, the PHOSPHATE TRANSPORTER1 (PHT1) family encodes the high-affinity phosphate transporters. They are transcriptionally induced by phosphate starvation and require PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR (PHF1) to exit the endoplasmic reticulum (ER), indicating intracellular traffic as an additional level of regulation of PHT1 activity. Our study revealed that PHF1 acts on PHT1, upstream of vesicle coat protein COPII formation, and that additional regulatory events occur during PHT1 trafficking and determine its ER exit and plasma membrane stability. Phosphoproteomic and mutagenesis analyses revealed modulation of PHT1;1 ER export by Ser-514 phosphorylation status. Confocal microscopy analysis of root tip cells showed that PHT1;1 is localized to the plasma membrane and is present in intracellular endocytic compartments. More precisely, PHT1;1 was localized to sorting endosomes associated with prevacuolar compartments. Kinetic analysis of PHT1;1 stability and targeting suggested a modulation of PHT1 internalization from the plasma membrane to the endosomes, followed by either subsequent recycling (in low Pi) or vacuolar degradation (in high Pi). For the latter condition, we identified a rapid mechanism that reduces the pool of PHT1 proteins present at the plasma membrane. This mechanism is regulated by the Pi concentration in the medium and appears to be independent of degradation mechanisms potentially regulated by the PHO2 ubiquitin conjugase. We propose a model for differential trafficking of PHT1 to the plasma membrane or vacuole as a function of phosphate concentration.

Proteomics investigation of endogenous S-nitrosylation in Arabidopsis.

S-Nitrosylation emerges as an important protein modification in many processes. However, most data were obtained at the protein level after addition of a NO donor, particularly in plants where information about the cysteines nitrosylated in these proteins is scarce. An adapted work-flow, combining the classical biotin switch method and labeling with isotope-coded affinity tags (ICAT), is proposed. Without addition of NO donor, a total of 53 endogenous nitrosocysteines was identified in Arabidopsis cells, in proteins belonging to all cell territories, including membranes, and covering a large panel of functions. This first repertoire of nitrosothiols in plants enabled also preliminary structural description. Three apolar motifs, not located in close vicinity of cysteines and accounting for half the dataset, were detected and are proposed to complement nitrosylation prediction algorithms, poorly trained with plant data to date. Analysis of changes induced by a brief salt stress showed that NaCl modified the nitrosylation level of a small proportion of endogenously nitrosylated proteins and did not concern all nitrosothiols in these proteins. The possible role of some NO targets in the response to salt stress was discussed.

Exocytosis and protein secretion in Trypanosoma

BackgroundHuman African trypanosomiasis is a lethal disease caused by the extracellular parasite Trypanosoma brucei. The proteins secreted by T. brucei inhibit the maturation of dendritic cells and their ability to induce lymphocytic allogenic responses. To better understand the pathogenic process, we combined different approaches to characterize these secreted proteins.ResultsOverall, 444 proteins were identified using mass spectrometry, the largest parasite secretome described to date. Functional analysis of these proteins revealed a strong bias toward folding and degradation processes and to a lesser extent toward nucleotide metabolism. These features were shared by different strains of T. brucei, but distinguished the secretome from published T. brucei whole proteome or glycosome. In addition, several proteins had not been previously described in Trypanosoma and some constitute novel potential therapeutic targets or diagnostic markers. Interestingly, a high proportion of these secreted proteins are known to have alternative roles once secreted. Furthermore, bioinformatic analysis showed that a significant proportion of proteins in the secretome lack transit peptide and are probably not secreted through the classical sorting pathway. Membrane vesicles from secretion buffer and infested rat serum were purified on sucrose gradient and electron microscopy pictures have shown 50- to 100-nm vesicles budding from the coated plasma membrane. Mass spectrometry confirmed the presence of Trypanosoma proteins in these microvesicles, showing that an active exocytosis might occur beyond the flagellar pocket.ConclusionsThis study brings out several unexpected features of the secreted proteins and opens novel perspectives concerning the survival strategy of Trypanosoma as well as possible ways to control the disease. In addition, concordant lines of evidence support the original hypothesis of the involvement of microvesicle-like bodies in the survival strategy allowing Trypanosoma to exchange proteins at least between parasites and/or to manipulate the host immune system.

Analysis of the plant proteome.

For many years the analysis of plant proteomes has been restricted to the construction of descriptive catalogues or the search for markers. The analysis of plant proteomes is now gaining a functional dimension, however, because the focus has shifted onto well-defined plant-specific tissues and organelles, the simultaneous mining of proteomic and physiological data and specific methodological efforts.

Complexity of the human whole saliva proteome

Recent characterization of the whole saliva proteome led to contradictory pictures concerning the complexity of its proteome. In this work, 110 proteins were analysed by mass spectrometry allowing the identification of 10 accessions previously not detected on protein two-dimensional maps, including myosin heavy chain (fast skeletal muscle, IIA and IIB), phosphatidylethanolamine binding protein, secretory actin-binding protein precursor and triosephosphate isomerase. Further comparison with available data demonstrated simultaneously a low diversity in terms of variety of accessions and a high complexity in terms of number of protein spots identifying the same accession, the two thirds of identified spots corresponding to amylases, cystatins and immunoglobulins. This diversity may be of interest in the development of non invasive diagnostic tool for several disease.ResumenLas recientes caracterizaciones del proteoma salival completo han llevado a resultados contradictorios. En este trabajo, se han analizado 110 proteínas por espectrometría de masas, lo que ha permitido la identificación de 10 nuevas no detectadas anteriormente en los mapas proteínicos bi-dimensionales. Incluyen cadena pesada de miosina (músculo esquelético rápido, IIa y IIb); proteína de unión a fosfatidiletanolamina, precursor de la proteína secretora de la unión a la actina y triosafosfato isomerasa. Una comparación más precisa con los datos de los estudios precedentes demuestra una baja diversidad en la variedad de accesos y una alta complejidad en el número de bandas correspondientes al mismo acceso. Los dos tercios de las bandas identificadas corresponden a amilasas, cistatinas e inmunoglobulinas. Esta diversidad puede ser de interés en el desarrollo de técnicas de diagnósticos no invasivas.

Comparative analysis of proteome changes induced by the two spotted spider mite Tetranychus urticae and methyl jasmonate in citrus leaves.

Citrus plants are currently facing biotic and abiotic stresses. Therefore, the characterization of molecular traits involved in the response mechanisms to stress could facilitate selection of resistant varieties. Although large cDNA microarray profiling has been generated in citrus tissues, the available protein expression data are scarce. In this study, to identify differentially expressed proteins in Citrus clementina leaves after infestation by the two-spotted spider mite Tetranychus urticae, a proteome comparison was undertaken using two-dimensional gel electrophoresis. The citrus leaf proteome profile was also compared with that of leaves treated over 0-72h with methyl jasmonate, a compound playing a key role in the defense mechanisms of plants to insect/arthropod attack. Significant variations were observed for 110 protein spots after spider mite infestation and 67 protein spots after MeJA treatments. Of these, 50 proteins were successfully identified by liquid chromatography-mass spectrometry-tandem mass spectrometry. The majority constituted photosynthesis- and metabolism-related proteins. Five were oxidative stress associated enzymes, including phospholipid glutathione peroxidase, a salt stressed associated protein, ascorbate peroxidase and Mn-superoxide dismutase. Seven were defense-related proteins, such as the pathogenesis-related acidic chitinase, the protease inhibitor miraculin-like protein, and a lectin-like protein. This is the first report of differentially regulated proteins after T. urticae attack and exogenous MeJA application in citrus leaves.

Proteomic analysis of MON810 and comparable non-GM maize varieties grown in agricultural fields

Worldwide maize is the second major agricultural commodity and around one-fourth is currently biotech, with significant application of the insect resistant event MON810 particularly in the European Union. Grains are the major commercialized part of the plant, and can be harvested after maturity (for food and feed purposes) or at late milky-starchy stage (for forage uses, with the whole plant). We assessed possible proteomic unintended effects of the MON810 transgene using two-dimensional gel electrophoresis coupled to mass spectrometry. To keep in a realistic scenario we used plants grown in agricultural fields in a region where ~50% of maize was MON810, and analyzed grains at milky-starchy stage. In maize, differential transcripts and metabolites between GM and comparable non-GM varieties tend to be variety specific. Thus, we analyzed two variety pairs, DKC6575/Tietar and PR33P67/PR33P66 which are considered representative of Food and Agriculture Organization 700 and 600 varieties commercially grown in the region. MON810 and non-GM milky-starchy grains had virtually identical proteomic patterns, with a very small number of spots showing fold-variations in the 1-1.8 range. They were all variety specific and had divergent identities and functions. Although 2DE allows the analysis of a limited dataset our results support substantial equivalence between MON810 and comparable non-GM varieties.

CDK-dependent potentiation of MPS1 kinase activity is essential to the mitotic checkpoint.

Accurate chromosome segregation relies upon a mitotic checkpoint that monitors kinetochore attachment toward opposite spindle poles before enabling chromosome disjunction [1]. The MPS1/TTK protein kinase is a core component of the mitotic checkpoint that lies upstream of MAD2 and BubR1 both at the kinetochore and in the cytoplasm [2, 3]. To gain insight into the mechanisms underlying the regulation of MPS1 kinase, we undertook the identification of Xenopus MPS1 phosphorylation sites by mass spectrometry. We mapped several phosphorylation sites onto MPS1 and we show that phosphorylation of S283 in the noncatalytic region of MPS1 is required for full kinase activity. This phosphorylation potentiates MPS1 catalytic efficiency without impairing its affinity for the substrates. By using Xenopus egg extracts depleted of endogenous MPS1 and reconstituted with single point mutants, we show that phosphorylation of S283 is essential to activate the mitotic checkpoint. This phosphorylation does not regulate the localization of MPS1 to the kinetochore but is required for the recruitment of MAD1/MAD2, demonstrating its role at the kinetochore. Constitutive phosphorylation of S283 lowers the number of kinetochores required to hold the checkpoint, which suggests that CDK-dependent phosphorylation of MPS1 is essential to sustain the mitotic checkpoint when few kinetochores remain unattached.

Towards the profiling of the Arabidopsis thaliana plasma membrane transportome by targeted proteomics

Plant membranes bear a variety of transporters belonging to multigene families that are affected by environmental and nutritional conditions. In addition, they often display high‐sequence identity, making difficult in‐depth investigation by current shot‐gun strategies. In this study, we set up a targeted proteomics approach aimed at identifying and quantifying within single experiments the five major proton pumps of the autoinhibited H+ATPases (AHA) family, the 13 plasma membrane intrinsic proteins (PIP) water channels (PIPs), and ten members of ammonium transporters (AMTs) and nitrate transporter (NRT) families. Proteotypic peptides were selected and isotopically labeled heavy versions were used for technical optimization and for quantification of the corresponding light version in biological samples. This approach allowed to quantify simultaneously nine PIPs in leaf membranes and 13 PIPs together with three autoinhibited H+ATPases, two ammonium transporters, and two NRTs in root membranes. Similarly, it was used to investigate the effect of a salt stress on the expression of these latter 20 transporters in roots. These novel isoform‐specific data were compared with published transcriptome information and revealed a close correlation between PIP isoforms and transcripts levels. The obtained resource is reusable and can be expanded to other transporter families for large‐scale profiling of membrane transporters.