Laboratoire Physiologie Cellulaire & Végétale

JASPAR (http://jaspar.genereg.net/) is an open-access database containing manually curated, non-redundant transcription factor (TF) binding profiles for TFs across six taxonomic groups. In this 9th release, we expanded the CORE collection with 341 new profiles (148 for plants, 101 for vertebrates, 85 for urochordates, and 7 for insects), which corresponds to a 19% expansion over the previous release. We added 298 new profiles to the Unvalidated collection when no orthogonal evidence was found in the literature. All the profiles were clustered to provide familial binding profiles for each taxonomic group. Moreover, we revised the structural classification of DNA binding domains to consider plant-specific TFs. This release introduces word clouds to represent the scientific knowledge associated with each TF. We updated the genome tracks of TFBSs predicted with JASPAR profiles in eight organisms; the human and mouse TFBS predictions can be visualized as native tracks in the UCSC Genome Browser. Finally, we provide a new tool to perform JASPAR TFBS enrichment analysis in user-provided genomic regions. All the data is accessible through the JASPAR website, its associated RESTful API, the R/Bioconductor data package, and a new Python package, pyJASPAR, that facilitates serverless access to the data.

Tight coupling between biochemical and mechanical properties of the actin cytoskeleton drives a large range of cellular processes including polarity establishment, morphogenesis, and motility. This is possible because actin filaments are semi-flexible polymers that, in conjunction with the molecular motor myosin, can act as biological active springs or "dashpots" (in laymen's terms, shock absorbers or fluidizers) able to exert or resist against force in a cellular environment. To modulate their mechanical properties, actin filaments can organize into a variety of architectures generating a diversity of cellular organizations including branched or crosslinked networks in the lamellipodium, parallel bundles in filopodia, and antiparallel structures in contractile fibers. In this review we describe the feedback loop between biochemical and mechanical properties of actin organization at the molecular level in vitro, then we integrate this knowledge into our current understanding of cellular actin organization and its physiological roles.

The filamentous fungus Aspergillus niger is widely exploited by the fermentation industry for the production of enzymes and organic acids, particularly citric acid. We sequenced the 33.9-megabase genome of A. niger CBS 513.88, the ancestor of currently used enzyme production strains. A high level of synteny was observed with other aspergilli sequenced. Strong function predictions were made for 6,506 of the 14,165 open reading frames identified. A detailed description of the components of the protein secretion pathway was made and striking differences in the hydrolytic enzyme spectra of aspergilli were observed. A reconstructed metabolic network comprising 1,069 unique reactions illustrates the versatile metabolism of A. niger. Noteworthy is the large number of major facilitator superfamily transporters and fungal zinc binuclear cluster transcription factors, and the presence of putative gene clusters for fumonisin and ochratoxin A synthesis.

Forest-driven water and energy cycles are poorly integrated into regional, national, continental and global decision-making on climate change adaptation, mitigation, land use and water management. This constrains humanity’s ability to protect our planet’s climate and life-sustaining functions. The substantial body of research we review reveals that forest, water and energy interactions provide the foundations for carbon storage, for cooling terrestrial surfaces and for distributing water resources. Forests and trees must be recognized as prime regulators within the water, energy and carbon cycles. If these functions are ignored, planners will be unable to assess, adapt to or mitigate the impacts of changing land cover and climate. Our call to action targets a reversal of paradigms, from a carbon-centric model to one that treats the hydrologic and climate-cooling effects of trees and forests as the first order of priority. For reasons of sustainability, carbon storage must remain a secondary, though valuable, by-product. The effects of tree cover on climate at local, regional and continental scales offer benefits that demand wider recognition. The forest- and tree-centered research insights we review and analyze provide a knowledge-base for improving plans, policies and actions. Our understanding of how trees and forests influence water, energy and carbon cycles has important implications, both for the structure of planning, management and governance institutions, as well as for how trees and forests might be used to improve sustainability, adaptation and mitigation efforts.

JASPAR (https://jaspar.elixir.no/) is a widely-used open-access database presenting manually curated high-quality and non-redundant DNA-binding profiles for transcription factors (TFs) across taxa. In this 10th release and 20th-anniversary update, the CORE collection has expanded with 329 new profiles. We updated three existing profiles and provided orthogonal support for 72 profiles from the previous release's UNVALIDATED collection. Altogether, the JASPAR 2024 update provides a 20% increase in CORE profiles from the previous release. A trimming algorithm enhanced profiles by removing low information content flanking base pairs, which were likely uninformative (within the capacity of the PFM models) for TFBS predictions and modelling TF-DNA interactions. This release includes enhanced metadata, featuring a refined classification for plant TFs' structural DNA-binding domains. The new JASPAR collections prompt updates to the genomic tracks of predicted TF binding sites (TFBSs) in 8 organisms, with human and mouse tracks available as native tracks in the UCSC Genome browser. All data are available through the JASPAR web interface and programmatically through its API and the updated Bioconductor and pyJASPAR packages. Finally, a new TFBS extraction tool enables users to retrieve predicted JASPAR TFBSs intersecting their genomic regions of interest.

JASPAR (http://jaspar.genereg.net) is an open-access database storing curated, non-redundant transcription factor (TF) binding profiles representing transcription factor binding preferences as position frequency matrices for multiple species in six taxonomic groups. For this 2016 release, we expanded the JASPAR CORE collection with 494 new TF binding profiles (315 in vertebrates, 11 in nematodes, 3 in insects, 1 in fungi and 164 in plants) and updated 59 profiles (58 in vertebrates and 1 in fungi). The introduced profiles represent an 83% expansion and 10% update when compared to the previous release. We updated the structural annotation of the TF DNA binding domains (DBDs) following a published hierarchical structural classification. In addition, we introduced 130 transcription factor flexible models trained on ChIP-seq data for vertebrates, which capture dinucleotide dependencies within TF binding sites. This new JASPAR release is accompanied by a new web tool to infer JASPAR TF binding profiles recognized by a given TF protein sequence. Moreover, we provide the users with a Ruby module complementing the JASPAR API to ease programmatic access and use of the JASPAR collection of profiles. Finally, we provide the JASPAR2016 R/Bioconductor data package with the data of this release.

JASPAR (http://jaspar.genereg.net) is the largest open-access database of matrix-based nucleotide profiles describing the binding preference of transcription factors from multiple species. The fifth major release greatly expands the heart of JASPAR-the JASPAR CORE subcollection, which contains curated, non-redundant profiles-with 135 new curated profiles (74 in vertebrates, 8 in Drosophila melanogaster, 10 in Caenorhabditis elegans and 43 in Arabidopsis thaliana; a 30% increase in total) and 43 older updated profiles (36 in vertebrates, 3 in D. melanogaster and 4 in A. thaliana; a 9% update in total). The new and updated profiles are mainly derived from published chromatin immunoprecipitation-seq experimental datasets. In addition, the web interface has been enhanced with advanced capabilities in browsing, searching and subsetting. Finally, the new JASPAR release is accompanied by a new BioPython package, a new R tool package and a new R/Bioconductor data package to facilitate access for both manual and automated methods.

In situ, cells are highly sensitive to geometrical and mechanical constraints from their microenvironment. These parameters are, however, uncontrolled under classic culture conditions, which are thus highly artefactual. Micro-engineering techniques provide tools to modify the chemical properties of cell culture substrates at sub-cellular scales. These can be used to restrict the location and shape of the substrate regions, in which cells can attach, so-called micropatterns. Recent progress in micropatterning techniques has enabled the control of most of the crucial parameters of the cell microenvironment. Engineered micropatterns can provide a micrometer-scale, soft, 3-dimensional, complex and dynamic microenvironment for individual cells or for multi-cellular arrangements. Although artificial, micropatterned substrates allow the reconstitution of physiological in situ conditions for controlled in vitro cell culture and have been used to reveal fundamental cell morphogenetic processes as highlighted in this review. By manipulating micropattern shapes, cells were shown to precisely adapt their cytoskeleton architecture to the geometry of their microenvironment. Remodelling of actin and microtubule networks participates in the adaptation of the entire cell polarity with respect to external constraints. These modifications further impact cell migration, growth and differentiation.

Proteomics has traditionally used the separating power of two-dimensional electrophoresis for the quantitative analysis of protein amounts in complex extracts. However, the limitations of this approach in terms of throughput and analyzable protein range have elicited the development of other proteomics approaches, based either on peptide separations instead of protein separations, or based on direct protein recognition and selection on dedicated arrays (protein chips). These recent methods seem very promising, and probably look more promising than they will ultimately be, just because their weaknesses are not fully characterized yet. The purpose of this paper is thus to highlight the strengths and weaknesses of all the proteomics approaches proposed to date and to try to deduce the respective niches in proteomics that these approaches will have in the future.

Demand for organic foods is partially driven by consumers' perceptions that they are more nutritious. However, scientific opinion is divided on whether there are significant nutritional differences between organic and non-organic foods, and two recent reviews have concluded that there are no differences. In the present study, we carried out meta-analyses based on 343 peer-reviewed publications that indicate statistically significant and meaningful differences in composition between organic and non-organic crops/crop-based foods. Most importantly, the concentrations of a range of antioxidants such as polyphenolics were found to be substantially higher in organic crops/crop-based foods, with those of phenolic acids, flavanones, stilbenes, flavones, flavonols and anthocyanins being an estimated 19 (95 % CI 5, 33) %, 69 (95 % CI 13, 125) %, 28 (95 % CI 12, 44) %, 26 (95 % CI 3, 48) %, 50 (95 % CI 28, 72) % and 51 (95 % CI 17, 86) % higher, respectively. Many of these compounds have previously been linked to a reduced risk of chronic diseases, including CVD and neurodegenerative diseases and certain cancers, in dietary intervention and epidemiological studies. Additionally, the frequency of occurrence of pesticide residues was found to be four times higher in conventional crops, which also contained significantly higher concentrations of the toxic metal Cd. Significant differences were also detected for some other (e.g. minerals and vitamins) compounds. There is evidence that higher antioxidant concentrations and lower Cd concentrations are linked to specific agronomic practices (e.g. non-use of mineral N and P fertilisers, respectively) prescribed in organic farming systems. In conclusion, organic crops, on average, have higher concentrations of antioxidants, lower concentrations of Cd and a lower incidence of pesticide residues than the non-organic comparators across regions and production seasons.

Contents: Preface. Introductory overviews: Research on bacterial wilt: a perspective on international linkages and access to the literature; the current bacterial wilt situation: a global overview - Epidemiology of Ralstonia solanacearum: introduction and prospectus on the survival of introduction and prospectus on the survival; Fate of Ralstonia solanacearum Biovar 2 as affected by conditions and soil treatments in temperate climate zones; Mechanization has contributed to the spread of bacterial wilt on flue-cured tobacco in the Southeastern USA; Processes in the development of a biocontrol agent against bacterial wilt; Colonization capacity of Ralstonia solanacearum; Tomato strains differing in aggressiveness on tomatoes and weeds; Introduction to Europe of Ralstonia solanacearum Biovar 2, Race 3 in Pelargonium zonale cuttings from Kenya; Seeds from infected tomato plants appear to be free from contamination by Ralstonia solanacearum when tested by PCR or microbiological assays; The viable but non-culturable state in Ralstonia solanacearum: is there a realistic threat to our strategic concepts? - Bacterial wilt disease management: Management of bacterial wilt disease; Management of bacterial wilt in tomato with essential oils and systemic acquired resistance inducers; Monitoring of bacterial wilt in potato propagation material: a success story; Integrated control of potato bacterial wilt in Eastern Africa: the experience of African highlands initiative; Using Brassica spp. as biofumigants to reduce the population of Ralstonia solanacearum; Seed-plot technique: empowerment of farmers in production of bacterial wilt-free seed potato in Kenya and Uganda; Primary bacterial wilt study on tomato in vegetable areas of Ho Chi Minh City, Vietnam; Rhizome solarization and microwave treatment: ecofriendly methods for disinfecting ginger seed rhizomes; Management of bacterial wilt of potato using one-season rotation crops in South-Western Uganda; Potato bacterial wilt management: new prospects for an old problem - Breeding and deployment of wilt-resistant crops: A broad review and perspective on breeding for resistance to bacterial wilt; Progress on genetic enhancement for resistance to groundnut bacterial wilt in China; Search for resistance to bacterial wilt in a Brazilian Capsicum germplasm collection; Solanum phureja and S. stenotomum are sources of resistance to Ralstonia solanacearum for somatic hybrids of potato; Assessment of resistance to bacterial wilt in CIP advanced potato clones; Screening long pepper (Piper spp.) resistance to bacterial wilt caused by Ralstonia solanacearum - Host plant response and disease development: Host resistance to Ralstonia solanacearum; Microscopic studies of root infection in resistant tomato cv. Hawaii 7996; Development of bacterial wilt resistant varieties and basis of resistance in eggplant (Solanum melongena); QTL mapping for bacterial wilt resistance in Hawaii 7996 using AFLP, RGA, and SSR markers; Genetic basis of resistance to bacterial wilt in Arabidopsi thaliana; Roles of the Hrp-secreted PopA protein in Ralstonia solanacearum interactions with plants - Pathogen genetics: A short history of the biochemical and genetic research on Ralstonia solanacearum pathogenesis; The Ralstonia solanacearum complete genome sequence: outputs and prospects; Genes involved in early bacterial wilt pathogenesis; Phase reversion from phenotype conversion mutants to wild type may be induced in Ralstonia solanacearum by a susceptible host-plant; Insertions in the avirulence gene AvrA alter the virulence of Ralstonia solanacearum on Nicotiana tabacum; Ralstonia solanacearum requires type-4 Pili for twitching motility, adherence, natural transformation and virulence; Understanding the molecular basis of bacterial wilt disease: a view from the inside out - R. solanacearum in banana and plantains: Bacterial wilt diseases of banana: evolution and ecology; Comparative genome plasticity of tomato and banana strains o

Tumor necrosis factor (TNF) is an important factor in various acute and chronic neurodegenerative disorders. In retinal ischemia, we show early, transient upregulation of TNF, TNF receptor 1 (TNF-R1), and TNF-R2 6 hr after reperfusion preceding neuronal cell loss. To assess the specific role of TNF and its receptors, we compared ischemia-reperfusion-induced retinal damage in mice deficient for TNF-R1, TNF-R2, or TNF by quantifying neuronal cell loss 8 d after the insult. Surprisingly, TNF deficiency did not affect overall cell loss, yet absence of TNF-R1 led to a strong reduction of neurodegeneration and lack of TNF-R2 led to an enhancement of neurodegeneration, indicative of TNF-independent and TNF-dependent processes in the retina, with TNF-R1 augmenting neuronal death and TNF-R2 promoting neuroprotection. Western blot analyses of retinas revealed that reduction of neuronal cell loss in TNF-R1/ animals correlated with the presence of activated Akt/protein kinase B (PKB). Inhibition of the phosphatidylinositol 3-kinase signaling pathway reverted neuroprotection in TNF-R1-deficient mice, indicating an instrumental role of Akt/PKB in neuroprotection and TNF-R2 dependence of this pathway. Selective inhibition of TNF-R1 function may represent a new approach to reduce ischemia-induced neuronal damage, being potentially superior to strategies aimed at suppression of TNF activity in general.

Seeds represent the main source of nutrients for animals and humans, and knowledge of their biology provides tools for improving agricultural practices and managing genetic resources. There is also tremendous interest in using seeds as a sustainable alternative to fossil reserves for green chemistry. Seeds accumulate large amounts of storage compounds such as carbohydrates, proteins and oils. It would be useful for agro-industrial purposes to produce seeds that accumulate these storage compounds more specifically and at higher levels. The main metabolic pathways necessary for oil, starch or protein accumulation are well characterized. However, the overall regulation of partitioning between the various pathways remains unclear. Such knowledge could provide new molecular tools for improving the qualities of crop seeds (Focks and Benning, 1998, Plant Physiol. 118, 91). Studies to improve understanding of the genetic controls of seed development and metabolism therefore remain a key area of research. In the model plant Arabidopsis, genetic analyses have demonstrated that LEAFY COTYLEDON genes, namely LEC1, LEC2 and FUSCA3 (FUS3), are key transcriptional regulators of seed maturation, together with ABSCISIC ACID INSENSITIVE 3 (ABI3). Interestingly, LEC2, FUS3 and ABI3 are related proteins that all contain a 'B3' DNA-binding domain. In recent years, genetic and molecular studies have shed new light on the intricate regulatory network involving these regulators and their interactions with other factors such as LEC1, PICKLE, ABI5 or WRI1, as well as with sugar and hormonal signaling. Here, we summarize the most recent advances in our understanding of this complex regulatory network and its role in the control of seed maturation.

Recent advances in the proteomics field have allowed a series of high throughput experiments to be conducted on chloroplast samples, and the data are available in several public databases. However, the accurate localization of many chloroplast proteins often remains hypothetical. This is especially true for envelope proteins. We went a step further into the knowledge of the chloroplast proteome by focusing, in the same set of experiments, on the localization of proteins in the stroma, the thylakoids, and envelope membranes. LC-MS/MS-based analyses first allowed building the AT_CHLORO database (http://www.grenoble.prabi.fr/protehome/grenoble-plant-proteomics/), a comprehensive repertoire of the 1323 proteins, identified by 10,654 unique peptide sequences, present in highly purified chloroplasts and their subfractions prepared from Arabidopsis thaliana leaves. This database also provides extensive proteomics information (peptide sequences and molecular weight, chromatographic retention times, MS/MS spectra, and spectral count) for a unique chloroplast protein accurate mass and time tag database gathering identified peptides with their respective and precise analytical coordinates, molecular weight, and retention time. We assessed the partitioning of each protein in the three chloroplast compartments by using a semiquantitative proteomics approach (spectral count). These data together with an in-depth investigation of the literature were compiled to provide accurate subplastidial localization of previously known and newly identified proteins. A unique knowledge base containing extensive information on the proteins identified in envelope fractions was thus obtained, allowing new insights into this membrane system to be revealed. Altogether, the data we obtained provide unexpected information about plastidial or subplastidial localization of some proteins that were not suspected to be associated to this membrane system. The spectral counting-based strategy was further validated as the compartmentation of well known pathways (for instance, photosynthesis and amino acid, fatty acid, or glycerolipid biosynthesis) within chloroplasts could be dissected. It also allowed revisiting the compartmentation of the chloroplast metabolism and functions.

The development of chloroplasts and the integration of their function within a plant cell rely on the presence of a complex biochemical machinery located within their limiting envelope membranes. To provide the most exhaustive view of the protein repertoire of chloroplast envelope membranes, we analyzed this membrane system using proteomics. To this purpose, we first developed a procedure to prepare highly purified envelope membranes from Arabidopsis chloroplasts. We then extracted envelope proteins using different methods, i.e. chloroform/methanol extraction and alkaline or saline treatments, in order to retrieve as many proteins as possible, from the most to least hydrophobic ones. Liquid chromatography tandem mass spectrometry analyses were then performed on each envelope membrane subfraction, leading to the identification of more than 100 proteins. About 80% of the identified proteins are known to be, or are very likely, located in the chloroplast envelope. The validation of localization in the envelope of two phosphate transporters exemplifies the need for a combination of strategies to perform the most exhaustive identification of genuine chloroplast envelope proteins. Interestingly, some of the identified proteins are found to be Nalpha-acetylated, which indicates the accurate location of the N terminus of the corresponding mature protein. With regard to function, more than 50% of the identified proteins have functions known or very likely to be associated with the chloroplast envelope. These proteins are a) involved in ion and metabolite transport, b) components of the protein import machinery, and c) involved in chloroplast lipid metabolism. Some soluble proteins, like proteases, proteins involved in carbon metabolism, or proteins involved in responses to oxidative stress, were associated with envelope membranes. Almost one-third of the proteins we identified have no known function. The present work helps understanding chloroplast envelope metabolism at the molecular level and provides a new overview of the biochemical machinery of the chloroplast envelope membranes.

In the previous paper (Block, M. A., Dorne, A.-J., Joyard, J., and Douce, R. (1983) J. Biol. Chem. 258, 13273-13280), we have described a method for the separation of membrane fractions enriched in outer and inner envelope membranes from spinach chloroplasts. The two envelope membranes have a different weight ratio of acyl lipid to protein (2.5-3 for the outer envelope membrane and 0.8-1 for the inner envelope membrane). The two membranes also differ in their polar lipid composition. However, in order to prevent the functioning of the galactolipid:galactolipid galactosyltransferase during the course of envelope membrane separation, we have analyzed the polar lipid composition of each envelope membrane after thermolysin treatment of the intact chloroplasts. The outer envelope membrane is characterized by the presence of high amounts of phosphatidylcholine and digalactosyldiacylglycerol whereas the inner envelope membrane has a polar lipid composition almost identical with that of the thykaloids. No phosphatidylethanolamine or cardiolipin could be detected in either envelope membranes, thus demonstrating that the envelope membranes, and especially the outer membrane, do not resemble extrachloroplastic membranes. No striking differences were found in the fatty acid composition of the polar lipids from either the outer or the inner envelope membrane. The two envelope membranes also differ in their carotenoid composition. Among the different enzymatic activities associated with the chloroplast envelope, we have shown that the Mg2+-dependent ATPase, the UDP-Gal:diacylglycerol galactosyltransferase, the phosphatidic acid phosphatase, and the acyl-CoA thioesterase are associated with the inner envelope from spinach chloroplasts whereas the acyl-CoA synthetase is located on the outer envelope membrane.

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Na+/H+ exchanger reverse transcription-polymerase chain reaction mitogen-activated protein kinase NHE regulatory factor Maintaining intracellular pH values close to neutrality is a crucial task for a wide variety of cells. Hence, various mechanisms for pH regulation have been selected early in evolution and are ubiquitously distributed. Among the actors in this scene, the members of the Na+/H+ exchanger gene family (NHE1 isoforms) are widely expressed and constitute extremely efficient systems for protecting cells against internal acidification. To date, at least six genes have been identified in mammalian cells, and to various extents, the corresponding proteins have been molecularly and functionally characterized. In this short review, we will update our current knowledge on these NHE family members and highlight the most important aspects of the basic function of these transporters. Then, in a broader physiological context, we will present what we think are the most prominent specific features of the different NHE isoforms.Structural and Functional Domains of NHEsThe first cDNA encoding the NHE-1 isoform was cloned using an expression strategy based on the ability of Na+/H+ exchangers to protect antiporter-deficient cells (1Pouysségur J. Sardet C. Franchi A. L'Allemain G. Paris S. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 4833-4837Crossref PubMed Scopus (438) Google Scholar) against otherwise lethal intracellular acidification (2Sardet C. Franchi A. Pouysségur J. Cell. 1989; 56: 271-280Abstract Full Text PDF PubMed Scopus (668) Google Scholar). Variations in the hormonal regulation and pharmacological features of Na+/H+ exchange were the first indications that a large family of Na+/H+ exchange molecules existed (3Clark J.D. Limbird L.E. Am. J. Physiol. 1991; 261: C945-C953Crossref PubMed Google Scholar). Therefore, using the NHE-1 cDNA as a probe led to the molecular identification of the NHE-2, -3, and -4 (4Tse C.M. Brant S.R. Walker M.S. Pouysségur J. Donowitz M. J. Biol. Chem. 1992; 267: 9340-9346Abstract Full Text PDF PubMed Google Scholar, 5Tse C.M. Levine S.A. Yun C.H. Montrose M.H. Little P.J. Pouysségur J. Donowitz M. J. Biol. Chem. 1993; 268: 11917-11924Abstract Full Text PDF PubMed Google Scholar, 6Wang Z. Orlowski J. Shull G.E. J. Biol. Chem. 1993; 268: 11925-11928Abstract Full Text PDF PubMed Google Scholar, 7Orlowski J. Kandasamy R.A. Shull G.E. J. Biol. Chem. 1992; 267: 9331-9339Abstract Full Text PDF PubMed Google Scholar) isoforms. In addition, non-epithelial isoforms such as NHE-5 (8Klanke C.A. Su Y.R. Callen D.F. Wang Z. Meneton P. Baird N. Kandasamy R.A. Orlowski J. Otterud B.E. Leppert M. et al.Genomics. 1995; 25: 615-622Crossref PubMed Scopus (141) Google Scholar, 9Baird N.R. Orlowski J. Szabo E.Z. Zaun H.C. Schultheis P.J. Menon A.G. Shull G.E. J. Biol. Chem. 1999; 274: 4377-4382Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 10Sun A.M. Liu Y. Centracchio J. Dworkin L.D. J. Membr. Biol. 1998; 164: 293-300Crossref PubMed Scopus (24) Google Scholar) and NHE-6 have been cloned recently. By contrast to the other Na+/H+ exchangers, NHE-6 is not expressed at the plasma membrane but in the mitochondria (11Numata M. Petrecca K. Lake N. Orlowski J. J. Biol. Chem. 1998; 273: 6951-6959Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar).Topological Features and Sequence ConservationThe highly hydrophobic N-terminal region of the protein is predicted to span the membrane 10–12 times depending on the algorithm used to calculate the hydropathy plot of the protein. In particular, the region situated in the central part of the transmembrane domain (residues 226–281 in the human NHE-1) is quite hydrophobic but contains several negatively charged residues. By contrast the C-terminal region of the Na+/H+ exchangers is hydrophilic and has been shown to be located in the cell cytoplasm, at least for the NHE-1 isoform (12Shrode L.D. Gan B.S. D'Souza S.J. Orlowski J. Grinstein S. Am. J. Physiol. 1998; 275: C431-C439Crossref PubMed Google Scholar, 13Sardet C. Counillon L. Franchi A. Pouysségur J. Science. 1990; 247: 723-726Crossref PubMed Scopus (375) Google Scholar). Interestingly, recent experiments on the NHE-3 isoform seem to indicate that epitopes within the C-terminal region of this protein are extracellularly exposed (14Biemesderfer D. DeGray B. Aronson P.S. J. Biol. Chem. 1998; 273: 12391-12396Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). For NHE-1 and -2, the loop between the putative transmembrane segments 1 and 2 is glycosylated and therefore extracellular (15Counillon L. Pouysségur J. Reithmeier R.A. Biochemistry. 1994; 33: 10463-10469Crossref PubMed Scopus (123) Google Scholar, 16Tse C.M. Levine S.A. Yun C.H. Khurana S. Donowitz M. Biochemistry. 1994; 33: 12954-12961Crossref PubMed Scopus (75) Google Scholar). Methods such as scanning N-glycosylation mutagenesis (17Popov M. Tam L.Y. Li J. Reithmeier R.A. J. Biol. Chem. 1997; 272: 18325-18332Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar) will be necessary to gain further topological information.Sequence Comparison between NHE IsoformsThe transmembrane domain exhibits from 45 to 65% amino acid identity, although this score drops to about 25–35% for the cytoplasmic domain. A more detailed analysis of the sequence homology clusters reveals the presence of two subfamilies of isoforms that have probably diverged later in evolution: NHE-2 and -4 as well as NHE-3 and -5. The central part of the domain (putative transmembrane segments 5a and 5b between residues 226 and 281 in human NHE-1) is nearly identical between all the NHE isoforms. This part of the polypeptide possesses negatively charged residues (aspartates 226, 238, and 267 and glutamates 247, 248, 253, and 262 in the human NHE-1) included in a highly hydrophobic stretch of sequence, and the substitution of Glu-262 in the NHE-1 isoform results in the inactivation of the transporter (18Fafournoux P. Noel J. Pouysségur J. J. Biol. Chem. 1994; 269: 2589-2596Abstract Full Text PDF PubMed Google Scholar). Although it is not possible to rule out an indirect effect of this mutation, this result in association with the extreme sequence conservation of this region among the NHE members strongly suggests that these two transmembrane segments of the exchangers constitute the catalytic core of the Na+/H+ exchangers.By contrast, the first putative transmembrane segment of the Na+/H+ exchangers and the first extracellular loop are not well conserved in the NHE family. These N-terminal sequences are divergent even in the same isoform cloned from various mammalian species (19Counillon L. Pouysségur J. Biochim. Biophys. Acta. 1993; 1172: 343-345Crossref PubMed Scopus (23) Google Scholar), indicating that sequence conservation in the first extracellular loop is not crucial for the function of the protein. A closer analysis of the first stretch of hydrophobic residues using the von Heijne rules (20von Heijne G. Nucleic Acids Res. 1986; 14: 4683-4690Crossref PubMed Scopus (3686) Google Scholar, 21von Heijne G. J. Membr. Biol. 1990; 115: 195-201Crossref PubMed Scopus (854) Google Scholar) reveals that this first putative transmembrane segment has the features of a signal peptide, including a positively charged N-terminal end and a relatively short hydrophobic stretch.Although the cytosolic domain sequence seems to be more poorly conserved, alignment methods based on the presence of hydrophobic secondary structures (hydrophobic score analysis) (22Gaboriaud C. Bissery V. Benchetrit T. Mornon J.P. FEBS Lett. 1987; 224: 149-155Crossref PubMed Scopus (541) Google Scholar, 23Callebaut I. Labesse G. Durand P. Poupon A. Canard L. Chomilier J. Henrissat B. Mornon J.P. Cell. Mol. Life Sci. 1997; 53: 621-645Crossref PubMed Scopus (430) Google Scholar) show that these C-terminal domains clearly exhibit structural similarities. 2L. Counillon, unpublished results. Recently, circular dichroism measurements performed on the Escherichia coli-expressed NHE-1 C-terminal region confirmed that this part of the protein possesses a high degree of structural organization (24Gebreselassie D. Rajarathnam K. Fliegel L. Biochem. Cell Biol. 1998; 76: 837-842Crossref PubMed Scopus (20) Google Scholar).Physiological Roles and Regulation of NHE IsoformsThe NHE-1 isoform is expressed in virtually all cells and tissues, although the expression pattern of the other NHE isoforms exhibits striking variation among different tissues. The least ambiguous expression pattern is that of NHE-3, which is highly expressed in the kidney (proximal tubule, thin and thick limbs of the loop of Henle) (59Soleimani M. Singh G. Bizal G.L. Gullans S.R. McAteer J.A. J. Biol. Chem. 1994; 269: 27973-27978Abstract Full Text PDF PubMed Google Scholar) and intestine (jejunum, ileum, ascending and descending colon, and rectum) (60Dujeda P.K. Rao D.D. Syed I. Joshi V. Dahdal R.Y. Gardner C. Risk M.C. Schmidt L. Bavishi D. Kim K.E. Harig J.M. Goldstein J.L. Layden T.J. Ramaswamy K. Am. J. Physiol. 1996; 271: G438-G493PubMed Google Scholar). Whereas NHE-1 is found mostly on the basolateral membrane of epithelial cells (61Coupaye-Gerard B. Bookstein C. Duncan P. Chen X.Y. Smith P.R. Musch M. Ernst S.A. Chang E.B. Kleyman T.R. Am. J. Physiol. 1996; 271: C1639-C1645Crossref PubMed Google Scholar) or both in the basolateral and apical membranes in epithelial cell lines such as opossum kidney or Madin-Darby canine kidney cells (62Noel J. Roux D. Pouysségur J. J. Cell Sci. 1996; 109: 929-939PubMed Google Scholar), NHE-3 is specifically targeted to the apical membrane (62Noel J. Roux D. Pouysségur J. J. Cell Sci. 1996; 109: 929-939PubMed Google Scholar, 63Hoogerwerf W.A. Tsao S.C. Devuyst O. Levine S.A. Yun C.H. Yip J.W. Cohen M.E. Wilson P.D. Lazenby A.J. Tse C.M. Donowitz M. Am. J. Physiol. 1996; 270: G29-G41PubMed Google Scholar).NHE-2, like NHE-3, has been detected both in intestine and kidney and is also targeted to the apical membrane of epithelial cells. However, whereas the presence of NHE-2 in the intestine has been confirmed by independent investigations (see for example Ref. 60Dujeda P.K. Rao D.D. Syed I. Joshi V. Dahdal R.Y. Gardner C. Risk M.C. Schmidt L. Bavishi D. Kim K.E. Harig J.M. Goldstein J.L. Layden T.J. Ramaswamy K. Am. J. Physiol. 1996; 271: G438-G493PubMed Google Scholar), the expression of this protein in the kidney is somewhat controversial (59Soleimani M. Singh G. Bizal G.L. Gullans S.R. McAteer J.A. J. Biol. Chem. 1994; 269: 27973-27978Abstract Full Text PDF PubMed Google Scholar, 64Sun A.M. Liu Y. Dworkin L.D. Tse C.M. Donowitz M. Yip K.P. J. Membr. Biol. 1997; 160: 85-90Crossref PubMed Scopus (60) Google Scholar, 65Bookstein C. Xie Y. Rabenau K. Musch M.W. McSwine R.L. Rao M.C. Chang E.B. Am. J. Physiol. 1997; 273: C1496-C1505Crossref PubMed Google Scholar).NHE-4 mRNA can be found in the stomach, intestine, kidney, and in the cavi ammoni fields of the hippocampus. In the kidney, NHE-4 is mostly present in the inner medulla collecting duct and has also been found heterogeneously distributed on the basolateral membrane of cortical tubule cells (65Bookstein C. Xie Y. Rabenau K. Musch M.W. McSwine R.L. Rao M.C. Chang E.B. Am. J. Physiol. 1997; 273: C1496-C1505Crossref PubMed Google Scholar).The recently cloned NHE-5 isoform has been detected predominantly in brain but also in testis, spleen, and skeletal muscle by Northern blot (8Klanke C.A. Su Y.R. Callen D.F. Wang Z. Meneton P. Baird N. Kandasamy R.A. Orlowski J. Otterud B.E. Leppert M. et al.Genomics. 1995; 25: 615-622Crossref PubMed Scopus (141) Google Scholar), whereas NHE-6, which is expressed in mitochondria, has a wide tissue distribution.The common mechanism by which intracellular signaling pathways modulate the Na+/H+ exchangers involves the C-terminal region of these proteins, as shown in a series of key experiments. For example, the expression of a chimera consisting of the transmembrane region of the cAMP-insensitive human NHE-1 and the cytosolic region of the cAMP-activable β-NHE-1 of trout red cells results in a protein which is activated by cAMP, identical to the way the complete β-NHE-1 isoform behaves (66Borgese F. Sardet C. Cappadoro M. Pouysségur J. Motais R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6765-6769Crossref PubMed Scopus (124) Google Scholar, 67Borgese F. Malapert M. Fievet B. Pouysségur J. Motais R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5431-5435Crossref PubMed Scopus (37) Google Scholar). Conversely, NHE-3 is inhibited by cAMP in epithelial cells, and a chimeric construct between the transmembrane region of NHE-1 and the cytosolic region of NHE-3 becomes inhibited by cAMP (68Cabado A.G. Yu F.H. Kapus A. Lukacs G. Grinstein S. Orlowski J. J. Biol. Chem. 1996; 271: 3590-3599Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). These key findings indicate that the C-terminal domain dictates the type of hormonal regulation in a given cell.Molecular Dissection of NHE-1 ActivationNHE-1 activation by an extreme variety of extracellular stimuli, including hormones, integrins, and virtually all growth factors results from an increase in affinity of the transporter for intracellular protons. The simplest model that has been proposed is that the cytoplasmic tail cooperates with the central pH i sensor to decrease the pH i threshold value of NHE-1. In this regard, the cytoplasmic tail is seen as a signal integrator capable of transmitting hormonal signals to the transmembrane built-in pH i sensor (69Wakabayashi S. Fafournoux P. Sardet C. Pouysségur J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2424-2428Crossref PubMed Scopus (236) Google Scholar). Therefore, as for a promoter region of a regulated gene, it is not surprising to see that the NHE-1 cytoplasmic tail has “collected” regulatory boxes that convey specific extracellular signals. For example, all growth factors have been shown to induce a very rapid and transient rise in cytoplasmic calcium as well as a more or less sustained activation of the p42/p44 MAPK cascade. Interestingly and as presented below, the NHE-1 cytoplasmic domain intercepts these distinct signals for transmission into a cytoplasmic alkalinization. Bertrand et al. (70Bertrand B. Wakabayashi S. Ikeda T. Pouysségur J. Shigekawa M. J. Biol. Chem. 1994; 269: 13703-13709Abstract Full Text PDF PubMed Google Scholar) demonstrated that calmodulin physically interacts with a particular subdomain of the NHE-1 cytosolic region (71Wakabayashi S. Bertrand B. Ikeda T. Pouysségur J. Shigekawa M. J. Biol. Chem. 1994; 269: 13710-13715Abstract Full Text PDF PubMed Google Scholar) releasing a negative constraint, thus resulting in the activation of NHE-1 by increases in intracellular Ca2+. Therefore, this calmodulin-binding regulatory box is sufficient to account for the rapid and transient activation of NHE-1 in response to growth factors and other Ca2+-mobilizing agonists. By contrast, a similar sequence is not found in NHE-3, which is also regulated by calmodulin, both in a calmodulin kinase-dependent and -independent manner (72Levine S.A. Nath S.K. Yun C.H. Yip J.W. Montrose M. Donowitz M. Tse C.M. J. Biol. Chem. 1995; 270: 13716-13725Crossref PubMed Scopus (103) Google Scholar).Direct phosphorylation of NHE1 and/or phosphorylation of ancillary proteins could account for more robust and sustained activation of NHE-1. Both mechanisms have been well documented. First, it was demonstrated that NHE-1 is a phosphoprotein and that its level of phosphorylation is increased in mitogen-stimulated cells when compared with unstimulated controls (13Sardet C. Counillon L. Franchi A. Pouysségur J. Science. 1990; 247: 723-726Crossref PubMed Scopus (375) Google Scholar, 73Livne A.A. Sardet C. Pouysségur J. FEBS Lett. 1991; 284: 219-222Crossref PubMed Scopus (43) Google Scholar, 74Wang H. Silva N.L. Lucchesi P.A. Haworth R. Wang K. Michalak M. Pelech S. Fliegel L. Biochemistry. 1997; 36: 9151-9158Crossref PubMed Scopus (85) Google Scholar, 75Sardet C. Fafournoux P. Pouysségur J. J. Biol. Chem. 1991; 266: 19166-19171Abstract Full Text PDF PubMed Google Scholar). Phosphopeptide mapping carried out on wild-type and deletion mutants of the cytoplasmic region (76Wakabayashi S. Bertrand B. Shigekawa M. Fafournoux P. Pouysségur J. J. Biol. Chem. 1994; 269: 5583-5588Abstract Full Text PDF PubMed Google Scholar) revealed that the phosphorylation sites are located in the C-terminal cytoplasmic region of the protein. Ser-703 was recently demonstrated to be phosphorylated in vivo by the p42/p44 MAPK-activated target, p90RSK (77Takahashi E. Abe J. Gallis B. Aebersold R. Spring D.J. Krebs E.G. Berk B.C. J. Biol. Chem. 1999; 274: 20206-20214Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar), and to represent a major site for serum activation. This result is in agreement with our demonstration, using a Raf-activable construct, that p42/p44 MAPK plays a key role in NHE1 activation (78Bianchini L. L'Allemain G. Pouysségur J. J. Biol. Chem. 1997; 272: 271-279Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Besides the MAPK pathway, NHE-1 has been shown to be phosphorylated by p160 ROCK (79Sahai E. Alberts A.S. Treisman R. EMBO J. 1998; 17: 1350-1361Crossref PubMed Scopus (229) Google Scholar), a Rho effector associated with the assembly of stress fibers and focal adhesions. However, p42/p44 MAPK-mediated NHE-1 activation cannot be entirely explained by the direct phosphorylation of NHE-1. First, deletion of the distal cytoplasmic tail containing Ser-703 and other major phosphorylation sites attenuates but does not abolish growth factor activation. The residual activation (about 50%) remains sensitive to the MEK inhibitor PD98059 (78Bianchini L. L'Allemain G. Pouysségur J. J. Biol. Chem. 1997; 272: 271-279Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). The simplest working model taking into account this set of results is that additional regulatory proteins, which may themselves be phosphorylated, interact with various domains of the cytosolic region of the exchanger. Candidate proteins have been identified, such as p24 NHE-1 (80Goss G. Orlowski J. Grinstein S. Am. J. Physiol. 1996; 270: C1493-C1502Crossref PubMed Google Scholar), HSP70 (81Silva N.L. Haworth R.S. Singh D. Fliegel L. Biochemistry. 1995; 34: 10412-10420Crossref PubMed Scopus (84) Google Scholar), CHP (82Lin X. Barber D.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12631-12636Crossref PubMed Scopus (151) Google Scholar), and other proteins obtained by double hybrid screening, such as myosin light chain phosphatase. 4P. Fafournoux and J. Pouysségur, unpublished results. Additionally, NHE-1 can be activated by different mechanical stimuli such as osmotic stress or cell spreading. Grinstein et al. (83Grinstein S. Woodside M. Sardet C. Pouysségur J. Rotin D. J. Biol. Chem. 1992; 267: 23823-23828Abstract Full Text PDF PubMed Google Scholar) have demonstrated that the mechanism of this activation is phosphorylation-independent. In view of their finding that NHE-1 is associated with the actin cytoskeleton in focal adhesion plaques (84Grinstein S. Woodside M. Waddell T.K. Downey G.P. Orlowski J. Pouysségur J. EMBO J. 1993; PubMed Scopus Google Scholar), these results that this activation be by direct with This is by the that the presence of relatively high of is for NHE-1 activation N. Orlowski J. Grinstein S. J. Physiol. 1997; 109: PubMed Scopus Google Scholar) and that results in a more plasma membrane of NHE-1. the NHE-1 phosphorylation level is not NHE-1 interact in an manner with an ancillary protein NHE-1 with Woodside M. Wakabayashi S. Pouysségur J. Waddell T. Downey G.P. Grinstein S. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). al. (78Bianchini L. L'Allemain G. Pouysségur J. J. Biol. Chem. 1997; 272: 271-279Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar) demonstrated that the MAPK is a major for NHE-1 activation by growth stimuli, whereas the and stress kinase pathways and are not in the activation of NHE-1 by the C-terminal domain of NHE-1 can be as a series of regulatory phosphorylation or of regulatory proteins, the affinity of the transporter for intracellular is NHE-3 regulation mechanism is different from the NHE-1 activation the in of the of the transporter of in its affinity for intracellular protons. and it is possible to NHE-3 in S. A. Yu F. K. Lukacs G. K. Orlowski J. Grinstein S. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). and S. 1997; Google Scholar) have the and of a acid NHE regulatory factor review, see Ref. S. 1997; Google Scholar). This which can be cytoplasmic or negatively NHE-3 by direct in cells, is to cAMP to This protein is present in various of both the intestine and the kidney it has been detected in the NHE-3 is not indicating that it could cAMP regulation of proteins other the NHE-3 proteins, such as have been shown to other transmembrane including the or can also cAMP regulation NHE-3, indicating that the NHE regulatory proteins be C.H. S. M. D. Tsao S. Tse C.M. Donowitz M. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar). et al. R.A. J.A. A. S. Grinstein S. 1998; PubMed Scopus Google Scholar) demonstrated that the can with its a direct regulation of NHE-3 Yun et al. C.H. G. A. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar) have demonstrated that to an intracellular region within the C-terminal domain of NHE-3 and more the protein in this these results that or have a the of NHE-3 and kinase to the NHE-3 cytosolic region therefore to of various regulatory which seem to and signals from various signaling pathways (72Levine S.A. Nath S.K. Yun C.H. Yip J.W. Montrose M. Donowitz M. Tse C.M. J. Biol. Chem. 1995; 270: 13716-13725Crossref PubMed Scopus (103) Google Scholar). In an independent and (68Cabado A.G. Yu F.H. Kapus A. Lukacs G. Grinstein S. Orlowski J. J. Biol. Chem. 1996; 271: 3590-3599Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) that the region situated between residues and cAMP In this which contains and are crucial for cAMP K. Yu F.H. A.G. Szabo E.Z. Grinstein S. Orlowski J. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). Interestingly, although of is is an to into the physiological role of proteins to gene from this are for NHE isoforms. Schultheis et al. P.J. Meneton P. M. T. Wang T. G. Aronson P.S. Shull G.E. 1998; PubMed Scopus Google Scholar) have for NHE-3, an isoform which was to as well as kidney to these exhibit a decrease in are and present both in kidney and This important result the predicted physiological role of NHE-3 and that this when compared with -2, and the of the in kidney and contrast, et al. C.M. D. A. Aronson P.S. J.L. Cell. 1997; 91: Full Text Full Text PDF PubMed Scopus Google Scholar) have the molecular of the present in and that these a mutation, which a in the sequence of resulting in the of a The inactivation of NHE-1 gene function C.M. Schultheis P.J. R.L. Shull G.E. Am. J. Physiol. 1999; PubMed Google Scholar) confirmed the of this mutation, a finding which was in light of the expression of systems such as Interestingly, these have in their or in their kidney or intestine show in brain this which is highly sensitive to was not as the of the NHE-1 gene is therefore to as for the of the other isoforms may have surprising physiological Hence, the recent of the NHE-2 isoform in not result in in function but in a resulting in acid in the P.J. Meneton P. M. G.P. G. T. Shull G.E. J. 1998; PubMed Scopus Google Scholar). Therefore, as has been for gene the of the resulting be of the possible of the NHE isoforms of the and the presence of mechanisms in In this the recent of gene and of to be very for the physiological of Maintaining intracellular pH values close to neutrality is a crucial task for a wide variety of cells. Hence, various mechanisms for pH regulation have been selected early in evolution and are ubiquitously distributed. Among the actors in this scene, the members of the Na+/H+ exchanger gene family (NHE1 isoforms) are widely expressed and constitute extremely efficient systems for protecting cells against internal acidification. To date, at least six genes have been identified in mammalian cells, and to various extents, the corresponding proteins have been molecularly and functionally characterized. In this short review, we will update our current knowledge on these NHE family members and highlight the most important aspects of the basic function of these transporters. Then, in a broader physiological context, we will present what we think are the most prominent specific features of the different NHE isoforms. and Functional Domains of NHEsThe first cDNA encoding the NHE-1 isoform was cloned using an expression strategy based on the ability of Na+/H+ exchangers to protect antiporter-deficient cells (1Pouysségur J. Sardet C. Franchi A. L'Allemain G. Paris S. Proc. Natl. Acad. Sci. U. S. 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We show here that the pvr2 locus in pepper, conferring recessive resistance against strains of potato virus Y (PVY), corresponds to a eukaryotic initiation factor 4E (eIF4E) gene. RFLP analysis on the PVY-susceptible and resistant pepper cultivars, using an eIF4E cDNA from tobacco as probe, revealed perfect map co-segregation between a polymorphism in the eIF4E gene and the pvr2 alleles, pvr2(1) (resistant to PVY-0) and pvr2(2) (resistant to PVY-0 and 1). The cloned pepper eIF4E cDNA encoded a 228 amino acid polypeptide with 70-86% nucleotide sequence identity with other plant eIF4Es. The sequences of eIF4E protein from two PVY-susceptible cultivars were identical and differed from the eIF4E sequences of the two PVY-resistant cultivars Yolo Y (YY) (pvr2(1)) and FloridaVR2 (F) (pvr2(2)) at two amino acids, a mutation common to both resistant genotypes and a second mutation specific to each. Complementation experiments were used to show that the eIF4E gene corresponds to pvr2. Thus, potato virus X-mediated transient expression of eIF4E from susceptible cultivar Yolo Wonder (YW) in the resistant genotype YY resulted in loss of resistance to subsequent PVY-0 inoculation and transient expression of eIF4E from YY (resistant to PVY-0; susceptible to PVY-1) rendered genotype F susceptible to PVY-1. Several lines of evidence indicate that interaction between the potyvirus genome-linked protein (VPg) and eIF4E are important for virus infectivity, suggesting that the recessive resistance could be due to incompatibility between the VPg and eIF4E in the resistant genotype.

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