Spatiotemporal resolution of BDNF neuroprotection against glutamate excitotoxicity in cultured hippocampal neurons.

Brain-derived neurotrophic factor (BDNF) protects hippocampal neurons from glutamate excitotoxicity as determined by analysis of chromatin condensation, through activation of extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3-K) signaling pathways. However, it is still unknown whether BDNF also prevents the degeneration of axons and dendrites, and the functional demise of synapses, which would be required to preserve neuronal activity. Herein, we have studied the time-dependent changes in several neurobiological markers, and the regulation of proteolytic mechanisms in cultured rat hippocampal neurons, through quantitative western blot and immunocytochemistry. Calpain activation peaked immediately after the neurodegenerative input, followed by a transient increase in ubiquitin-conjugated proteins and increased abundance of cleaved-caspase-3. Proteasome and calpain inhibition did not reproduce the protective effect of BDNF and caspase inhibition in preventing chromatin condensation. However, proteasome and calpain inhibition did protect the neuronal markers for dendrites (MAP-2), axons (Neurofilament-H) and the vesicular glutamate transporters (VGLUT1-2), whereas caspase inhibition was unable to mimic the protective effect of BDNF on neurites and synaptic markers. BDNF partially prevented the downregulation of synaptic activity measured by the KCl-evoked glutamate release using a Förster (Fluorescence) resonance energy transfer (FRET) glutamate nanosensor. These results translate a time-dependent activation of proteases and spatial segregation of these mechanisms, where calpain activation is followed by proteasome deregulation, from neuronal processes to the soma, and finally by caspase activation in the cell body. Moreover, PI3-K and PLCγ small molecule inhibitors significantly blocked the protective action of BDNF, suggesting an activity-dependent mechanism of neuroprotection. Ultimately, we hypothesize that neuronal repair after a degenerative insult is initiated at the synaptic level.

A cytosolic trans-activation domain essential for ammonium uptake.

Polytopic membrane proteins are essential for cellular uptake and release of nutrients. To prevent toxic accumulation, rapid shut-off mechanisms are required. Here we show that the soluble cytosolic carboxy terminus of an oligomeric ammonium transporter from Arabidopsis thaliana serves as an allosteric regulator essential for function; mutations in the C-terminal domain, conserved between bacteria, fungi and plants, led to loss of transport activity. When co-expressed with intact transporters, mutants inactivated functional subunits, but left their stability unaffected. Co-expression of two inactive transporters, one with a defective pore, the other with an ablated C terminus, reconstituted activity. The crystal structure of an Archaeoglobus fulgidus ammonium transporter (AMT) suggests that the C terminus interacts physically with cytosolic loops of the neighbouring subunit. Phosphorylation of conserved sites in the C terminus are proposed as the cognate control mechanism. Conformational coupling between monomers provides a mechanism for tight regulation, for increasing the dynamic range of sensing and memorizing prior events, and may be a general mechanism for transporter regulation.

Development and use of fluorescent nanosensors for metabolite imaging in living cells.

To understand metabolic networks, fluxes and regulation, it is crucial to be able to determine the cellular and subcellular levels of metabolites. Methods such as PET and NMR imaging have provided us with the possibility of studying metabolic processes in living organisms. However, at present these technologies do not permit measuring at the subcellular level. The cameleon, a fluorescence resonance energy transfer (FRET)-based nanosensor uses the ability of the calcium-bound form of calmodulin to interact with calmodulin binding polypeptides to turn the corresponding dramatic conformational change into a change in resonance energy transfer between two fluorescent proteins attached to the fusion protein. The cameleon and its derivatives were successfully used to follow calcium changes in real time not only in isolated cells, but also in living organisms. To provide a set of tools for real-time measurements of metabolite levels with subcellular resolution, protein-based nanosensors for various metabolites were developed. The metabolite nanosensors consist of two variants of the green fluorescent protein fused to bacterial periplasmic binding proteins. Different from the cameleon, a conformational change in the binding protein is directly detected as a change in FRET efficiency. The prototypes are able to detect various carbohydrates such as ribose, glucose and maltose as purified proteins in vitro. The nanosensors can be expressed in yeast and in mammalian cell cultures and were used to determine carbohydrate homeostasis in living cells with subcellular resolution. One future goal is to expand the set of sensors to cover a wider spectrum of metabolites by using the natural spectrum of bacterial periplasmic binding proteins and by computational design of the binding pockets of the prototype sensors.

A nuclear gene encoding mitochondrial Delta-pyrroline-5-carboxylate dehydrogenase and its potential role in protection from proline toxicity.

Delta1-pyrroline-5-carboxylate (P5C), an intermediate in biosynthesis and degradation of proline (Pro), is assumed to play a role in cell death in plants and animals. Toxicity of external Pro and P5C supply to Arabidopsis suggested that P5C dehydrogenase (P5CDH; EC 1.2.1.12) plays a crucial role in this process by degrading the toxic Pro catabolism intermediate P5C. Also in a Deltaput2 yeast mutant, lacking P5CDH, Pro led to growth inhibition and formation of reactive oxygen species (ROS). Complementation of the Deltaput2 mutant allowed identification of the Arabidopsis P5CDH gene. AtP5CDH is a single-copy gene and the encoded protein was localized to the mitochondria. High homology of AtP5CDH to LuFIS1, an mRNA up-regulated during susceptible pathogen attack in flax, suggested a role for P5CDH in inhibition of hypersensitive reactions. An Arabidopsis mutant (cpr5) displaying a constitutive pathogen response was found to be hypersensitive to external Pro. In agreement with a role in prevention of cell death, AtP5CDH was expressed at a basal level in all tissues analysed. The highest expression was found in flowers that are known to contain the highest Pro levels under normal conditions. External supply of Pro induced AtP5CDH expression, but much more slowly than Pro dehydrogenase (AtProDH) expression. Uncoupled induction of the AtProDH and AtP5CDH genes further supports the hypothesis that P5C levels have to be tightly controlled. These results indicate that, in addition to the well-studied functions of Pro, for example in osmoregulation, the Pro metabolism intermediate P5C also serves as a regulator of cellular stress responses.

Rhesus factors and ammonium: a function in efflux?

Completion of fungal, plant and human genomes paved the way to the identification of erythrocytic rhesus proteins and their kidney homologs as ammonium transporters.

Function of the cytosolic N-terminus of sucrose transporter AtSUT2 in substrate affinity.

AtSUT2 was found to be a low-affinity sucrose transporter (K(M)=11.7 mM at pH 4). Chimeric proteins between AtSUT2 and the high-affinity StSUT1 were constructed in which the extended N-terminus and central loop of AtSUT2 were exchanged with those domains of StSUT1 and vice versa. Chimeras containing the N-terminus of AtSUT2 showed significantly lower affinity for sucrose compared to chimeras containing the N-terminus of StSUT1. The results indicate a significant function of the N-terminus but not the central cytoplasmic loop in determining substrate affinity. Expression of AtSUT2 in major veins of source leaves and in flowers is compatible with a role as a second low-affinity sucrose transporter or as a sucrose sensor.

A new subfamily of sucrose transporters, SUT4, with low affinity/high capacity localized in enucleate sieve elements of plants.

A new subfamily of sucrose transporters from Arabidopsis (AtSUT4), tomato (LeSUT4), and potato (StSUT4) was isolated, demonstrating only 47% similarity to the previously characterized SUT1. SUT4 from two plant species conferred sucrose uptake activity when expressed in yeast. The K(m) for sucrose uptake by AtSUT4 of 11.6 +/- 0.6 mM was approximately 10-fold greater than for all other plant sucrose transporters characterized to date. An ortholog from potato had similar kinetic properties. Thus, SUT4 corresponds to the low-affinity/high-capacity saturable component of sucrose uptake found in leaves. In contrast to SUT1, SUT4 is expressed predominantly in minor veins in source leaves, where high-capacity sucrose transport is needed for phloem loading. In potato and tomato, SUT4 was immunolocalized specifically to enucleate sieve elements, indicating that like SUT1, macromolecular trafficking is required to transport the mRNA or the protein from companion cells through plasmodesmata into the sieve elements.

SUT2, a putative sucrose sensor in sieve elements.

In leaves, sucrose uptake kinetics involve high- and low-affinity components. A family of low- and high-affinity sucrose transporters (SUT) was identified. SUT1 serves as a high-affinity transporter essential for phloem loading and long-distance transport in solanaceous species. SUT4 is a low-affinity transporter with an expression pattern overlapping that of SUT1. Both SUT1 and SUT4 localize to enucleate sieve elements of tomato. New sucrose transporter-like proteins, named SUT2, from tomato and Arabidopsis contain extended cytoplasmic domains, thus structurally resembling the yeast sugar sensors SNF3 and RGT2. Features common to these sensors are low codon bias, environment of the start codon, low expression, and lack of detectable transport activity. In contrast to LeSUT1, which is induced during the sink-to-source transition of leaves, SUT2 is more highly expressed in sink than in source leaves and is inducible by sucrose. LeSUT2 protein colocalizes with the low- and high-affinity sucrose transporters in sieve elements of tomato petioles, indicating that multiple SUT mRNAs or proteins travel from companion cells to enucleate sieve elements. The SUT2 gene maps on chromosome V of potato and is linked to a major quantitative trait locus for tuber starch content and yield. Thus, the putative sugar sensor identified colocalizes with two other sucrose transporters, differs from them in kinetic properties, and potentially regulates the relative activity of low- and high-affinity sucrose transport into sieve elements.

Hypersensitivity of an Arabidopsis sugar signaling mutant toward exogenous proline application.

In transgenic Arabidopsis a patatin class I promoter from potato is regulated by sugars and proline (Pro), thus integrating signals derived from carbon and nitrogen metabolism. In both cases a signaling cascade involving protein phosphatases is involved in induction. Other endogenous genes are also regulated by both Pro and carbohydrates. Chalcone synthase (CHS) gene expression is induced by both, whereas the Pro biosynthetic Delta(1)-pyrroline-5-carboxylate synthetase (P5CS) is induced by high Suc concentrations but repressed by Pro, and Pro dehydrogenase (ProDH) is inversely regulated. The mutant rsr1-1, impaired in sugar dependent induction of the patatin promoter, is hypersensitive to low levels of external Pro and develops autofluorescence and necroses. Toxicity of Pro can be ameliorated by salt stress and exogenously supplied metabolizable carbohydrates. The rsr1-1 mutant shows a reduced response regarding sugar induction of CHS and P5CS expression. ProDH expression is de-repressed in the mutant but still down-regulated by sugar. Pro toxicity seems to be mediated by the degradation intermediate Delta(1)-pyrroline-5-carboxylate. Induction of the patatin promoter by carbohydrates and Pro, together with the Pro hypersensitivity of the mutant rsr1-1, demonstrate a new link between carbon/nitrogen and stress responses.

The molecular physiology of ammonium uptake and retrieval.

Plants are able to take up ammonium from the soil, or through symbiotic interactions with microorganisms, via the root system. Using functional complementation of yeast mutants, it has been possible to identify a new class of membrane proteins, the ammonium transporter/methylammonium permease (AMT/MEP) family, that mediate secondary active ammonium uptake in eukaryotic and prokaryotic organisms. In plants, the AMT gene family can be subdivided according to their amino-acid sequences into three subfamilies: a large subfamily of AMT1 genes and two additional subfamilies each with single members (LeAMT1;3 from tomato and AtAMT2;1 from Arabidopsis thaliana). These transporters vary especially in their kinetic properties and regulatory mechanism. High-affinity transporters are induced in nitrogen-starved roots, whereas other transporters may be considered as the 'work horses' that are active when conditions are conducive to ammonium assimilation. The expression of several AMTs in root hairs further supports a role in nutrient acquisition. These studies provide basic information that will be needed for the dissection of nitrogen uptake by plants at the molecular level and for determining the role of individual AMTs in nutrient uptake and potentially in nutrient efficiency.

Differential regulation of three functional ammonium transporter genes by nitrogen in root hairs and by light in leaves of tomato.

To elucidate the role of NH4+ transporters in N nutrition of tomato, two new NH4+ transporter genes were isolated from cDNA libraries of root hairs or leaves of tomato. While LeAMT1;2 is closely related to LeAMT1;1 (75.6% amino acid identity), LeAMT1;3 is more distantly related (62.8% identity) and possesses two short upstream open reading frames in the 5' end of the mRNA and a particularly short N-terminus of the protein as unique features. When expressed in yeast mutants defective in NH4+ uptake, all three genes complemented NH4+ uptake. In roots of hydroponically grown plants, transcript levels of LeAMT1;2 increased after NH4+ or NO3- supply, while LeAMT1;1 was induced by N deficiency coinciding with low glutamine concentrations, and LeAMT1;3 was not detected. In aeroponic culture, expression of LeAMT1;1 and LeAMT1;2 was higher in root hairs than in the remaining root fraction. Growth of plants at elevated CO2 slightly decreased expression of LeAMT1;2 and LeAMT1;3 in leaves, but strongly repressed transcript levels of chloroplast glutamine synthetase and photorespiratory serine hydroxymethyl-transferase. Expression of LeAMT1;2 and LeAMT1;3 showed a reciprocal diurnal regulation with highest transcript levels of LeAMT1;3 in darkness and highest levels of LeAMT1;2 after onset of light. These results indicate that in tomato at least two high-affinity NH4+ transporters, LeAMT1;1 and LeAMT1;2, are differentially regulated by N and contribute to root hair-mediated NH4+ acquisition from the rhizosphere. In leaves, the reciprocally expressed transporters LeAMT1;2 and LeAMT1;3 are supposed to play different roles in N metabolism, NH4+ uptake and/or NH3 retrieval during photorespiration.

Amino acid transporters are localized to transfer cells of developing pea seeds.

To determine the nature and cellular localization of amino acid transport in pea seeds, two cDNA clones belonging to the AAP family of H(+)/amino acid co-transporters (PsAAP1 and PsAAP2) were isolated from a cotyledon cDNA library of pea (Pisum sativum L.). Functional expression in the yeast amino acid uptake mutants 22Delta6AAL and 22Delta8AA showed that PsAAP1 mediates transport of neutral, acidic, and basic amino acids. RNA-blot analyses showed that PsAAP1 is expressed in seeds and vegetative organs, including amino acid sinks and sources, whereas PsAAP2 could not be detected. For developing seeds, transcripts of PsAAP1 were detected in coats and cotyledons, with seed coats giving a weak signal. In cotyledons, expression was highest in epidermal-transfer-cell-enriched tissue. RNA in situ hybridization analysis showed that PsAAP1 was predominantly present in epidermal transfer cells forming the outer surface of cotyledons, which abuts the seed coats. Overall, our observations suggest that this transporter, which is localized in transfer cells of cotyledons, might play a role in the uptake of the full spectrum of amino acids released from seed coats.

Hypersensitivity of an Arabidopsis sugar signaling mutant toward exogenous proline application.

In transgenic Arabidopsis a patatin class I promoter from potato is regulated by sugars and proline (Pro), thus integrating signals derived from carbon and nitrogen metabolism. In both cases a signaling cascade involving protein phosphatases is involved in induction. Other endogenous genes are also regulated by both Pro and carbohydrates. Chalcone synthase (CHS) gene expression is induced by both, whereas the Pro biosynthetic Delta(1)-pyrroline-5-carboxylate synthetase (P5CS) is induced by high Suc concentrations but repressed by Pro, and Pro dehydrogenase (ProDH) is inversely regulated. The mutant rsr1-1, impaired in sugar dependent induction of the patatin promoter, is hypersensitive to low levels of external Pro and develops autofluorescence and necroses. Toxicity of Pro can be ameliorated by salt stress and exogenously supplied metabolizable carbohydrates. The rsr1-1 mutant shows a reduced response regarding sugar induction of CHS and P5CS expression. ProDH expression is de-repressed in the mutant but still down-regulated by sugar. Pro toxicity seems to be mediated by the degradation intermediate Delta(1)-pyrroline-5-carboxylate. Induction of the patatin promoter by carbohydrates and Pro, together with the Pro hypersensitivity of the mutant rsr1-1, demonstrate a new link between carbon/nitrogen and stress responses.

A new family of high-affinity transporters for adenine, cytosine, and purine derivatives in Arabidopsis.

In many organisms, including plants, nucleic acid bases and derivatives such as caffeine are transported across the plasma membrane. Cytokinins, important hormones structurally related to adenine, are produced mainly in root apices, from where they are translocated to shoots to control a multitude of physiological processes. Complementation of a yeast mutant deficient in adenine uptake (fcy2) with an Arabidopsis cDNA expression library enabled the identification of a gene, AtPUP1 (for Arabidopsis thaliana purine permease1), belonging to a large gene family (AtPUP1 to AtPUP15) encoding a new class of small, integral membrane proteins. AtPUP1 transports adenine and cytosine with high affinity. Uptake is energy dependent, occurs against a concentration gradient, and is sensitive to protonophores, potentially indicating secondary active transport. Competition studies show that purine derivatives (e.g., hypoxanthine), phytohormones (e.g., zeatin and kinetin), and alkaloids (e.g., caffeine) are potent inhibitors of adenine and cytosine uptake. Inhibition by cytokinins is competitive (competitive inhibition constant K(i) = 20 to 35 microM), indicating that cytokinins are transported by this system. AtPUP1 is expressed in all organs except roots, indicating that the gene encodes an uptake system for root-derived nucleic acid base derivatives in shoots or that it exports nucleic acid base analogs from shoots by way of the phloem. The other family members may have different affinities for nucleic acid bases, perhaps functioning as transporters for nucleosides, nucleotides, and their derivatives.

Cross-talk between ammonium transporters in yeast and interference by the soybean SAT1 protein.

Ammonium uptake in the yeast Saccharomyces cerevisiae involves three membrane transporters (Mep1, -2 and -3) belonging to an evolutionarily conserved protein family that also includes the rhesus (Rh) blood group polypeptides of erythrocytes. We show here that, in the 26972c mutant defective in NH4+ transport, the Mep1 protein carrying an amino acid substitution in its cytoplasmic C-terminus trans-inhibits the closely related Mep3 protein. The same mutation introduced into Mep3 leads to loss of transport activity and this inactive form also trans-inhibits native Mep3. Inhibition of Mep3 is post-translational and can be overcome by overexpression. These results are consistent with a direct interaction between Mep proteins, as is the case for the Rh polypeptides. The soybean GmSAT1 gene, recently cloned for its ability to complement the NH4+ transport defect of strain 26972c, has been described as an NH4+ channel protein involved in the transfer of fixed nitrogen from the bacteroid to the host plant. We show here that GmSAT1 contains a sequence homologous to the DNA-binding domain of basic helix-loop-helix (bHLH) transcription factors. We also show that GmSAT1 restores NH4+ uptake in the yeast mutant by interfering with the inhibition of Mep3. Our results are not consistent with a direct role of GmSAT1 in ammonium transport.

Cloning and expression of amino acid transporters from broad bean.

This work describes the isolation of a full-length (VfAAP2) and three partial amino acid transporter genes (VfAAPa, VfAAPb, VfAAPc) from broad bean (Vicia faba L.). The function of VfAAP2 was tested by heterologous expression in a yeast mutant deficient in proline uptake. VfAAP2 mediates proton-dependent proline uptake with an apparent Km of about 1 mM. Analysis of substrate specificity by competition experiments showed that aromatic amino acids, neutral aliphatic acids and L-citrulline are the best competitors, whereas basic amino acids do not compete with proline. Northern analysis indicates that all VfAAPs exhibit different patterns of expression. VfAAP2 is most strongly expressed in the stem and at a lower level in sink leaves and pods. VfAAPa, VfAAPb and VfAAPc are most strongly expressed in the flowers, but their expression in the other organs varies.

Identification of a pollen-specific sucrose transporter-like protein NtSUT3 from tobacco.

Pollen cells are symplasmically isolated during maturation and germination. Pollen therefore needs to take up nutrients via membrane carriers. Physiological measurements on pollen indicate sucrose transport in the pollen tube. A cDNA encoding a pollen-specific sucrose transporter-like protein NtSUT3 was isolated from a tobacco pollen cDNA library. NtSUT3 expression is detected only in pollen and is restricted to late pollen development, pollen germination and pollen tube growth. Altogether these data indicate that pollen is supplied not only with glucose, but also with sucrose through a specific sucrose transporter. The respective contribution of each transport pathway may change during pollen tube growth.

Sucrose transport into developing seeds of Pisum sativum L.

The anatomy of developing pea seeds is characterized by transfer cells present in both coats and cotyledons at the maternal/filial interface. To determine the nature and cellular localization of sucrose transporters in pea seeds, a full-length clone of a sucrose/H+ symporter (PsSUT1) was isolated from a cotyledon cDNA library. Northern blot analyses of different organs showed that PsSUT1 is expressed in non-seed tissues, including sucrose sinks and sources. Within developing seeds, transcripts of PsSUT1 and PsAHA1 genes were detected in all tissues, while transcripts of a sucrose binding protein (GmSBP) were confined to cotyledon epidermal transfer cells. Signal intensities of PsSUT1 and PsAHA1 transcripts and protein products were most pronounced in the thin-walled parenchyma cells of seed coats and epidermal transfer cells of cotyledons. For cotyledons, the highest transporter densities were localized to those portions of plasma membranes lining the wall ingrowth regions of epidermal transfer cells. Responses of [14C]sucrose influx to metabolic inhibitors indicated that proton-coupled sucrose transport was operative in both seed coats and cotyledons. Cotyledon epidermal transfer cells were shown to support the highest sucrose flux. Maximal transport activity was found to account for the sucrose flux differences between seed tissues. Intercellular movement of the symplasmic tracer, 5-(6)-carboxyfluorescein (CF), demonstrated that symplasmic pathways interconnect the vascular tissues to thin-walled parenchyma transfer cells of seed coats and, for cotyledons, epidermal transfer cells to storage parenchyma cells.

Transporters for ammonium, amino acids and peptides are expressed in pitchers of the carnivorous plant Nepenthes.

Insect capture and digestion contribute substantially to the nitrogen budget of carnivorous plants. In Nepenthes, insect-derived nitrogenous compounds are imported from the pitcher fluid and transported throughout the plant via the vascular tissue to support growth. Import and distribution of nutrients may require transmembrane nitrogen transporters. Representatives of three classes of genes encoding transporters for the nitrogenous compounds ammonium, amino acids and peptides were identified in Nepenthes pitchers. The expression at the cellular level of an ammonium transporter gene, three amino acid transporter genes, and one peptide transporter gene were investigated in the insect trapping organs of Nepenthes. Expression of the ammonium transporter gene NaAMT1 was detected in the head cells of digestive glands in the lower part of the pitcher where NaAMT1 may function in ammonium uptake from the pitcher fluid. One amino acid transporter gene, NaAAP1, was expressed in bundle sheath cells surrounding the vascular tissue. To understand the locations where transmembrane transport could be required within the pitcher, symplasmic and apoplasmic continuity was probed using fluorescent dyes. Symplasmic connections were not found between cortical cells and vascular bundles. Therefore, the amino acid transporter encoded by NaAAP1 may be involved in transport of amino acids into the vascular tissue. In contrast, expression of the peptide transporter gene NaNTR1 was detected in phloem cells of the vascular tissue within pitchers. NaNTR1 may function in the export of nitrogen from the pitcher by loading peptides into the phloem.