High-affinity Amino Acid Transport Research Articles

Overexpression of NtLHT1 affects the development of leaf morphology and abiotic tolerance in tobacco

LYSINE HISTIDINE TRANSPORTER1 (LHT1) is a crucial broad-specificity and high-affinity amino acid transporter affecting the uptake of nitrogen and probably the tolerance to abiotic stress in plants. However, little is known about the phenotypic functions of LHT1 in plant growth and development and abiotic stress tolerance. In this study, we identified the NtLHT1 gene from the tobacco variety Honghuadajinyuan (HD) and determined its important roles in leaf morphological development and plant resistance to abiotic stress. Comprehensive functional analyses using knockout and overexpression transgenic lines (ntlht1 and OE) revealed overexpression of NtLHT1 accelerated leave senescence and increased plant height, leaf number and plant tolerance under cold, salt and drought stresses. In addition, NtLHT1 overexpression significantly decreased the leaf elongation of HD, causing the leaves to change from a long-elliptical shape to an elliptical shape. However silencing NtLHT1 decreased the seed germination rate under NaCl and PEG stresses. Moreover, NtLHT1 significantly affected the contents of various amino acids, such as the neutral, acidic, non-polar and aromatic amino acids, ethylene precursor (ACC), GA3 and IAA in tobacco. These results suggested that the amino acid and ethylene precursor ACC transport activities of NtLHT1 provide fine regulatory function for plant growth and development and plant tolerance to abiotic stress.

A single amino acid transporter controls the uptake of priming-inducing beta-amino acids and the associated tradeoff between induced resistance and plant growth.

Selected β-amino acids, such as β-aminobutyric acid (BABA) and R-β-homoserine (RBH), can prime plants for resistance against a broad spectrum of diseases. Here, we describe a genome-wide screen of fully annotated Arabidopsis thaliana T-DNA insertion lines for impaired in RBH-induced immunity (iri) mutants against the downy mildew pathogen Hyaloperonospora arabidopsidis, yielding 104 lines that were partially affected and four lines that were completely impaired in RBH-induced resistance (IR). We confirmed the iri1-1 mutant phenotype with an independent T-DNA insertion line in the same gene, encoding the high-affinity amino acid transporter LYSINE HISTIDINE TRANSPORTER 1 (LHT1). Uptake experiments with yeast cells expressing LHT1 and mass spectrometry-based quantification of RBH and BABA in leaves of lht1 mutant and LHT1 overexpression lines revealed that LHT1 acts as the main transporter for cellular uptake and systemic distribution of RBH and BABA. Subsequent characterization of lht1 mutant and LHT1 overexpression lines for IR and growth responses revealed that the levels of LHT1-mediated uptake determine the tradeoff between IR and plant growth by RBH and BABA.

The role of nutrients in B lymphocyte growth and survival responses.

Abstract B cell activation is an energetically demanding process during which B lymphocytes shift from a resting state to a proliferative, metabolically active state. Little is known about the metabolic reprogramming process or the role extracellular nutrients play in the activation response. We demonstrate that there are distinct requirements for the nutrients L-glutamine and glucose during activation. We show that cells activated in glucose-depleted conditions are still able to undergo growth and signaling events. In contrast, we show that extracellular L-glutamine is essential for all but the earliest activation events, and cells cultured in L-glutamine-deprived conditions are unable to enter the cell cycle. Consistently, we show that extracellular supplementation of the cell-permeable derivative of a-ketoglutarate (a-KG) is able to rescue cell activation in the absence of glutamine. We also show the induction of the high affinity amino acid transporter ASCT2 is required for glutamine uptake following BCR crosslinking. Specifically, we found that halting glutamine uptake or processing by inhibiting ASCT2 or the glutaminolytic enzyme glutaminase causes activation defects that parallel those observed in glutamine deprived conditions, indicating a requirement for glutaminolysis during the very early stages of activation. Finally, we found that a-KG does not contribute to epigenetic remodeling, but is necessary for mTORC1 activation. In turn, mTORC1 activity is required for upregulation of the glucose transporter Glut1 during activation, as well as increased glucose uptake. These findings indicate a distinct metabolic profile that begins with glutamine uptake, and acts through mTORC1 signaling to later promote glucose uptake.

P2X7 receptor activation downmodulates Na+-dependent high-affinity GABA and glutamate transport into rat brain cortex synaptosomes

Sodium-dependent high-affinity amino-acid transporters play crucial roles in terminating synaptic transmission in the central nervous system (CNS). However, there is lack of information about the mechanisms underlying the regulation of amino-acid transport by fast-acting neuromodulators, like ATP. Here, we investigated whether activation of the ATP-sensitive P2X7 receptor modulates Na+-dependent high-affinity γ-aminobutyric acid (GABA) and glutamate uptake into nerve terminals (synaptosomes) of the rat cerebral cortex. Radiolabeled neurotransmitter accumulation was evaluated by liquid scintillation spectrometry. The cell-permeant sodium-selective fluorescent indicator, SBFI-AM, was used to estimate Na+ influx across plasma membrane. 2′(3′)-O-(4-benzoylbenzoyl)ATP (BzATP, 3–300μM), a prototypic P2X7 receptor agonist, concentration-dependently decreased [3H]GABA (14%) and [14C]glutamate (24%) uptake; BzATP decreased transport maximum velocity (Vmax) without affecting the Michaelis constant (Km) values. The selective P2X7 receptor antagonist, A-438079 (3μM), prevented inhibition of [3H]GABA and [14C]glutamate uptake by BzATP (100μM). The inhibitory effect of BzATP coincided with its ability to increase intracellular Na+ and was mimicked by Na+ ionophores, like gramicidin and monensin. Increases in intracellular Na+ (with veratridine or ouabain) or substitution of extracellular Na+ by N-methyl-d-glucamine (NMDG)+ all decreased [3H]GABA and [14C]glutamate uptake and attenuated BzATP effects. Uptake inhibition by BzATP (100μM) was also attenuated by calmidazolium, which selectively inhibits Na+ currents through the P2X7 receptor pore. In conclusion, disruption of the Na+ gradient by P2X7 receptor activation downmodulates high-affinity GABA and glutamate uptake into rat cortical synaptosomes. Interference with amino-acid transport efficacy may constitute a novel target for therapeutic management of cortical excitability.

LjLHT1.2—a mycorrhiza-inducible plant amino acid transporter from Lotus japonicus

In mycorrhizal associations, the fungal partner assists its plant host by providing nitrogen (N) in addition to phosphate. Arbuscular mycorrhizal (AM) fungi have access to inorganic and organic forms of N and translocate them, via arginine, from the extra- to the intraradical mycelium, where N is transferred to the plant as inorganic N compounds such as ammonium. However, several putative amino acid transporters (AATs) with an altered expression in Lotus japonicus mycorrhizal roots were recorded in a previous microarray-based investigation, which led to the question of whether a transfer of organic N, mainly in the form of amino acids, could occur in AM roots. Here, we have characterized an AAT gene (LjLHT1.2) that encodes for lysine–histidine–transporter (LHT)-type amino acid transporter. We show that it is induced in mycorrhizas, but not in nodulated roots. By using in situ hybridization and laser microdissection technology, the corresponding transcripts have been demonstrated to be located above all in arbusculated cells but also in the non-colonized cells of the root cortex. The gene expression resulted to be differentially regulated by the availability of the N sources. Furthermore, functional experiments, via heterologous expression in yeast, have demonstrated that the protein was a high-affinity amino acid transporter. Taken together, the results show that LjLHT1.2 may allow the uptake of energy-rich N compounds, such as amino acids, towards the cortical cells. We suggest that LjLHT1.2 could be involved in complex mechanisms that guarantee the re-uptake and recycle of amino acids and which are particularly efficient in mycorrhizal roots.

PtAAP11, a high affinity amino acid transporter specifically expressed in differentiating xylem cells of poplar

Amino acids are the currency of nitrogen exchange between source and sink tissues in plants and constitute a major source of the components used for cellular growth and differentiation. The characterization of a new amino acid transporter belonging to the amino acid permease (AAP) family, AAP11, expressed in the perennial species Populus trichocarpa is reported here. PtAAP11 expression analysis was performed by semi-quantitative RT-PCR and GUS activity after poplar transformation. PtAAP11 function was studied in detail by heterologous expression in yeast. The poplar genome contains 14 putative AAPs which is quite similar to other species analysed except Arabidopsis. PtAAP11 was mostly expressed in differentiating xylem cells in different organs. Functional characterization demonstrated that PtAAP11 was a high affinity amino acid transporter, more particularly for proline. Compared with other plant amino acid transporters, PtAAP11 represents a novel high-affinity system for proline. Thus, the functional characterization and expression studies suggest that PtAAP11 may play a major role in xylogenesis by providing proline required for xylem cell wall proteins. The present study provides important information highlighting the role of a specific amino acid transporter in xylogenesis in poplar.

Plasma membrane expression of the neuronal glutamate transporter EAAC1 is regulated by glial factors: Evidence for different regulatory pathways associated with neuronal maturation

At the glutamatergic synapse the neurotransmitter is removed from the synaptic cleft by high affinity amino acid transporters located on neurons (EAAC1) and astrocytes (GLAST and GLT1), and a coordinated action of these cells is necessary in order to regulate glutamate extracellular concentration. We show here that treatment of neuronal cultures with glial soluble factors (GCM) is associated with a redistribution of EAAC1 and GLAST to the cell membrane and we analysed the effect of membrane cholesterol depletion on this regulation. In enriched neuronal culture (90% neurons and 10% astrocytes), GCM treatment for 10 days increases EAAC1 and GLAST cell surface expression with no change in total expression. In opposite, GLT1 surface expression is not modified by GCM but total expression is increased. When cholesterol is acutely depleted from the membrane by 10 mM methyl-beta-cyclodextrin (β5-MCD, 30 min), glutamate transport activity and cell surface expressions of EAAC1 and GLAST are decreased in the enriched neuronal culture treated by GCM. In pure neuronal culture addition of GCM also increases EAAC1 cell membrane expression but surprisingly acute treatment with β5-MCD decreases glutamate uptake activity but not EAAC1 cell membrane expression. By immunocytochemistry a modification in the distribution of EAAC1 within neurons was undetectable whatever the treatment but we show that EAAC1 was no more co localized with Thy-1 in the enriched neuronal culture treated by GCM suggesting that GCM have stimulated polarity formation in neurons, an index of maturation. In conclusion we suggest that different regulatory mechanisms are involved after GCM treatment, glutamate transporter trafficking to and from the plasma membrane in enriched neuronal culture and modulation of EAAC1 intrinsic activity and/or association with regulatory proteins at the cell membrane in the pure neuronal culture. These different regulatory pathways of EAAC1 are associated with different neuronal maturation stages.

Arabidopsis LHT1 Is a High-Affinity Transporter for Cellular Amino Acid Uptake in Both Root Epidermis and Leaf Mesophyll

Amino acid transport in plants is mediated by at least two large families of plasma membrane transporters. Arabidopsis thaliana, a nonmycorrhizal species, is able to grow on media containing amino acids as the sole nitrogen source. Arabidopsis amino acid permease (AAP) subfamily genes are preferentially expressed in the vascular tissue, suggesting roles in long-distance transport between organs. We show that the broad-specificity, high-affinity amino acid transporter LYSINE HISTIDINE TRANSPORTER1 (LHT1), an AAP homolog, is expressed in both the rhizodermis and mesophyll of Arabidopsis. Seedlings deficient in LHT1 cannot use Glu or Asp as sole nitrogen sources because of the severe inhibition of amino acid uptake from the medium, and uptake of amino acids into mesophyll protoplasts is inhibited. Interestingly, lht1 mutants, which show growth defects on fertilized soil, can be rescued when LHT1 is reexpressed in green tissue. These findings are consistent with two major LHT1 functions: uptake in roots and supply of leaf mesophyll with xylem-derived amino acids. The capacity for amino acid uptake, and thus nitrogen use efficiency under limited inorganic N supply, is increased severalfold by LHT1 overexpression. These results suggest that LHT1 overexpression may improve the N efficiency of plant growth under limiting nitrogen, and the mutant analyses may enhance our understanding of N cycling in plants.

Antibody production and immunocytochemical localization of amino acid transporters.

This chapter details the procedural and conceptual issues that underpin the localization of high-affinity amino acid transporters by immunocytochemistry, with particular reference to the mammalian nervous system. Defining a role for transporters in any individual tissue often requires an understanding of the spatial distribution of a specific transport system. Techniques such as in situ hybridization provide clear evidence for the potential expression of a transporter by a specific cell type. In many cases however, properties such as targeting to basal or apical surfaces of, for instance, a transport epithelium or targeting to distinct plasmalemmal or cytoplasmic compartments in response to stimulation are most readily determined by the use of antibodies to detect such transporters, in intact tissues, isolated cells, or subcellular fractions of tissues. Methodologies for creating antibodies for the direct immunocytochemical localization of transporters are presented in this chapter. Issues that influence the applicability of methodologies such as postmortem delay, especially in human tissues, are also addressed.

High Affinity Amino Acid Transporters Specifically Expressed in Xylem Parenchyma and Developing Seeds of Arabidopsis

Arabidopsis amino acid transporters (AAPs) show individual temporal and spatial expression patterns. A new amino acid transporter, AAP8 was isolated by reverse transcription-PCR. Growth and transport assays in comparison to AAP1-5 characterize AAP8 and AAP6 as high affinity amino acid transport systems from Arabidopsis. Histochemical promoter-beta-glucuronidase (GUS) studies identified AAP6 expression in xylem parenchyma, cells requiring high affinity transport due to the low amino acid concentration in xylem sap. AAP6 may thus function in uptake of amino acids from xylem. Histochemical analysis of AAP8 revealed stage-dependent expression in siliques and developing seeds. Thus AAP8 is probably responsible for import of organic nitrogen into developing seeds. The only missing transporter of the family AAP7 was nonfunctional in yeast with respect to amino acid transport, and expression was not detectable. Therefore, AAP6 and -8 are the only members of the family able to transport aspartate with physiologically relevant affinity. AAP1, -6 and -8 are the closest AAP paralogs. Although AAP1 and AAP8 originate from a duplicated region on chromosome I, biochemical properties and expression pattern diverged. Overlapping substrate specificities paired with individual properties and expression patterns point to specific functions of each of the AAP genes in nitrogen distribution rather than to mere redundancy.

Cochlear nerve projections to the small cell shell of the cochlear nucleus: the neuroanatomy of extremely thin sensory axons.

Labeling cochlear nerve fibers in the inner ear of chinchillas with biotinylated dextran polyamine was used to trace the thin fibers (Type II), which likely innervate outer hair cells. These axons, 0. 1-0.5 microm in diameter, were distinguished from the thicker Type I, fibers innervating inner hair cells, and traced to small-cell clusters in the cochlear nucleus. This study provided two major new insights into the outer hair cell connections in the cochlear nucleus and the potential significance of very thin axons and synaptic nests, which are widespread in the CNS. 1) EM serial reconstructions of labeled and unlabeled material revealed that Type II axons rarely formed synapses with conventional features (vesicles gathered at junctions). Rather, their endings contained arrays of endoplasmic reticulum and small spherical vesicles without junctions. 2) Type II axons projected predominantly to synaptic nests, where they contacted other endings and dendrites of local interneurons (small stellate and mitt cells, but not granule cells). Synaptic nests lacked intrinsic glia and, presumably, their high-affinity amino acid transporters. As functional units, nests and their Type II inputs from outer hair cells may contribute to an analog processing mode, which is slower, more diffuse, longer-lasting, and potentially more plastic than the digital processors addressed by inner hair cells.

Cochlear nerve projections to the small cell shell of the cochlear nucleus: The neuroanatomy of extremely thin sensory axons

Labeling cochlear nerve fibers in the inner ear of chinchillas with biotinylated dextran polyamine was used to trace the thin fibers (Type II), which likely innervate outer hair cells. These axons, 0.1–0.5 μm in diameter, were distinguished from the thicker Type I, fibers innervating inner hair cells, and traced to small-cell clusters in the cochlear nucleus. This study provided two major new insights into the outer hair cell connections in the cochlear nucleus and the potential significance of very thin axons and synaptic nests, which are widespread in the CNS. 1) EM serial reconstructions of labeled and unlabeled material revealed that Type II axons rarely formed synapses with conventional features (vesicles gathered at junctions). Rather, their endings contained arrays of endoplasmic reticulum and small spherical vesicles without junctions. 2) Type II axons projected predominantly to synaptic nests, where they contacted other endings and dendrites of local interneurons (small stellate and mitt cells, but not granule cells). Synaptic nests lacked intrinsic glia and, presumably, their high-affinity amino acid transporters. As functional units, nests and their Type II inputs from outer hair cells may contribute to an analog processing mode, which is slower, more diffuse, longer-lasting, and potentially more plastic than the digital processors addressed by inner hair cells. Synapse 33:83–117, 1999. © 1999 Wiley-Liss, Inc.

The raz1 mutant of Arabidopsis thaliana lacks the activity of a high-affinity amino acid transporter

The raz1 mutant of Arabidopsis thaliana (L.) Heynh. has been selected as resistant to the toxic proline analogue, azetidine-2-carboxylic acid (2AZ). Seedlings of the mutant tolerated fivefold higher concentrations of 2AZ (ED50 = 0.25 mM) than the wild-type seedlings (EC50 = 0.05 mM). The mutant gene was found to be semi-dominant and the corresponding RAZ1 locus was mapped on chromosome 5 at 69.6 +/- 1.8 cM. The resistance to 2AZ could be fully and exclusively accounted for by the lower uptake rate of the proline analogue in the mutant. The influx of L-proline in roots of wild-type seedlings could be dissected into two components: (i) a component with a high affinity and a low capacity for L-proline (Km) approximately 20 microM, Vmax approximately 60 nmol.(g FW)-1.h-1) and also a high affinity for L-2AZ (Ki approximately 40 microM) and (ii) a low-affinity, high-capacity component (Km) approximately 5 mM: Vmax = 1300 nmol.(g FW)-1.h-1). Clearly, the raz1 mutation affects the activity of a high-affinity transporter, because the high-affinity uptake of proline in the mutant was at least fivefold lower than in the wild-type, whereas the low-affinity uptake was unchanged.

Amino acid transport in isolated hepatocytes after partial hepatectomy in the rat.

The effect of partial (70%) hepatectomy on amino acid transport was investigated by measuring the uptake of the nonmetabolizable amino acid analogue alpha-amino-[1(-14C]isobutyric acid in hepatocytes freshly isolated from the liver remnant. In hepatocytes from partially hepatectomized rats, both the influx and the efflux of alpha-aminoisobutyric acid were increased. Quantitative analysis of the relationship between the initial rate of uptake and the substrate concentration indicated that a high-affinity component was responsible for the increased rate of amino acid entry in hepatocytes from partially hepatectomized rats. This component could be detected 90 min after operation, but its Vmax increased with time. The high-affinity component was strictly dependent on sodium and had the properties of a pure A system; its mode of energization partly involved cationic transmembrane gradients. The L system of amino acid transport was not affected after partial hepatectomy. The rapid emergence of a high-affinity amino acid transport system in the liver remnant following partial hepatectomy may play an important role in the regulation of liver regeneration.

Amino acid-conferred protection against melphalan—Characterization of melphalan transport and correlation of uptake with cytotoxicity in cultured L1210 murine leukemia cells

The uptake of melphalan by L1210 cells was reduced to one-sixth of controls by physiological concentrations of the l-isomers of leucine and glutamine, and this decrease was accompanied by a corresponding decrease in cytotoxicity. Cytotoxicity was estimated by treatment of cells with melphalan for 35 min in phosphate-buffered saline containing bovine serum albumin and glucose followed by clonal growth of the surviving cells in soft agar. It was prominent within a critical region of net melphalan uptake of 2–5 pmoles/10 5 cells. Inhibition analysis revealed that at cytotoxic concentrations melphalan is transported by a high-affinity amino acid transport system of the leucine ( l) type. The values of the Michaelis constants ( K m ) for l-leucine, a protective amino acid, l-valine, a minimally protective amino acid, and melphalan were 6, 58 and 19 μM respectively. These results suggest that the ability of amino acids to protect L1210 cells from melphalan cytotoxicity is related to their affinities for the leucine carrier sites.