Transcriptome-Wide Analysis of Nitrogen-Regulated Genes in Tea Plant (Camellia sinensis L. O. Kuntze) and Characterization of Amino Acid Transporter CsCAT9.1

The vigor of tea plants (Camellia sinensis) and tea quality are strongly influenced by the abundance and forms of nitrogen, principally NO3−, NH4+, and amino acids. Mechanisms to access different nitrogen sources and the regulatory cues remain largely elusive in tea plants. A transcriptome analysis was performed to categorize differentially expressed genes (DEGs) in roots and young leaves during the early response to four nitrogen treatments. Relative to the continuously nitrogen-replete control, the three nitrogen-deprived and resupplied treatments shared 237 DEGs in the shoots and 21 DEGs in the root. Gene-ontology characterization revealed that transcripts encoding genes predicted to participate in nitrogen uptake, assimilation, and translocation were among the most differentially expressed after exposure to the different nitrogen regimes. Because of its high transcript level regardless of nitrogen condition, a putative amino acid transporter, TEA020444/CsCAT9.1, was further characterized in Arabidopsis and found to mediate the acquisition of a broad spectrum of amino acids, suggesting a role in amino acid uptake, transport, and deposition in sinks as an internal reservoir. Our results enhance our understanding of nitrogen-regulated transcript level patterns in tea plants and pinpoint candidate genes that function in nitrogen transport and metabolism, allowing tea plants to adjust to variable nitrogen environments.

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The amino acid transporter SNAT2 mediates l-proline-induced differentiation of ES cells

AJP Cell Physiology ◽

10.1152/ajpcell.00235.2010 ◽

2011 ◽

Vol 300 (6) ◽

pp. C1270-C1279 ◽

Cited By ~ 29

Author(s):

Boon Siang Nicholas Tan ◽

Ana Lonic ◽

Michael B. Morris ◽

Peter D. Rathjen ◽

Joy Rathjen

Keyword(s):

Amino Acids ◽

Cell Differentiation ◽

Amino Acid ◽

Biological Activities ◽

Es Cells ◽

Embryonic Stem ◽

Amino Acid Transporter ◽

Acid Uptake ◽

Es Cell ◽

Acid Transporter

There is an increasing appreciation that amino acids can act as signaling molecules in the regulation of cellular processes through modulation of intracellular cell signaling pathways. In culture, embryonic stem (ES) cells can be differentiated to a second, pluripotent cell population, early primitive ectoderm-like cells in response to biological activities within the conditioned medium MEDII. The amino acid l-proline has been identified as a component of MEDII required for ES cell differentiation. Here, we define the primary l-proline transporter on ES and early primitive ectoderm-like cells as sodium-coupled neutral amino acid transporter 2 (SNAT2). SNAT2 uptake of l-proline can be inhibited by the addition of millimolar concentrations of other substrates. The addition of excess amino acids was used to regulate the uptake of l-proline by ES cells, and the effect on differentiation was analyzed. The ability of SNAT2 substrates, but not other amino acids, to prevent changes in morphology, gene expression, and differentiation kinetics suggested that l-proline uptake through SNAT2 was required for ES cell differentiation. These data reveal an unexpected role for amino acid uptake and the amino acid transporter SNAT2 in regulation of pluripotent cells in culture and provides a number of specific, inexpensive, and nontoxic culture additives with the potential to improve the quality of ES cell culture.

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The Regulation and Function of the L-Type Amino Acid Transporter 1 (LAT1) in Cancer

International Journal of Molecular Sciences ◽

10.3390/ijms19082373 ◽

2018 ◽

Vol 19 (8) ◽

pp. 2373 ◽

Cited By ~ 24

Author(s):

Travis Salisbury ◽

Subha Arthur

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Cancer Cells ◽

Amino Acid Uptake ◽

Essential Amino Acids ◽

Amino Acid Transporter ◽

Amino Acid Transporters ◽

Acid Uptake ◽

Cancer Pathways ◽

Acid Transporter

The progression of cancer is associated with increases in amino acid uptake by cancer cells. Upon their entry into cells through specific transporters, exogenous amino acids are used to synthesize proteins, nucleic acids and lipids and to generate ATP. The essential amino acid leucine is also important for maintaining cancer-associated signaling pathways. By upregulating amino acid transporters, cancer cells gain greater access to exogenous amino acids to support chronic proliferation, maintain metabolic pathways, and to enhance certain signal transduction pathways. Suppressing cancer growth by targeting amino acid transporters will require an in-depth understanding of how cancer cells acquire amino acids, in particular, the transporters involved and which cancer pathways are most sensitive to amino acid deprivation. L-Type Amino Acid Transporter 1 (LAT1) mediates the uptake of essential amino acids and its expression is upregulated during the progression of several cancers. We will review the upstream regulators of LAT1 and the downstream effects caused by the overexpression of LAT1 in cancer cells.

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Cloning, Expression, and Functional Characterization of Secondary Amino Acid Transporters of Lactococcus lactis

Journal of Bacteriology ◽

10.1128/jb.01948-12 ◽

2012 ◽

Vol 195 (2) ◽

pp. 340-350 ◽

Cited By ~ 23

Author(s):

Hein Trip ◽

Niels L. Mulder ◽

Juke S. Lolkema

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Lactococcus Lactis ◽

Aromatic Amino Acids ◽

Amino Acid Transporter ◽

Amino Acid Transporters ◽

Acid Uptake ◽

Content Type ◽

Secondary Amino ◽

Acid Transporter

ABSTRACTFourteen genes encoding putative secondary amino acid transporters were identified in the genomes ofLactococcus lactissubsp.cremorisstrains MG1363 and SK11 andL. lactissubsp. lactisstrains IL1403 and KF147, 12 of which were common to all four strains. Amino acid uptake inL. lactiscells overexpressing the genes revealed transporters specific for histidine, lysine, arginine, agmatine, putrescine, aromatic amino acids, acidic amino acids, serine, and branched-chain amino acids. Substrate specificities were demonstrated by inhibition profiles determined in the presence of excesses of the other amino acids. Four knockout mutants, lacking the lysine transporter LysP, the histidine transporter HisP (formerly LysQ), the acidic amino acid transporter AcaP (YlcA), or the aromatic amino acid transporter FywP (YsjA), were constructed. The LysP, HisP, and FywP deletion mutants showed drastically decreased rates of uptake of the corresponding substrates at low concentrations. The same was observed for the AcaP mutant with aspartate but not with glutamate. In rich M17 medium, the deletion of none of the transporters affected growth. In contrast, the deletion of the HisP, AcaP, and FywP transporters did affect growth in a defined medium with free amino acids as the sole amino acid source. HisP was essential at low histidine concentrations, and AcaP was essential in the absence of glutamine. FywP appeared to play a role in retaining intracellularly synthesized aromatic amino acids when these were not added to the medium. Finally, HisP, AcaP, and FywP did not play a role in the excretion of accumulated histidine, glutamate, or phenylalanine, respectively, indicating the involvement of other transporters.

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Identification and Characterization of Inhibitors of a Neutral Amino Acid Transporter, SLC6A19, Using Two Functional Cell-Based Assays

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/2472555218794627 ◽

2018 ◽

Vol 24 (2) ◽

pp. 111-120 ◽

Cited By ~ 6

Author(s):

Sanjay J. Danthi ◽

Beirong Liang ◽

Oanh Smicker ◽

Benjamin Coupland ◽

Jill Gregory ◽

...

Keyword(s):

Amino Acid ◽

High Throughput Screening ◽

Amino Acid Transporter ◽

Neutral Amino Acid ◽

Imaging Plate ◽

Acid Uptake ◽

Functional Assays ◽

Neutral Amino Acid Transporter ◽

Acid Transporter ◽

Pharmacologically Active

SLC6A19 (B0AT1) is a neutral amino acid transporter, the loss of function of which results in Hartnup disease. SLC6A19 is also believed to have an important role in amino acid homeostasis, diabetes, and weight control. A small-molecule inhibitor of human SLC6A19 (hSLC6A19) was identified using two functional cell-based assays: a fluorescence imaging plate reader (FLIPR) membrane potential (FMP) assay and a stable isotope-labeled neutral amino acid uptake assay. A diverse collection of 3440 pharmacologically active compounds from the Microsource Spectrum and Tocriscreen collections were tested at 10 µM in both assays using MDCK cells stably expressing hSLC6A19 and its obligatory subunit, TMEM27. Compounds that inhibited SLC6A19 activity in both assays were further confirmed for activity and selectivity and characterized for potency in functional assays against hSLC6A19 and related transporters. A single compound, cinromide, was found to robustly, selectively, and reproducibly inhibit SLC6A19 in all functional assays. Structurally related analogs of cinromide were tested to demonstrate structure–activity relationship (SAR). The assays described here are suitable for carrying out high-throughput screening campaigns to identify modulators of SLC6A19.

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Neutral amino acid transporter ASCT2 displays substrate-induced Na+ exchange and a substrate-gated anion conductance

Biochemical Journal ◽

10.1042/bj3460705 ◽

2000 ◽

Vol 346 (3) ◽

pp. 705-710 ◽

Cited By ~ 58

Author(s):

Angelika BRÖER ◽

Carsten WAGNER ◽

Florian LANG ◽

Stefan BRÖER

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Anion Channel ◽

Amino Acid Transporter ◽

Neutral Amino Acid ◽

Xenopus Laevis Oocytes ◽

Anion Conductance ◽

Neutral Amino Acid Transporter ◽

Inward Currents ◽

Acid Transporter

The neutral amino acid transporter ASCT2 mediates electroneutral obligatory antiport but at the same time requires Na+ for its function. To elucidate the mechanism, ASCT2 was expressed in Xenopus laevis oocytes and transport was analysed by flux studies and two-electrode voltage clamp recordings. Flux studies with 22NaCl indicated that the uptake of one molecule of glutamine or alanine is accompanied by the uptake of four to seven Na+ ions. Similarly to the transport of amino acids, the Na+ uptake was mediated by an obligatory Na+ exchange mechanism that depended on the presence of amino acids but was not stoichiometrically coupled to the amino acid transport. Other cations could not replace Na+ in this transport mechanism. When NaCl was replaced by NaSCN in the transport buffer, the superfusion of oocytes with amino acid substrates resulted in large inward currents, indicating the presence of a substrate-gated anion channel in the ASCT2 transporter. The Km for glutamine derived from these experiments is in good agreement with the Km derived from flux studies; it varied between 40 and 90 μM at holding potentials of -60 and -20 mV respectively. The permeability of the substrate-gated anion conductance decreased in the order SCN- NO3- > I- > Cl- and also required the presence of Na+.

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Molecular Identification and Characterization of Hydroxycinnamoyl Transferase in Tea Plants (Camellia sinensis L.)

International Journal of Molecular Sciences ◽

10.3390/ijms19123938 ◽

2018 ◽

Vol 19 (12) ◽

pp. 3938 ◽

Cited By ~ 2

Author(s):

Chi-Hui Sun ◽

Chin-Ying Yang ◽

Jason Tzen

Keyword(s):

Abiotic Stress ◽

Amino Acid ◽

Camellia Sinensis ◽

Polymerase Chain Reaction Analysis ◽

Coffea Canephora ◽

Tea Plant ◽

Amino Acid Sequences ◽

Hibiscus Cannabinus ◽

Expression Level ◽

Tea Plants

Tea (Camellia sinensis L.) contains abundant secondary metabolites, which are regulated by numerous enzymes. Hydroxycinnamoyl transferase (HCT) is involved in the biosynthesis pathways of polyphenols and flavonoids, and it can catalyze the transfer of hydroxyconnamoyl coenzyme A to substrates such as quinate, flavanol glycoside, or anthocyanins, thus resulting in the production of chlorogenic acid or acylated flavonol glycoside. In this study, the CsHCT gene was cloned from the Chin-Shin Oolong tea plant, and its protein functions and characteristics were analyzed. The full-length cDNA of CsHCT contains 1311 base pairs and encodes 436 amino acid sequences. Amino acid sequence was highly conserved with other HCTs from Arabidopsis thaliana, Populus trichocarpa, Hibiscus cannabinus, and Coffea canephora. Quantitative real-time polymerase chain reaction analysis showed that CsHCT is highly expressed in the stem tissues of both tea plants and seedlings. The CsHCT expression level was relatively high at high altitudes. The abiotic stress experiment suggested that low temperature, drought, and high salinity induced CsHCT transcription. Furthermore, the results of hormone treatments indicated that abscisic acid (ABA) induced a considerable increase in the CsHCT expression level. This may be attributed to CsHCT involvement in abiotic stress and ABA signaling pathways.

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Intestinal peptidases form functional complexes with the neutral amino acid transporter B0AT1

Biochemical Journal ◽

10.1042/bj20120307 ◽

2012 ◽

Vol 446 (1) ◽

pp. 135-148 ◽

Cited By ~ 40

Author(s):

Stephen J. Fairweather ◽

Angelika Bröer ◽

Megan L. O'Mara ◽

Stefan Bröer

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Brush Border ◽

Brush Border Membrane ◽

Amino Acid Transporter ◽

The Body ◽

Site Directed Mutagenesis ◽

Substrate Affinity ◽

Xenopus Laevis Oocytes ◽

Acid Transporter

The brush-border membrane of the small intestine and kidney proximal tubule are the major sites for the absorption and re-absorption of nutrients in the body respectively. Transport of amino acids is mediated through the action of numerous secondary active transporters. In the mouse, neutral amino acids are transported by B0AT1 [broad neutral (0) amino acid transporter 1; SLC6A19 (solute carrier family 6 member 19)] in the intestine and by B0AT1 and B0AT3 (SLC6A18) in the kidney. Immunoprecipitation and Blue native electrophoresis of intestinal brush-border membrane proteins revealed that B0AT1 forms complexes with two peptidases, APN (aminopeptidase N/CD13) and ACE2 (angiotensin-converting enzyme 2). Physiological characterization of B0AT1 expressed together with these peptidases in Xenopus laevis oocytes revealed that APN increased the substrate affinity of the transporter up to 2.5-fold and also increased its surface expression (Vmax). Peptide competition experiments, in silico modelling and site-directed mutagenesis of APN suggest that the catalytic site of the peptidase is involved in the observed changes of B0AT1 apparent substrate affinity, possibly by increasing the local substrate concentration. These results provide evidence for the existence of B0AT1-containing digestive complexes in the brush-border membrane, interacting differentially with various peptidases, and responding to the dynamic needs of nutrient absorption in the intestine and kidney.

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