Cloning, functional expression and dietary regulation of the mouse neutral and basic amino acid transporter (NBAT)

The Na+-independent dibasic and neutral amino acid transporter NBAT is among the least hydrophobic of mammalian amino acid transporters. The transporter contains one to four transmembrane domains and induces amino acid transport activity via a b0,+-like system when expressed in Xenopus oocytes. However, the physiological role of NBAT remains unclear. Complementary DNA clones encoding mouse NBAT have now been isolated. The expression of mouse NBAT in Xenopus oocytes also induced an obligatory amino acid exchange activity similar to that of the b0,+-like system. The amount of NBAT mRNA in mouse kidney increased during postnatal development, consistent with the increase in renal cystine and dibasic transport activity. Dietary aspartate induced a marked increase in cystine transport via the b0,+ system in mouse ileum. A high-aspartate diet also increased the amount of NBAT mRNA in mouse ileum. In the ileum of mice fed on the aspartate diet, the extent of cystine transport was further increased by preloading brush border membrane vesicles with lysine. Hybrid depletion of NBAT mRNA from ileal polyadenylated RNA revealed that the increase in cystine transport activity induced by the high-aspartate diet, as measured in Xenopus oocytes, was attributable to NBAT. These results demonstrate that mouse NBAT has an important role in cystine transport.

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Multiple components of arginine and phenylalanine transport induced in neutral and basic amino acid transporter-cRNA-injected Xenopus oocytes

Biochemical Journal ◽

10.1042/bj3180915 ◽

1996 ◽

Vol 318 (3) ◽

pp. 915-922 ◽

Cited By ~ 13

Author(s):

George J PETER ◽

Iain G. DAVIDSON ◽

Aamir AHMED ◽

Lynn McILROY ◽

Alexander R. FORRESTER ◽

...

Keyword(s):

Amino Acid ◽

Xenopus Oocytes ◽

Competitive Inhibition ◽

Protein Complexes ◽

Basic Amino Acid ◽

Amino Acid Transporter ◽

Transport Processes ◽

Arginine Transport ◽

Acid Transporter ◽

Phenylalanine Transport

The induced uptakes of l-[3H]phenylalanine and l-[3H]arginine in oocytes injected with clonal NBAT (neutral and basic amino acid transporter) cRNA show differential inactivation by pre-treatment with N-ethylmaleimide (NEM), revealing at least two distinct transport processes. NEM-resistant arginine transport is inhibited by leucine and phenylalanine but not by alanine or valine; mutual competitive inhibition of NEM-resistant uptake of arginine and phenylalanine indicates that the two amino acids share a single transporter. NEM-senstive arginine transport is inhibited by leucine, phenylalanine, alanine and valine. At least two NEM-sensitive transporters may be expressed because we have been unable to confirm mutual competitive inhibition between arginine and phenylalanine transport. The NEM-resistant transport mechanism appears to involve distinct but overlapping binding sites for cationic and zwitterionic substrates. NBAT is known to form oligomeric protein complexes in cell membranes, and its functional roles when expressed in Xenopus oocytes may include interaction with oocyte proteins, leading to increased native amino acid transport activities; these resemble NBAT-expressed activities in terms of NEM-sensitivity and apparent substrate range (including an unusual inhibition by β-phenylalanine).

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Mutations of the basic amino acid transporter gene associated with cystinuria

Biochemical Journal ◽

10.1042/bj3100951 ◽

1995 ◽

Vol 310 (3) ◽

pp. 951-955 ◽

Cited By ~ 30

Author(s):

K Miyamoto ◽

K Katai ◽

S Tatsumi ◽

K Sone ◽

H Segawa ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Transport ◽

Basic Amino Acid ◽

Amino Acid Transporter ◽

Regulatory Subunit ◽

Acid Transport ◽

Transport Activity ◽

Wild Type ◽

Neutral Amino Acid Transporter ◽

Acid Transporter

To investigate the function of a basic and neutral amino acid transporter-like protein (rBAT) which is a candidate gene for cystinuria, we analysed the rBAT gene in cystinuric patients. Patient 1 is a compound heterozygote with mutations in the rBAT gene causing a glutamine-to-lysine transition at amino acid 268, and a threonine-to-alanine transition at amino acid 341, who inherited these alleles from his mother (E268K) and father (T341A), respectively. Injection of T341A and E268K mutant cRNAs into oocytes decreased transport activity to 53.9% and 62.5% of control (L-cystine transport activity in oocytes injected with wild-type rBAT cRNA), respectively. Co-injection of E268K and T341A into oocytes strongly decreased amino acid transport activity to 28% of control. On the other hand, co-injection of wild-type and mutant rBAT did not decrease transport activity. Furthermore, immunological studies have demonstrated that the reduction of amino acid transport is not due to a decrease in the amount of rBAT protein expressed in oocyte membranes. These results indicate that mutations in the rBAT gene are crucial disease-causing lesions in cystinuria. In addition, co-injection experiments suggest that rBAT may function as a transport activator or regulatory subunit by homo- or hetero-multimer complex formation.

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B0AT1 amino acid transporter complexed with SARS-CoV-2 receptor ACE2 forms a heterodimer functional unit: in situ conformation using radiation inactivation analysis

Function ◽

10.1093/function/zqab027 ◽

2021 ◽

Author(s):

Bruce R Stevens ◽

J Clive Ellory ◽

Robert L Preston

Keyword(s):

Amino Acid ◽

Membrane Trafficking ◽

Functional Unit ◽

Membrane Vesicles ◽

High Energy ◽

Amino Acid Transporter ◽

Neutral Amino Acid ◽

Transport Activity ◽

Acid Transporter

Abstract The SARS-CoV-2 receptor, Angiotensin Converting Enzyme-2 (ACE2), is expressed at levels of greatest magnitude in the small intestine as compared to all other human tissues. Enterocyte ACE2 is co-expressed as the apical membrane trafficking partner obligatory for expression and activity of the B0AT1 sodium-dependent neutral amino acid transporter. These components are assembled as an [ACE2: B0AT1]2 dimer-of-heterodimers quaternary complex that putatively steers SARS-CoV-2 tropism in the gastrointestinal (GI) tract. GI clinical symptomology is reported in about half of COVID-19 patients, and can be accompanied by gut shedding of virion particles. We hypothesized that within this 4-mer structural complex, each [ACE2: B0AT1] heterodimer pair constitutes a physiological “functional unit.” This was confirmed experimentally by employing purified lyophilized enterocyte brush border membrane vesicles that were exposed to increasing doses of high-energy electron radiation from a 16 MeV linear accelerator. Based on established target theory, the results indicated the presence of Na+-dependent neutral amino acid influx transport activity functional unit with target size mw = 183.7 ± 16.8 kDa in situ in intact apical membranes. Each thermodynamically stabilized [ACE2: B0AT1] heterodimer functional unit manifests the transport activity within the whole ∼345 kDa [ACE2: B0AT1]2 dimer-of-heterodimers quaternary structural complex. The results are consistent with our prior molecular docking modeling and gut-lung axis approaches to understanding COVID-19. These findings advance the understanding of the physiology of B0AT1 interaction with ACE2 in the gut, and thereby potentially contribute to translational developments designed to treat or mitigate COVID-19 variant outbreaks and/or GI symptom persistence in long-haul Post-Acute Sequelae of SARS-CoV-2 (PASC).

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Pichia pastoris and the Recombinant Human Heterodimeric Amino Acid Transporter 4F2hc-LAT1: From Clone Selection to Pure Protein

Methods and Protocols ◽

10.3390/mps4030051 ◽

2021 ◽

Vol 4 (3) ◽

pp. 51

Author(s):

Satish Kantipudi ◽

Daniel Harder ◽

Sara Bonetti ◽

Dimitrios Fotiadis ◽

Jean-Marc Jeckelmann

Keyword(s):

Amino Acid ◽

Pichia Pastoris ◽

Protein Complexes ◽

Amino Acid Transporter ◽

Amino Acid Transporters ◽

Expression Host ◽

Amino Acids And Derivatives ◽

Heterodimeric Complex ◽

Acid Transporter ◽

And Function

Heterodimeric amino acid transporters (HATs) are protein complexes composed of two subunits, a heavy and a light subunit belonging to the solute carrier (SLC) families SLC3 and SLC7. HATs transport amino acids and derivatives thereof across the plasma membrane. The human HAT 4F2hc-LAT1 is composed of the type-II membrane N-glycoprotein 4F2hc (SLC3A2) and the L-type amino acid transporter LAT1 (SLC7A5). 4F2hc-LAT1 is medically relevant, and its dysfunction and overexpression are associated with autism and tumor progression. Here, we provide a general applicable protocol on how to screen for the best membrane transport protein-expressing clone in terms of protein amount and function using Pichia pastoris as expression host. Furthermore, we describe an overexpression and purification procedure for the production of the HAT 4F2hc-LAT1. The isolated heterodimeric complex is pure, correctly assembled, stable, binds the substrate L-leucine, and is thus properly folded. Therefore, this Pichia pastoris-derived recombinant human 4F2hc-LAT1 sample can be used for downstream biochemical and biophysical characterizations.

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Estradiol inhibits the activity of proton-coupled amino acid transporter PAT1 expressed in Xenopus oocytes

European Journal of Pharmacology ◽

10.1016/j.ejphar.2012.08.006 ◽

2012 ◽

Vol 695 (1-3) ◽

pp. 34-39

Author(s):

Linlin Shan ◽

Yujie Yang ◽

Jin Wang ◽

Ji Zuo ◽

Xianhong Dong ◽

...

Keyword(s):

Amino Acid ◽

Xenopus Oocytes ◽

Amino Acid Transporter ◽

Acid Transporter

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Characterization of the rat neutral and basic amino acid transporter utilizing anti-peptide antibodies.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.90.9.4022 ◽

1993 ◽

Vol 90 (9) ◽

pp. 4022-4026 ◽

Cited By ~ 18

Author(s):

R. Mosckovitz ◽

N. Yan ◽

E. Heimer ◽

A. Felix ◽

S. S. Tate ◽

...

Keyword(s):

Amino Acid ◽

Basic Amino Acid ◽

Amino Acid Transporter ◽

Acid Transporter ◽

Peptide Antibodies

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Downregulation of Placental Amino Acid Transporter Expression and mTORC1 Signaling Activity Contributes to Fetal Growth Retardation in Diabetic Rats

International Journal of Molecular Sciences ◽

10.3390/ijms21051849 ◽

2020 ◽

Vol 21 (5) ◽

pp. 1849

Author(s):

Jie Xu ◽

Jiao Wang ◽

Yang Cao ◽

Xiaotong Jia ◽

Yujia Huang ◽

...

Keyword(s):

Amino Acid ◽

Rat Model ◽

Amino Acid Transporter ◽

Amino Acid Transporters ◽

Amino Acid Transport System ◽

Intrauterine Growth ◽

System L ◽

Acid Transporter ◽

Transporter Expression

Alterations in placental transport may contribute to abnormal fetal intrauterine growth in pregnancies complicated by diabetes, but it is not clear whether the placental amino acid transport system is altered in diabetic pregnancies. We therefore studied the changes in the expressions of placental amino acid transporters in a rat model of diabetes induced by streptozotocin, and tested the effects of hyperglycemia on trophoblast amino acid transporter in vitro. Our results showed that the expressions for key isoforms of system L amino acid transporters were significantly reduced in the placentas of streptozotocin-induced diabetic pregnant rats, which was associated with the decreased birthweight in the rats. A decreased placental efficiency and decreased placental mammalian target of rapamycin (mTOR) complex 1 (mTORC1) activity were also found in the rat model. In addition, hyperglycemia in vitro could inhibit amino acid transporter expression and mTORC1 activity in human trophoblast. Inhibition of mTORC1 activity led to reduced amino acid transporter expression in placental trophoblast. We concluded that reduced placental mTORC1 activity during pregnancy resulted in decreased placental amino acid transporter expression and, subsequently, contributed to fetal intrauterine growth restriction in pregnancies complicated with diabetes.

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Amino Acid Transport Defects in Human Inherited Metabolic Disorders

International Journal of Molecular Sciences ◽

10.3390/ijms21010119 ◽

2019 ◽

Vol 21 (1) ◽

pp. 119 ◽

Cited By ~ 3

Author(s):

Raquel Yahyaoui ◽

Javier Pérez-Frías

Keyword(s):

Amino Acid ◽

Metabolic Disorders ◽

Strong Association ◽

Cell Types ◽

Amino Acid Transporter ◽

Molecular Defect ◽

Amino Acid Transporters ◽

Inherited Disorders ◽

Laboratory Findings ◽

Acid Transporter

Amino acid transporters play very important roles in nutrient uptake, neurotransmitter recycling, protein synthesis, gene expression, cell redox balance, cell signaling, and regulation of cell volume. With regard to transporters that are closely connected to metabolism, amino acid transporter-associated diseases are linked to metabolic disorders, particularly when they involve different organs, cell types, or cell compartments. To date, 65 different human solute carrier (SLC) families and more than 400 transporter genes have been identified, including 11 that are known to include amino acid transporters. This review intends to summarize and update all the conditions in which a strong association has been found between an amino acid transporter and an inherited metabolic disorder. Many of these inherited disorders have been identified in recent years. In this work, the physiological functions of amino acid transporters will be described by the inherited diseases that arise from transporter impairment. The pathogenesis, clinical phenotype, laboratory findings, diagnosis, genetics, and treatment of these disorders are also briefly described. Appropriate clinical and diagnostic characterization of the underlying molecular defect may give patients the opportunity to avail themselves of appropriate therapeutic options in the future.

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