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

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

Biochemical Journal ◽

10.1042/bj3280657 ◽

1997 ◽

Vol 328 (2) ◽

pp. 657-664 ◽

Cited By ~ 14

Author(s):

Hiroko SEGAWA ◽

Ken-ichi MIYAMOTO ◽

Yoshio OGURA ◽

Hiromi HAGA ◽

Kyoko MORITA ◽

...

Keyword(s):

Amino Acid ◽

Xenopus Oocytes ◽

Physiological Role ◽

Basic Amino Acid ◽

Functional Expression ◽

Amino Acid Transporter ◽

Amino Acid Transporters ◽

Transport Activity ◽

Acid Transporter ◽

Mouse Ileum

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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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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l-Arginine and Cationic Amino Acid Transporter 2B Regulate Growth and Survival of Leishmania amazonensis Amastigotes in Macrophages

Infection and Immunity ◽

10.1128/iai.00026-07 ◽

2007 ◽

Vol 75 (6) ◽

pp. 2802-2810 ◽

Cited By ~ 44

Author(s):

Nanchaya Wanasen ◽

Carol L. MacLeod ◽

Lesley G. Ellies ◽

Lynn Soong

Keyword(s):

Amino Acid ◽

Amino Acid Transporter ◽

Parasite Growth ◽

Leishmania Amazonensis ◽

Growth Enhancement ◽

Cationic Amino Acid Transporter ◽

Cationic Amino Acid ◽

Arginine Transport ◽

Ifn Γ ◽

Acid Transporter

ABSTRACT Leishmania spp. are obligate intracellular parasites, requiring a suitable microenvironment for their growth within host cells. We previously reported that the growth of Leishmania amazonensis amastigotes in murine macrophages (Mφs) was enhanced in the presence of gamma interferon (IFN-γ), a Th1 cytokine normally associated with classical Mφ activation and killing of intracellular pathogens. In this study, we provided several lines of evidence suggesting that IFN-γ-mediated parasite growth enhancement was associated with l-arginine transport via mouse cationic amino acid transporter 2B (mCAT-2B). (i) mRNA expression of Slc7A2, the gene encoding for mCAT-2B, as well as l-arginine transport was increased in IFN-γ-treated Mφs. (ii) Supplementation of l-arginine in Mφ cultures increased parasite growth. (iii) Parasite growth enhancement in wild-type Mφs was inhibited in the presence of nonmetabolized l-arginine analogues. (iv) IFN-γ-mediated parasite growth was absent in Mφs derived from mCAT-2B-deficient mice. Although we detected a clear upregulation of mCAT-2B and l-arginine transport, no measurable iNOS or arginase activities were observed in IFN-γ-treated, infected Mφs. Together, these data suggest an involvement of a novel l-arginine usage independent of iNOS and arginase activities during IFN-γ-mediated parasite growth enhancement. A possible role of mCAT-2B in supplying l-arginine directly to the parasites for their proliferation is discussed.

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