scholarly journals Expression of membrane transporters in cane toad Bufo marinus oocytes

1999 ◽  
Vol 202 (16) ◽  
pp. 2217-2223
Author(s):  
D. Markovich ◽  
R.R. Regeer
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Expression System ◽  
Bufo Marinus ◽  
Membrane Transporters ◽  
Xenopus Laevis Oocytes ◽  
Amino Acid Transporters ◽  
Acid Uptake ◽  
Cane Toad ◽  
Toad Bufo

Membrane transport proteins (transporters and ion channels) have been extensively expressed in amphibian oocytes. The aims of this study were to determine whether oocytes from the cane toad Bufo marinus could be used as an alternative expression system to the broadly used Xenopus laevis oocytes. mRNAs encoding plasma membrane transporters NaSi-1 and sat-1 (sulphate transporters), NaDC-1 (dicarboxylate transporter), SGLT-1 (Na(+)/glucose cotransporter) and rBAT and 4F2 hc (amino acid transporters) were injected into B. marinus oocytes. All led to significant induction of their respective transport activities. Uptake rates were comparable with those in X. laevis oocytes, with the exception of rBAT, which was able to induce amino acid uptake only in X. laevis oocytes, suggesting that rBAT may require an endogenous X. laevis oocyte protein that is absent from B. marinus oocytes. Transport kinetics were determined for the NaSi-1 cotransporter in B. marinus oocytes, with identical results to those obtained in X. laevis oocytes. NaSi-1 specificity for the Na(+) cation was determined, and the anions selenate, molybdate, tungstate, oxalate and thiosulphate could all inhibit NaSi-1-induced sulphate transport. This study demonstrates that cane toad oocytes can be used successfully to express plasma membrane proteins, making this a viable heterologous system for the expression of proteins.

scholarly journals Expression of solute carrier 7A4 (SLC7A4) in the plasma membrane is not sufficient to mediate amino acid transport activity

2002 ◽  
Vol 364 (3) ◽  
pp. 767-775 ◽  
Author(s):  
Sabine WOLF ◽  
Annette JANZEN ◽  
Nicole VÉKONY ◽  
Ursula MARTINÉ ◽  
Dennis STRAND ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Protein Expression ◽  
Amino Acid Transport ◽  
Fluorescent Protein ◽  
Xenopus Laevis Oocytes ◽  
Amino Acid Transporters ◽  
Acid Transport ◽  
Transport Activity ◽  
Solute Carrier

Member 4 of human solute carrier family 7 (SLC7A4) exhibits significant sequence homology with the SLC7 subfamily of human cationic amino acid transporters (hCATs) [Sperandeo, Borsani, Incerti, Zollo, Rossi, Zuffardi, Castaldo, Taglialatela, Andria and Sebastio (1998) Genomics 49, 230–236]. It is therefore often referred to as hCAT-4 even though no convincing transport activity has been shown for this protein. We expressed SLC7A4 in Xenopus laevis oocytes, but could not detect any transport activity for cationic, neutral or anionic amino acids or for the polyamine putrescine. In addition, human glioblastoma cells stably overexpressing a fusion protein between SLC7A4 and the enhanced green fluorescent protein (EGFP) did not exhibit an increased transport activity for l-arginine. The lack of transport activity was not due to a lack of SLC7A4 protein expression in the plasma membrane, as in both cell types SLC7A4-EGFP exhibited a similar subcellular localization and level of protein expression as functional hCAT-EGFP proteins. The expression of SLC7A4 can be induced in NT2 teratocarcinoma cells by treatment with retinoic acid. However, also for this endogenously expressed SLC7A4, we could not detect any transport activity for l-arginine. Our data demonstrate that the expression of SLC7A4 in the plasma membrane is not sufficient to induce an amino acid transport activity in X. laevis oocytes or human cells. Therefore, SLC7A4 is either not an amino acid transporter or it needs additional (protein) factor(s) to be functional.

Is gamma-actin a regulator of amino acid transport?

1996 ◽  
Vol 270 (6) ◽  
pp. C1647-C1655 ◽  
Author(s):  
G. Lin ◽  
J. I. McCormick ◽  
R. M. Johnstone
Keyword(s):  
Amino Acid ◽  
Cell Line ◽  
Amino Acid Transport ◽  
Normal Growth ◽  
Amino Acid Uptake ◽  
Xenopus Laevis Oocytes ◽  
Amino Acid Transporters ◽  
Acid Uptake ◽  
Acid Transport ◽  
Dependent Transport

A mutated yeast cell line incapable of growth in minimal medium with proline as the sole nitrogen source was restored to normal growth by transfection with a cDNA from mouse Ehrlich cells. The cloned cDNA (E51) was found to be 90% homologous to gamma-actin. Immediately after transfection with E51 cDNA, both alpha-aminoisobutyric acid (AIB) and proline uptake in the mutated yeast were increased, particularly at pH 5. The expression of the same E51 cDNA also enhanced amino acid uptake in Xenopus laevis oocytes after injection into the Xenopus nuclei. A mutated mammalian lymphocyte cell line (GF-17), deficient in system A transport, also showed increased Na(+)-dependent transport after transfection with E51 cDNA. Whereas the mock transfected GF-17 cells failed to grow in the selection medium, the transfectants with E51 cDNA grew better than the untransfected cells. The data are consistent with the conclusion that expression of E51 cDNA can modify inactive, endogenous amino acid transporters, permitting substantial amino acid uptake in cells deficient in amino acid transporter(s) and permitting rapid cell growth. The data suggest that the gamma-actin-like protein coded for by E51 cDNA may play a significant regulatory role in amino acid transport.

scholarly journals The Heavy Chain 4F2hc Modulates the Substrate Affinity and Specificity of the Light Chains LAT1 and LAT2

2020 ◽  
Vol 21 (20) ◽  
pp. 7573
Author(s):  
Satish Kantipudi ◽  
Jean-Marc Jeckelmann ◽  
Zöhre Ucurum ◽  
Patrick D. Bosshart ◽  
Dimitrios Fotiadis
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Heavy Chain ◽  
Mammalian Cells ◽  
Plasma Membranes ◽  
Expression System ◽  
Functional Characterization ◽  
Light Chains ◽  
Substrate Affinity ◽  
Amino Acid Transporters

The human L-type amino acid transporters LAT1 and LAT2 mediate the transport of amino acids and amino acid derivatives across plasma membranes in a sodium-independent, obligatory antiport mode. In mammalian cells, LAT1 and LAT2 associate with the type-II membrane N-glycoprotein 4F2hc to form heteromeric amino acid transporters (HATs). The glycosylated ancillary protein 4F2hc is known to be important for successful trafficking of the unglycosylated transporters to the plasma membrane. The heavy (i.e., 4F2hc) and light (i.e., LAT1 and LAT2) chains belong to the solute carrier (SLC) families SLC3 and SLC7, and are covalently linked by a conserved disulfide bridge. Overexpression, absence, or malfunction of certain HATs is associated with human diseases and HATs are therefore considered therapeutic targets. Here, we present a comparative, functional characterization of the HATs 4F2hc-LAT1 and 4F2hc-LAT2, and their light chains LAT1 and LAT2. For this purpose, the HATs and the light chains were expressed in the methylotrophic yeast Pichia pastoris and a radiolabel transport assay was established. Importantly and in contrast to mammalian cells, P. pastoris has proven useful as eukaryotic expression system to successfully express human LAT1 and LAT2 in the plasma membrane without the requirement of co-expressed trafficking chaperone 4F2hc. Our results show a novel function of the heavy chain 4F2hc that impacts transport by modulating the substrate affinity and specificity of corresponding LATs. In addition, the presented data confirm that the light chains LAT1 and LAT2 constitute the substrate-transporting subunits of the HATs, and that light chains are also functional in the absence of the ancillary protein 4F2hc.

Structural domains involved in substrate selectivity in two neutral amino acid transporters

2004 ◽  
Vol 287 (3) ◽  
pp. C754-C761 ◽  
Author(s):  
Andrea Soragna ◽  
Stefania Anna Mari ◽  
Rossana Pisani ◽  
Antonio Peres ◽  
Michela Castagna ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Uptake ◽  
Transmembrane Domains ◽  
Neutral Amino Acid ◽  
Xenopus Laevis Oocytes ◽  
Amino Acid Transporters ◽  
Acid Uptake ◽  
Substrate Selectivity ◽  
Organic Substrates ◽  
Structural Determinants

The ability of the two highly homologous Na+/Cl−-dependent neutral amino acid transporters KAAT1 and CAATCH1, cloned from the midgut epithelium of the larva Manduca sexta, to transport different amino acids depends on the cotransported ion, on pH, and on the membrane voltage. Different organic substrates give rise to transport-associated currents with their own characteristics, which are notably distinct between the two proteins. Differences in amplitude, kinetics, and voltage dependence of the transport-associated currents have been observed, as well as different substrate selectivity patterns measured by radioactive amino acid uptake assays. These diversities represent useful tools to investigate the structural determinants involved in the substrate selectivity. To identify these regions, we built four chimeric proteins between the two transporters. These proteins, heterologously expressed in Xenopus laevis oocytes, were analyzed by two-electrode voltage clamp and uptake measurements. Initially, we exchanged the first three domains, obtaining the chimeras C3K9 and K3C9 (where numbers indicate the transmembrane domains and letters represent the original proteins), which showed electrophysiological and [3H]amino acid uptake characteristics resembling those of KAAT1 and CAATCH1, respectively. Subsequent substitution of the last four domains in C3K9 and K3C9 gave the proteins C3K5C4 and K3C5K4, which showed the same behavior as KAAT1 and CAATCH1 in electrophysiological and transport determinations. These results suggest that in KAAT1 and CAATCH1, only the central transmembrane domains (from 4 to 8) of the protein are responsible for substrate selectivity.

scholarly journals Transport of a neurotoxicant by molecular mimicry: the methylmercury–l-cysteine complex is a substrate for human L-type large neutral amino acid transporter (LAT) 1 and LAT2

2002 ◽  
Vol 367 (1) ◽  
pp. 239-246 ◽  
Author(s):  
Tracey A. SIMMONS-WILLIS ◽  
Albert S. KOH ◽  
Thomas W. CLARKSON ◽  
Nazzareno BALLATORI
Keyword(s):  
Amino Acid ◽  
Metal Ion ◽  
Molecular Mimicry ◽  
Neutral Amino Acid ◽  
Xenopus Laevis Oocytes ◽  
Amino Acid Transporters ◽  
Acid Uptake ◽  
Target Tissue ◽  
Large Neutral Amino Acid ◽  
Simple Diffusion

Methylmercury (MeHg) readily crosses cell membrane barriers to reach its target tissue, the brain. Although it is generally assumed that this rapid transport is due to simple diffusion, recent studies have demonstrated that MeHg is transported as a hydrophilic complex, and possibly as an l-cysteine complex on the ubiquitous L-type large neutral amino acid transporters (LATs). To test this hypothesis, studies were carried out in Xenopus laevis oocytes expressing two of the major L-type carriers in humans, LAT1—4F2 heavy chain (4F2hc) and LAT2—4F2hc. Oocytes expressing LAT1—4F2hc or LAT2—4F2hc demonstrated enhanced uptake of [14C]MeHg when administered as the l-cysteine or d,l-homocysteine complexes, but not when administered as the d-cysteine, N-acetyl-l-cysteine, penicillamine or GSH complexes. Kinetic analysis of transport indicated that the apparent affinities (Km) of MeHg—l-cysteine uptake by LAT1 and LAT2 (98±8 and 64±8μM respectively) were comparable with those for methionine (99±9 and 161±11μM), whereas the Vmax values were higher for MeHg—l-cysteine, indicating that it may be a better substrate than the endogenous amino acid. Uptake and efflux of [3H]methionine and [14C]MeHg—l-cysteine were trans-stimulated by leucine and phenylalanine, but not by glutamate, indicating that MeHg—l-cysteine is both a cis- and trans-substrate. In addition, [3H]methionine efflux was trans-stimulated by leucine and phenylalanine even in the presence of an inwardly directed methionine gradient, demonstrating concentrative transport by both LAT1 and LAT2. The present results describe a major molecular mechanism by which MeHg is transported across cell membranes and indicate that metal complexes may form a novel class of substrates for amino acid carriers. These transport proteins may therefore participate in metal ion homoeostasis and toxicity.

scholarly journals Amino Acid Transporters on the Guard of Cell Genome and Epigenome

Cancers ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 125
Author(s):  
Uğur Kahya ◽  
Ayşe Sedef Köseer ◽  
Anna Dubrovska
Keyword(s):  
Amino Acid ◽  
Cancer Metabolism ◽  
Tumor Imaging ◽  
Redox Homeostasis ◽  
Amino Acid Uptake ◽  
Metabolic Reprogramming ◽  
Tumor Evolution ◽  
Amino Acid Transporters ◽  
Acid Uptake ◽  
Growth And Survival

Tumorigenesis is driven by metabolic reprogramming. Oncogenic mutations and epigenetic alterations that cause metabolic rewiring may also upregulate the reactive oxygen species (ROS). Precise regulation of the intracellular ROS levels is critical for tumor cell growth and survival. High ROS production leads to the damage of vital macromolecules, such as DNA, proteins, and lipids, causing genomic instability and further tumor evolution. One of the hallmarks of cancer metabolism is deregulated amino acid uptake. In fast-growing tumors, amino acids are not only the source of energy and building intermediates but also critical regulators of redox homeostasis. Amino acid uptake regulates the intracellular glutathione (GSH) levels, endoplasmic reticulum stress, unfolded protein response signaling, mTOR-mediated antioxidant defense, and epigenetic adaptations of tumor cells to oxidative stress. This review summarizes the role of amino acid transporters as the defender of tumor antioxidant system and genome integrity and discusses them as promising therapeutic targets and tumor imaging tools.

Z and W sex chromosomes in the cane toad (Bufo marinus)

2009 ◽  
Vol 17 (8) ◽  
pp. 1015-1024 ◽  
Author(s):  
John Abramyan ◽  
Tariq Ezaz ◽  
Jennifer A. Marshall Graves ◽  
Peter Koopman
Keyword(s):  
Sex Chromosomes ◽  
Bufo Marinus ◽  
Cane Toad ◽  
Toad Bufo

scholarly journals Interaction of Excitatory Amino Acid Transporters 1 – 3 (EAAT1, EAAT2, EAAT3) with N-Carbamoylglutamate and N-Acetylglutamate

2017 ◽  
Vol 43 (5) ◽  
pp. 1907-1916 ◽  
Author(s):  
Birgitta C. Burckhardt ◽  
Gerhard Burckhardt
Keyword(s):  
Amino Acid ◽  
Excitatory Amino Acid ◽  
Hepatic Uptake ◽  
Xenopus Laevis Oocytes ◽  
Amino Acid Transporters ◽  
Synthetic Analog ◽  
Excitatory Amino Acid Transporters ◽  
Sodium Dependent ◽  
Inward Currents ◽  
Acetylglutamate Synthase

Background/Aims: Inborn deficiency of the N-acetylglutamate synthase (NAGS) impairs the urea cycle and causes neurotoxic hyperammonemia. Oral administration of N-carbamoylglutamate (NCG), a synthetic analog of N-acetylglutamate (NAG), successfully decreases plasma ammonia levels in the affected children. Due to structural similarities to glutamate, NCG may be absorbed in the intestine and taken up into the liver by excitatory amino acid transporters (EAATs). Methods: Using Xenopus laevis oocytes expressing either human EAAT1, 2, or 3, or human sodium-dependent dicarboxylate transporter 3 (NaDC3), transport-associated currents of NAG, NCG, and related dicarboxylates were assayed. Results: L-aspartate and L-glutamate produced saturable inward currents with Km values below 30 µM. Whereas NCG induced a small inward current only in EAAT3 expressing oocytes, NAG was accepted by all EAATs. With EAAT3, the NAG-induced current was sodium-dependent and saturable (Km 409 µM). Oxaloacetate was found as an additional substrate of EAAT3. In NaDC3-expressing oocytes, all dicarboxylates induced much larger inward currents than did L-aspartate and L-glutamate. Conclusion: EAAT3 may contribute to intestinal absorption and hepatic uptake of NCG. With respect to transport of amino acids and dicarboxylates, EAAT3 and NaDC3 can complement each other.

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