scholarly journals The 4F2hc surface antigen is necessary for expression of system L-like neutral amino acid-transport activity in C6-BU-1 rat glioma cells: evidence from expression studies in Xenopus laevis oocytes

1995 ◽  
Vol 312 (3) ◽  
pp. 863-870 ◽  
Author(s):  
S Bröer ◽  
A Bröer ◽  
B Hamprecht
Keyword(s):  
Amino Acid ◽  
Xenopus Laevis ◽  
Amino Acid Transport ◽  
Glioma Cells ◽  
Xenopus Laevis Oocytes ◽  
Acid Transport ◽  
Transport Activity ◽  
Rat Glioma ◽  
Polyadenylated Rna ◽  
System L

Mammalian cells possess a variety of amino acid-transport systems with overlapping substrate specificity. System L is one of the major amino acid-transport systems in all non-epithelial cells. Its molecular structure is not known. To clone the neutral amino acid-transporter system L, we followed an expression cloning strategy using Xenopus laevis oocytes. A cDNA library derived from C6-BU-1 rat glioma cells was used as a source, because high expression of system L activity could be demonstrated with polyadenylated RNA isolated from these cells, when injected into Xenopus laevis oocytes [Bröer, Bröer and Hamprecht (1994) Biochim. Biophys. Acta 1192, 95-100]. A single clone (ILAT) was identified, the sense cRNA of which, on injection into Xenopus laevis oocytes, stimulated sodium-independent isoleucine transport by about 100-fold. Further characterization revealed that transport of cationic amino acids was also stimulated. Sequencing of the cDNA showed that the identified clone is the heavy chain of the rat 4F2 surface antigen, a marker of tumour cells and activated lymphocytes. Uptake of neutral and cationic amino acids was not stimulated by the presence of Na+ ions. Antisense cRNA transcribed from this clone or antisense oligonucleotides, when co-injected with polyadenylated RNA from C6-BU-1 rat glioma cells, completely suppressed system L-like isoleucine-transport activity. We conclude that ILAT is necessary for expression of system L-like amino acid-transport activity by polyadenylated RNA from C6-BU-1 rat glioma cells.

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.

scholarly journals A rapid method for the functional reconstitution of amino acid transport systems from rat liver plasma membranes. Partial purification of System A

1988 ◽  
Vol 255 (3) ◽  
pp. 963-969 ◽  
Author(s):  
A R Quesada ◽  
J D McGivan
Keyword(s):  
Amino Acid ◽  
Rapid Method ◽  
Amino Acid Transport ◽  
Plasma Membranes ◽  
Transport Systems ◽  
Partial Purification ◽  
Acid Transport ◽  
Transport Activity ◽  
System A ◽  
System L

A rapid method for the functional reconstruction of amino acid transport from liver plasma-membrane vesicles using the neutral detergent decanoyl-N-glucamide (‘MEGA-10’) is described. The method is a modification of that previously employed in this laboratory for reconstitution of amino acid transport systems from kidney brush-border membranes [Lynch & McGivan (1987) Biochem. J. 244, 503-508]. The transport activities termed ‘System A’, ‘System N’, and ‘System L’ are all reconstituted. The reconstitution procedure is rapid and efficient and is suitable as an assay for transport activity in studies involving membrane fractionation. By using this reconstitution procedure, System A transport activity was partially purified by lectin-affinity chromatography.

Amino-acid-dependent modulation of amino acid transport in Xenopus laevis oocytes.

1996 ◽  
Vol 199 (4) ◽  
pp. 923-931 ◽  
Author(s):  
P M Taylor ◽  
S Kaur ◽  
B Mackenzie ◽  
G J Peter
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Xenopus Laevis ◽  
Amino Acid Transport ◽  
Acid Mixture ◽  
Xenopus Laevis Oocytes ◽  
Amino Acid Transporters ◽  
Amino Acid Supplementation ◽  
Acid Transport ◽  
Acid Supplementation

We have measured rates of uptake of arginine, glutamine, glutamate, serine, phenylalanine and glycine in Xenopus laevis oocytes cultured for periods of up to 24h in saline in the presence or absence of a mixture of 20 amino acids at concentrations approximating those in Xenopus plasma. Amino acid supplementation increased the total intracellular amino acid concentration from 8.2 to 18.4 nmol per oocyte. Specific Na(+)-dependent amino acid transporters (systems B0,+, Xag-) exhibit 'adaptive regulation' (up-regulation during amino acid deprivation and down-regulation during amino acid supplementation). Na(+)-independent transporters of glutamate, glutamine and glycine (including system asc) display an opposite modulation in activity, which may help to combat amino-acid-induced oxidative stress by increasing the supply of glutathione precursors. Single amino acids at physiological plasma concentrations (0.47 mmol l-1 L-alanine, 0.08 mmol l-1 L-glutamate) mimicked at least some effects of the amino acid mixture. The mechanisms of transport modulation do not appear to include trans-amino acid or membrane potential effects and, in the case of Na(+)-independent transport, are independent of protein or mRNA synthesis. Furthermore, activation of protein kinase C by phorbol 12-myristate 13-acetate did not significantly affect endogenous glutamine and glutamate transport. The Xenopus oocyte appears to possess endogenous signalling mechanisms for selectively modulating the activity of amino acid transport proteins expressed in its surface membranes, a factor for consideration when using oocytes as an expression system for structure-function studies of cloned amino acid transporters.

scholarly journals Molecular cloning of the mouse IMINO system: an Na+- and Cl−-dependent proline transporter

2005 ◽  
Vol 386 (3) ◽  
pp. 417-422 ◽  
Author(s):  
Sonja KOWALCZUK ◽  
Angelika BRÖER ◽  
Michael MUNZINGER ◽  
Nadine TIETZE ◽  
Karin KLINGEL ◽  
...  
Keyword(s):  
Small Intestine ◽  
Amino Acid ◽  
Epithelial Cells ◽  
Xenopus Laevis ◽  
Molecular Cloning ◽  
Amino Acid Transport ◽  
Tissue Expression ◽  
Xenopus Laevis Oocytes ◽  
Acid Transport ◽  
Slc6 Family

Neurotransmitter transporters of the SLC6 family play an important role in the removal of neurotransmitters in brain tissue and in amino acid transport in epithelial cells. Here we demonstrate that the mouse homologue of slc6a20 has all properties of the long-sought IMINO system. The mouse has two homologues corresponding to the single human SLC6A20 gene: these have been named XT3 and XT3s1. Expression of mouse XT3s1, but not XT3, in Xenopus laevis oocytes induced an electrogenic Na+-and-Cl−-dependent transporter for proline, hydroxyproline, betaine, N-methylaminoisobutyric acid and pipecolic acid. Expression of XT3s1 was found in brain, kidney, small intestine, thymus, spleen and lung, whereas XT3 prevailed in kidney and lung. Accordingly we suggest that the two homologues be termed ‘XT3s1 IMINOB’ and ‘XT3 IMINOK’ to indicate the tissue expression of the two genes.

scholarly journals Expression of the surface antigen 4F2hc affects system-L-like neutral-amino-acid-transport activity in mammalian cells

1997 ◽  
Vol 324 (2) ◽  
pp. 535-541 ◽  
Author(s):  
Stefan BRÖER ◽  
Angelika BRÖER ◽  
Bernd HAMPRECHT
Keyword(s):  
Amino Acid ◽  
Surface Antigen ◽  
Amino Acid Transport ◽  
Cho Cells ◽  
Mammalian Cells ◽  
Primary Cultures ◽  
Transport Systems ◽  
Acid Transport ◽  
Transport Activity ◽  
System L

Mammalian cells possess a variety of amino acid-transport systems with overlapping substrate specificity. System L is one of the major amino acid-transport systems of non-epithelial cells. By expression cloning we have recently demonstrated that the surface antigen 4F2hc (CD98) is a necessary component for expression of system-L-like amino acid-transport activity in C6-BU-1 rat glioma cells [Bröer, Bröer and Hamprecht (1995) Biochem. J. 312, 863–870]. 4F2hc mRNA was detected in CHO cells, COS cells, activated lymphocytes isolated from mouse spleen and primary cultures of astrocytes. In all these cell types, Na+-independent isoleucine transport was mediated by system L. No contribution of system y+L to isoleucine or arginine transport was detected in C6-BU-1 cells. In lymphocytes, both system-L-like amino acid-transport activity and 4F2hc mRNA levels increased after treatment with phorbol ester plus ionomycin. Antisense oligonucleotides caused modest inhibition of Na+-independent isoleucine transport in C6-BU-1 cells and primary cultures of astroglial cells, whereas arginine transport was unaffected. Overexpression of 4F2hc cDNA in CHO cells resulted in an increase in Na+-independent isoleucine transport.

scholarly journals Discrimination of two amino acid transport activities in 4F2 heavy chain- expressing Xenopus laevis oocytes

1998 ◽  
Vol 333 (3) ◽  
pp. 549-554 ◽  
Author(s):  
Angelika BRÖER ◽  
Bernd HAMPRECHT ◽  
Stefan BRÖER
Keyword(s):  
Amino Acid ◽  
Transport System ◽  
Amino Acid Transport ◽  
Transport Systems ◽  
Xenopus Laevis Oocytes ◽  
Acid Transport ◽  
Transport Activity ◽  
Independent System ◽  
Leucine Transport ◽  
Dependent Transport

Expression of the type II membrane proteins of the rbAT/4F2hc family in Xenopus laevisoocytes results in the induction of amino acid transport activity. To elucidate the mechanism of action, amino acid transport was investigated in oocytes expressing the surface antigen 4F2hc. Leucine transport was mediated by a Na+-independent and a Na+-dependent transport mechanism. Both systems could be further discriminated by their stereochemical constraints. Isoleucine, with a branch at the β-position, shared only the Na+-independent transport system with leucine. Both transport systems were sensitive to inhibition by arginine, but only the Na+-independent system was sensitive to inhibition by 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid. When compared with known transport systems the two transport activities could be described as similar to, but not identical with, mammalian systems b0,+ and y+L. The Na+-independent b0,+-like transport system was found both in rbAT and 4F2hc expressing oocytes, indicating that both proteins act in a similar way.

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