©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Induction of High Affinity Glutamate Transport Activity by Amino Acid Deprivation in Renal Epithelial Cells Does Not Involve an Increase in the Amount of Transporter Protein (*)

(Received for publication, December 13, 1995; and in revised form, March 5, 1996)

Benjamin Nicholson (§) John D. McGivan

From the Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In renal epithelial cells amino acid deprivation induces an increase in L-Asp transport with a doubling of the V(max) and no change in K (4.5 µM) in a cycloheximide-sensitive process. The induction of sodium-dependent L-aspartate transport was inhibited by single amino acids that are metabolized to produce glutamate but not by those that do not produce glutamate. The transaminase inhibitor aminooxyacetate in glutamine-free medium caused a decrease in cell glutamate content and an induction of glutamate transport. In complete medium aminooxyacetate neither decreased cell glutamate nor increased transport activity. These results are consistent with a triggering of induction of transport by low intracellular glutamate concentrations. High affinity glutamate transport in these cells is mediated by the excitatory amino acid carrier 1 (EAAC1) gene product. Western blotting using antibodies to the C-terminal region of EAAC1 showed that there is no increase in the amount of EAAC1 protein on prolonged incubation in amino acid-free medium. Conversely, the induction of high affinity glutamate transport by hyperosmotic shock was accompanied by an increase in EAAC1 protein. It is proposed that low glutamate levels lead to the induction of a putative protein that activates the EAAC1 transporter. A model illustrating such a mechanism is described.


INTRODUCTION

In mammalian cells intracellular glutamate concentrations are maintained at a high level by the presence of active transport systems for glutamate in the plasma membrane. A number of different glutamate transporters have been kinetically characterized in various cell types including the Na-independent transporter System x

In 1992 three high affinity Na-dependent glutamate transporters were cloned. These are termed EAAC1 (^1)(the human homologue is excitatory amino acid transporter 3, EAAT3)(3) , GLT-1 (EAAT2)(4) , and GLAST-1 (EAAT1)(5) . The distribution of these EAATs has been characterized by immunochemistry. EAAT2 is confined to specific parts of the brain, central nervous system, and placenta(6) ; EAAT1 is confined to the brain(7) , retina(8) , heart, and skeletal muscle(6) . EAAC1 is found in brain but also occurs in other tissues particularly in kidney and gut(3, 6) . More recently a further transporter EAAT4 has been identified in the cerebellum and placenta(9) .

The renal bovine epithelial cell line (NBL-1) has been used in our laboratory as a model system to study the regulation of amino acid transport(10, 11, 12) . These cells, which are probably of distal tubule origin, express high activities of Na-dependent glutamate transport which have properties similar to System X

In this paper the induction of System X


EXPERIMENTAL PROCEDURES

Materials

[U-^14C]L-Aspartate, S-dATP, the Sequenase kit, and ECL reagents were from Amersham International (Amersham, UK). Sulfo-SMCC was obtained from Pierce (Chester, UK). pBluescript and pGEX2T were from Stratagene (Cambridge, UK) and Pharmacia (St. Albans, UK), respectively. Restriction enzymes were from Boehringer Mannheim (Lewes, UK). The synthetic peptide and oligonucleotides were synthesized in the Department of Biochemistry. Tissue culture reagents were purchased from Life Technologies, Inc. (Paisley, UK) except the dialyzed newborn calf serum which was from Sigma.

Cell Culture

Unless indicated otherwise the NBL-1 cells were seeded at a density of 6 times 10^3 cells/ml into 35-mm Petri dishes in Ham's F-12 medium supplemented with 10% (v/v) fetal bovine serum, 2 mM glutamine, and antibiotics as described previously(11) . The cells were fed every other day until used in experiments, typically after 4 days. The amino acid-free medium used contained the inorganic salts of Ham's F-12 medium together with 10 mM glucose and 0.1% bovine serum albumin(11) . The glutamine-free medium contained all the ingredients of Ham's F-12 except glutamine and was supplemented with 10% (v/v) dialyzed newborn calf serum.

Transport Measurements

System X

Anti-peptide Antibody

The anti-peptide antibody was raised against a synthetic C-terminal peptide CAVDKSDTISFTQTSQF (amino acids 509-524 of the EAAC1 sequence (3) with an cysteine added to the N-terminal end). The peptide was linked to keyhole limpet hemocyanin via the terminal cysteine using sulfo-SMCC. The antibody was purified by passing down a column of the synthetic peptide cross-linked to CNBr-activated Sepharose 4B beads.

Cloning of the Hydrophilic Loop Region of EAAC1 and Preparation of an Anti-fusion Protein Antibody

The cDNA encoding the putative extracellular loop between helices 3 and 4 (amino acids 117-215) was first amplified from NBL-1 cell cDNA using nested PCR. The flanking primers were 5`-GCT/C TTT/C CCC GGA/C GAG ATT/C CTG/T/C ATG and 5`-TCA/G TAA/G/C AGA/G GCG GTT/G CCA/G TCC AT.

The product of the first reaction was used as template for second round PCR. The nested primers were 5`-T TTT GGA TCC AAG CCC/T GGC/A/G GTG/C ACC CAG/A AAG and 5`-TT TTG GAT CCG CCC/G/A AGC/G ACA/G TTG/A ATG/T CCG/A TC.

The PCR product was sequenced and shown to have 83.75% identity with the rabbit intestine EAAC1 cDNA sequence and 78.5% identity at the protein level. Because problems were encountered in direct ligation into the prokaryotic expression vector pGEX2T, the DNA was blunt-ended and cloned into EcoRV-cut pBluescript, which had been pretreated with calf intestinal alkaline phosphatase. The construct was transformed into Ca-competent Escherichia coli XL-1 by heat shock. Using the BamHI sites engineered into the primers, the insert was excised from pBluescript and ligated into pGEX2T, which was used to transform XL-1 as before. The construct was sequenced across the insert site and shown not to have any frameshift mutations. Once the absorption of the transformed cells at 600 nm reached 0.3, glutathione-S-transferase-loop construct expression was induced for 1 h with 1 mM isopropyl-1-thio-beta-D-galactopyranoside. 10 out of 20 transformants expressed the glutathione-S-transferase-loop construct in the correct orientation. The bacterial proteins were separated on SDS-PAGE, and the fusion protein was electroeluted overnight at 50 mA.

Western Blots

Cell extracts were prepared as follows. Cell cultures were washed twice in ice-cold phosphate-buffered saline (PBS). The cells were scraped off into ice-cold PBS in the presence of protease inhibitors (2 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, and PAL (1 µg/ml each of pepstatin, antipain, and leupeptin)) and centrifuged. The PBS was removed, and the cells were resuspended in 20 mM Tris-HCl, pH 7.4, before dissolving in the neutral detergent MEGA-10 to a final concentration of 1% in the presence of protease inhibitors. 25 µg of the samples were then separated on SDS-PAGE under reducing conditions. The proteins were transferred in the absence of SDS using a semi-dry blotting apparatus, 1 h, 10 V.

The blots were blocked for 20 min in PBS/0.02% Tween containing 5% dried milk powder. The antipeptide antibody was added for 1 h in the same buffer, and the blots were then washed three times in PBS/0.02% Tween before anti-rabbit horseradish peroxidase conjugate was added at 1 µl/ml in 5% powdered milk/PBS/0.02% Tween for 45 min. The blots were finally washed twice in PBS/0.02% Tween and then once in PBS before development by enhanced chemiluminescence.

Measurement of Internal Glutamate Concentration

Glutamate in neutralized acid extracts of whole cells was measured by the method of Bernt and Bergmeyer(16) .

Sequencing

Sequencing of the DNA cloned into pBluescript and pGEX2T was carried out using S-dATP and the Sequenase sequencing kit according to the manufacturer's instructions. The KS and T7 primers were used for sequencing pBluescript. A specific primer GCATGGCCTTTGCAGGG was used for sequencing across the pGEX2T multiple cloning site.


RESULTS

Characterization of the Induction of System X

Total amino acid deprivation causes an increase in the V(max) of Na-dependent L-aspartate transport in NBL-1 cells. This process is sensitive to cycloheximide and is maximal after 10 h(13) . On repeating this work it was found that the magnitude of the stimulation depended on the degree of confluency of the cells (Fig. 1). There was a 300% induction by amino acid starvation 3 days after seeding the cells (confluency approximately 80%), but the degree of induction decreased as the cells became more confluent and was relatively low in confluent cells. Subsequent experiments were performed under conditions where the induction was maximal, as indicated in the figure legends.


Figure 1: Effect of cell confluency on the degree of induction of L-Asp transport by amino acid deprivation. Three days after seeding the NBL-1 cells, the culture medium was either changed to fresh normal medium or the cells were washed once with amino acid-free medium and cultured in the same amino acid-free medium. The degree of confluency was assessed at this stage, and the transport experiments were performed 24 h later. This was repeated on the two subsequent days. The initial rate of Na-dependent aspartate transport is shown as the mean ± S.E. of values obtained from three Petri dishes in each case.



Addition of certain single amino acids to the amino acid-free medium reduced the induction of System X



In order to confirm that the effect of added amino acids on the apparent induction of transport was not in fact due to trans-inhibition of uptake by differential internal concentrations of glutamate, cells were incubated with various single amino acids (1 mM) in amino acid-free medium for 1 h allowing intracellular glutamate accumulation but not allowing enough time for protein synthesis to occur. In all cases there was no increase above the rate in amino acid-free medium (not shown). It was previously shown, and is confirmed here, that the induction of transport activity by amino acid-free medium was completely inhibited by cycloheximide, confirming that the effect was due to protein synthesis.

The results in Table 1suggest that the intracellular glutamate concentration may determine the level of expression of System X


Figure 2: Effect of aminooxyacetic acid in the presence and absence of glutamine on the induction of L-aspartate transport activity. Three days after seeding the cells the medium was changed to either normal medium or glutamine-free medium in the presence or absence of 0.5 mM aminooxyacetic acid as indicated in the bar chart. Before incubation in glutamine-free medium, the cells were washed once with glutamine-free medium to remove any residual glutamine. 24 h after this, the initial rate of transport of L-aspartate was measured. Data presented are mean ± S.E. of three determinations in each case.



In order to confirm this interpretation, the cell glutamate content was determined enzymatically. Fig. 3shows that on switching cells to amino acid-free medium the cellular glutamate content fell from 30 nmol/mg protein to 13 nmol/mg protein over a period of 5 h. In cells transferred to fresh normal medium at zero time, the cellular glutamate concentration fell much more slowly. Under the conditions corresponding to the experiment shown in Fig. 2, the internal glutamate contents at 1 h were determined. In glutamine-free media + 0.5 mM AOA the cell glutamate content fell to 15.0 ± 0.6 nmol/mg protein, while in normal media in the presence of 0.5 mM AOA the cell glutamate content was 29.2 ± 1.0 nmol/mg protein, which is similar to the control values. These results suggest that the induction of System X


Figure 3: Time course of changes in cellular L-glutamate content. NBL-1 cells were seeded in T75 flasks at 3 times 10^3 cells/ml retaining the same cell density/cm^2 as the seeding in the 35-mm Petri dishes. Three days after seeding, the cells were incubated in normal medium or washed once in amino acid-free medium before incubation in amino acid-free medium for the times indicated. The glutamate contents in cells incubated in normal medium are shown as single points, and the glutamate contents of cells incubated in amino acid-free medium are the mean ± S.E. of values from three separate flasks of cells in each case.



Induction of System X

Tunicamycin is an inhibitor of protein glycosylation. Incubation of cells with 0.1 µg/ml tunicamycin in amino acid-free medium for 24 h apparently reduced the amino acid starvation-dependent induction of aspartate transport, suggesting that glycosylation of a protein is a necessary step in this process (Fig. 4). However, the addition of 0.1 µg/ml tunicamycin to cells in normal medium for 24 h itself induced transport activity. The induction was cycloheximide-sensitive (Fig. 4). This effect was characterized by an increase in the V(max) from 110 to 180 pmol/mg/min whereas the K(m) for L-aspartate was unchanged at 4.6 µM (results not shown). The half-maximal effect was at 0.055 µg/ml tunicamycin, which is a concentration at which the effect of this compound on protein glycosylation should be specific. Tunicamycin is known to exert a stress effect on cells that is characterized by an increase in the 78-kDa glucose-regulated protein (GRP78) mRNA(17) .


Figure 4: Effect of tunicamycin on the induction of L-Asp transport in NBL-1 cells incubated in normal and amino acid-free media. Three days after seeding NBL-1 cells the medium was changed to either normal medium or amino acid-free medium with the indicated additions. 24 h later the rate of L-Asp transport was measured. The values are the mean ± S.E. of initial rates of Na-dependent aspartate transport obtained from three dishes of cells in each case.



Changes in EAAC1 Protein Levels During Induction of Activity by Amino Acid Deprivation, Hyperosmotic Stress, and Tunicamycin

The kinetic parameters and substrate specificity of System X

The purified anti-peptide C-terminal antibody recognized a major band at 64 kDa in NBL-1 cells, together with some minor bands (Fig. 5). This is reasonably consistent with the molecular mass of the EAAC1 protein from rabbit intestine which is predicted to be 57 kDa. The molecule contains four potential glycosylation sites, but the extent of glycosylation is not known. Fig. 5also shows that the anti-loop antibody recognized a major band of the same size as that recognized by the C-terminal anti-peptide antibody. The fact that antibodies raised to different regions of the EAAC1 protein recognize the same protein is good evidence that this protein is the EAAC1 gene product. Western blots (not shown) indicate that the C-terminal antibody recognized a protein of the same molecular weight in rat brain. This is also consistent with the molecular weight of the protein recognized in rat brain by another antibody raised to the C-terminal of EAAC1(7) .


Figure 5: Duplicate Western blots showing that both the anti-peptide (lane 1) and anti-fusion protein (lane 2) antibody recognize a band of the same size. Three days after seeding, the medium on NBL-1 cells was changed to fresh complete medium. 24 h later whole NBL-1 cell extracts were prepared as described under ``Experimental Procedures,'' separated by SDS-PAGE, and transferred to nitrocellulose. Duplicate 25-µg samples were probed with either the anti-peptide antibody or anti-fusion protein antibody.



Fig. 6shows Western blots of NBL-1 cells incubated for 24 h in normal medium, amino acid-free medium, normal medium + 200 mM sucrose, and normal medium + 0.1 µg/ml tunicamycin. No change in the protein level was observed between normal and amino acid-starved cells, although an increase in transport under these conditions has been shown to occur. However, cells exposed to sucrose or tunicamycin (other conditions that induce transport activity) showed a very significant increase in the amount of protein detected by the anti-peptide antibody. Table 2quantifies the changes in protein over a number of different experiments.


Figure 6: A representative Western blot of NBL-1 cells incubated for 24 h in normal medium (lane 1), amino acid-free medium (lane 2), normal medium + 200 mM sucrose (lane 3), and normal medium + 0.1 µg/ml tunicamycin (lane 4). Three days after seeding, the medium was changed to that indicated. 24 h later whole cell extracts were prepared as described under ``Experimental Procedures,'' and 25-µg samples were separated on SDS-PAGE, transferred to nitrocellulose, and probed with the anti-peptide EAAC1 antibody.





Table 2also reports the results of a similar experiment where transport was induced by the addition of 0.5 mM AOA and in the absence of 1 mML-Gln. There was no change in the amount of protein even though an increase in L-Asp transport occurred under these conditions (see Fig. 2).


DISCUSSION

The results presented above indicate that the necessary condition for the induction of glutamate transport by amino acid deprivation in NBL-1 cells is the presence of a cellular glutamate content of less than about 15 nmol/mg. Since the intracellular volume in these cells is about 6.2 µl/mg protein(10) , this would correspond to an intracellular concentration of 2.5 mM. When glutamate is depleted from 30 to about 15 nmol/mg the transport activity starts increasing after a period of about 5-6 h. These conclusions follow from the following facts. (i) Incubation of cells in amino acid-free medium leads to a reduction of glutamate levels to 15 nmol/mg within 2-3 h. (ii) Single amino acids that can be metabolized to glutamate prevent induction whereas those that cannot be metabolized cannot prevent induction (Table 1). (iii) Addition of AOA in the absence of glutamine reduces glutamate levels to 15 nmol/mg and causes the induction of transport. (iv) AOA in the presence of glutamine neither reduces glutamate levels nor induces transport. This is likely to be a physiologically important mechanism for maintaining glutamate levels.

Western blots have shown that there is no change in the EAAC1 protein level during amino acid deprivation even though there is an increase in transport. Under other conditions where transport has been shown to be induced, i.e. exposure to hyperosmotic medium or tunicamycin, a clear increase in the amount of protein detected by the C-terminal anti-peptide antibody is observed. The increase in transport activity is dependent on protein synthesis and is not due to differential transinhibition as a result of different internal glutamate concentrations. Also it has been previously shown in this laboratory that EAAC1 mRNA levels do not increase during amino acid deprivation, although these mRNA levels do increase as a result of hyperosmotic shock (14) and tunicamycin treatment. (^2)Since the induction of transport activity requires protein synthesis but the amount of EAAC1 protein itself does not increase, we postulate that the induction of a putative EAAC1-activating protein is responsible for the increase in the rate of aspartate transport. As the K(m) is unchanged on incubating the cells in amino acid-free medium(13) , it is unlikely that this is due to the induction of a different glutamate transporter. Since the activation is reduced by tunicamycin, this suggests the putative activating protein may well be a glycoprotein.

Tunicamycin itself in normal medium caused increases in EAAC1 mRNA and protein. The mechanism of this effect is not clear but is likely to be related to a stress effect possibly triggered by the presence of malfolded proteins as has been suggested for GRP78(18) . As there is a 4-fold increase in EAAC1 protein, but only a doubling in the rate of transport, it appears that some of EAAC1 protein is not reaching the plasma membrane due to mistargetting in the absence of glycosylation.

The question arises as to how the cell is able to detect changes in the cellular glutamate level in the range of 30 to 15 nmol/mg. There are a number of proteins that regulate amino acid transport in bacteria such as the leucine-responsive regulatory protein in E. coli(19) and the glutamate uptake regulatory protein in Zymomonas mobilis. This protein has a helix-turn-helix motif that is typical of a transcription factor and has been shown by gel retardation assays to bind the regulatory region of the E. coli gene gltP, which encodes a proton symporter for glutamate and aspartate(20) . The results in this paper are consistent with the presence of a low affinity glutamate binding protein (GBP) that can act in one of two ways to switch on the synthesis of a protein that activates System X


Figure 7: A model to account for the induction of System X



In NBL-1 cells System A is also induced by amino acid deprivation in a process that is protein synthesis-dependent, sensitive to tunicamycin, and reversed or prevented by the addition of single amino acids(11) ; these results are consistent with earlier work on hepatocytes(21) . Since System A has not yet been cloned, no definitive mechanism for this effect has been established. There are indications that the induction of System A involves the synthesis of a hypothetical transport activating protein (21) rather than the System A transport protein itself. This conclusion has been reinforced by studies of Chinese hamster ovary cell mutants that do not induce System A activity on amino acid deprivation ((22) ; for review see (23) ). It is possible that amino acid deprivation leads to the synthesis of one or more glycoproteins that act as activators of amino acid transport proteins in cell membranes, thus assisting in maintenance of the intracellular amino acid pool under these conditions.


FOOTNOTES

*
This work was funded by a Wellcome Trust Prize Studentship (to B. N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 44-117-9289000 (Ext. 8322); Fax: 44-117-9288274; B.Nicholson{at}bris.ac.uk.

(^1)
The abbreviations used are: EAAC1, excitatory amino acid carrier 1; AOA, aminooxyacetic acid; EAAT, excitatory amino acid transporter; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; GRP78, 78-kDa glucose-regulated protein; GBP, glutamate-binding protein; Sulfo-SMCC, sulfosuccinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate.

(^2)
B. Nicholson, unpublished results.


ACKNOWLEDGEMENTS

We are grateful to Dr. C. R. Helps for assistance with the production of the fusion protein.


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©1996 by The American Society for Biochemistry and Molecular Biology, Inc.