(Received for publication, December 13, 1995; and in revised form, March 5, 1996)
From the
In renal epithelial cells amino acid deprivation induces an
increase in L-Asp transport with a doubling of the V 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.
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 (
)(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
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-
-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.
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.
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 10
cells/ml retaining the same cell
density/cm
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.
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.
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).
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. ()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
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.