Biosynthesis, intracellular targeting, and degradation of the EAAC1 glutamate/aspartate transporter in C6 glioma cells.

Rat C6 glioma cells were used as a model system to study the biosynthesis, intracellular targeting, and degradation of the EAAC1 transporter, a sodium-dependent glutamate/aspartate transport protein that encodes System X(-)A,G activity. At steady state, nearly 70% of the EAAC1 transporter was located at the cell surface. The newly synthesized EAAC1 protein was co-translationally N-glycosylated with high mannose oligosaccharide chains that were processed into complex-type sugar chains as the protein matured. The final maturation steps for EAAC1 protein coincided with its plasma membrane arrival, which was first detected at about 45 min after the initial synthesis. The newly synthesized EAAC1 protein was protected from degradation during the maturation and targeting process, as well as during the first 5 h after plasma membrane arrival. After this initial lag period, both the newly synthesized transporter and the total cellular EAAC1 pool were degraded by first order kinetics with a half-life of 6 h. These results represent the first analysis of the synthesis and degradation of the EAAC1 amino acid transporter.

Among the members of the glutamate/aspartate transporter family, the EAAC1 transporter appears to be the most ubiquitously expressed. Although quite abundant in brain, a significant level of expression of this transporter can also be detected outside the nervous system in small intestine, kidney, heart, skeletal muscle, lung, liver (9,10), and placenta (13). It is postulated that the EAAC1 transporter may play a role in keeping the neuronal intracellular glutamate at high levels for use as a precursor for ␥-aminobutyric acid synthesis or for other metabolic reactions in the brain (14). In addition, consistent with its ubiquitous expression among different tissues, EAAC1 transporter functions as a primary mechanism to provide glutamate and aspartate for general metabolism and other intracellular functions.
Rat C6 glioma cells exhibit several biochemical features of normal glial cells, such as expressing glial fibrillary acidic protein (15). These cells express a high level of System X Ϫ A,G transport activity (16). Although there are reports that GLT1 is expressed in C6 cells (17), most evidence indicates that C6 glioma cells express EAAC1 but not GLAST, GLT1, or EAAT4 (11,18).
Although the distribution, regulation, and mechanism of anionic amino acid transporters has been extensively studied, much less is known about their biosynthesis and intracellular trafficking. It has been shown that the surface expression of EAAC1 can be rapidly up-regulated by both protein kinase C and phosphatidylinositol 3-kinase pathways (19,20), suggesting the redistribution of EAAC1 from an intracellular compartment. Furthermore, Lin et al. (21) have made the interesting observation that EAAC1 activity can be modulated through protein-protein interactions with GTRAP3-18. For the studies described here, C6 glioma cells were used as a model system to study the biosynthesis, intracellular targeting, and degradation of EAAC1. The results reveal that the EAAC1 protein is N-glycosylated in a co-translational manner. Synthesis and trafficking to the plasma membrane required a minimum of 45 min, and there was a lag of about 5 h prior to degradation of transporters on the cell surface. With regard to turnover, the plasma membrane resident EAAC1 was endocytosed and then degraded with a half-life of about 6 h. Interestingly, the newly synthesized plasma membrane-associated transporter population was degraded at the same rate as the total pool of EAAC1, suggesting that both surface and intracellular EAAC1 proteins have similar half-life values. The results obtained provide the basis for studying the role of EAAC1 transporter synthesis and degradation during disease states.

MATERIALS AND METHODS
Cell Culture-C6 glioma cells were obtained from the American Type Culture Collection (CCL107) and maintained in supplemented Eagle's * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. medium (MEM) 1 containing 10% FBS as a monolayer culture under a humidified atmosphere of 5% CO 2 , 95% air (37°C) for a maximum of eight passages. The cultured cells were transferred to 24-well cluster dishes for whole cell transport assays or to 100 -150-mm culture dishes for metabolic labeling, cell surface biotinylation, and total cellular protein or membrane protein collection.
Transport Assay-Amino acid uptake by C6 glioma cells was measured using the cluster tray method (22). One hundred thousand C6 cells were placed into each well of a 24-well tray and cultured for 24 h. To partially deplete the intracellular pool of amino acids and thus minimize trans-effects on transport and remove extracellular Na ϩ , the cells were incubated at 37°C twice for 15 min each in sodium-free Krebs-Ringers phosphate buffer (choline-KRP). To initiate transport, [ 3 H]aspartate in 250 l of either NaKRP (sodium-containing Krebs-Ringers phosphate buffer) or choline-KRP (37°C) was added simultaneously to each of the 24 wells in the cluster tray for 1 min. The transport measurement was terminated by discarding the radioactivity and rapidly washing the cells five times with 2 ml of ice-cold choline-KRP. The Na ϩ -dependent transport is taken as the difference between uptake in NaKRP and choline-KRP. The data are expressed as pmol⅐mg Ϫ1 protein⅐min Ϫ1 and are presented as the averages of four assays on at least two different batches of cultured cells.
Gel Electrophoresis and Immunoblotting-Gel electrophoresis was performed in 7.5% polyacrylamide gels following the protocol originally described by Laemmli (23). The protein samples were diluted with an equal volume of sample dilution buffer (SDB) consisting of 2% (w/v) SDS, 5% ␤-mecaptoethanol, 30 g/ml bromphenol blue, 20% glycerol, 0.125 mM Tris-HCl, pH 6.6 -6.8. The amount of protein loaded per lane will be stated in each figure legend. For immunoblotting, the fractionated proteins were transferred electrophoretically onto nitrocellulose membrane in 4°C transfer buffer (25 mM Tris-base, 190 mM glycine, 20% methanol) at 299 mA and constant current for 20 h. After the transfer, the blot was stained briefly in Fast Green stain (0.1% Fast Green, 50% methanol, 10% acetic acid) and destained (50% methanol, 10% acetic acid) to check for the efficiency of transfer and the evenness of loading. The blots were blocked in TBS-T buffer (10 mM Tris, pH 7.5, 200 mM NaCl, and 0.1% Tween 20) containing 1% Carnation nonfat dry milk for 2 h at or overnight at 4°C with constant agitation on an orbital shaker. The blots then were incubated in the same blocking solution containing a rabbit polyclonal anti-EAAC1 (rat) antibody (1:2000 dilution of serum) for 2 h at room temperature in test tubes with constant rotation. After extensively washing in TBS-T with 1% nonfat dry milk, the blots were incubated for 1 h at room temperature in blocking buffer with secondary antibody conjugated to horseradish peroxidase (1:20,000 dilution of goat anti-rabbit IgG-horseradish peroxidase). The blots were extensively washed with TBS-T with 1% nonfat dry milk before being visualized with SuperSignal Chemiluminescence detection reagents (Pierce) following the manufacturer's instructions. Light emissions from the blots were captured on Hyperfilm MP (Amersham Biosciences), and the band intensity was quantitated in the linear range of the film on a Visage Bioscan video densitometer. The preparation and characterization of the rabbit anti-EAAC1 polyclonal antibody against a rat EAAC1-maltose-binding protein fusion protein as antigen has been described previously (13).
Pulse-Chase Metabolic Labeling of C6 Glioma Cells-To study the de novo biosynthesis and the intracellular targeting of the EAAC1 transporter protein in C6 cells, pulse-chase labeling with L-[ 35 S]methioninecysteine (ProMix; Amersham Biosciences) was used. After placing 9 ϫ 10 6 cells onto each 100-mm culture dish or 2.3 ϫ 10 7 cells onto each 150-mm dish, the cell monolayers were cultured for 24 h to permit growth to near confluence. The cells were washed once with sterile 37°C phosphate-buffered saline, pH 7.4, and incubated with 15 ml/58cm 2 surface area of methionine-and cysteine-free Dulbecco's modified Eagle's medium (Invitrogen) for 2 ϫ 15 min at 37°C to deplete the intracellular pool of free methionine and cysteine. The depletion medium then was aspirated, and the cells were incubated with 200 Ci/ml of [ 35 S]Met-Cys in methionine-and cysteine-free Dulbecco's modified Eagle's medium at 37°C for 15-30 min (see each figure legend). The cells were washed twice at 37°C with MEM containing 5 mM each of nonradioactive methionine and cysteine (chasing medium) and then transferred to fresh chasing medium and incubated for 0 -60 h (see each figure legend) followed by immunoprecipitation of the EAAC1 protein.
For experiments in which the chase period was longer than 24 h, 1% FBS was added to the medium.
Immunoprecipitation and Fluorography-Immunoprecipitation of the EAAC1 transporter protein was performed following the procedure outlined by Harlow and Lane (24) with modifications. After pulse-chase labeling with L-[ 35 S]Met-Cys, the cells were washed twice with ice-cold phosphate-buffered saline and once with SEB buffer (250 mM sucrose, 2 mM EDTA, 2 mM EGTA, and 10 mM HEPES, pH 7.5) and then frozen in 2.5 ml of SEB buffer containing proteinase inhibitors (1 mM phenylmethylsulfonyl fluoride and 2 g/ml each of leupeptin, aprotonin, pepstatin, N-tosyl-L-phenylalanine chloromethyl ketone, and N-p-tosyl-Llysine chloromethyl ketone) at Ϫ80°C. For analysis, the cells were thawed on ice, and another 2.5 ml of ice-cold hypotonic EB buffer (2 mM EDTA, 2 mM EGTA, and 10 mM HEPES, pH 7.5) was added. The cells were scraped from the plates and homogenized on ice with 15 passes through a prechilled steel block cell homogenizer with a clearance of 0.0025 inches (Auburn Tool & Dye, Warwick, RI). The cell homogenate was centrifuged at 400 ϫ g for 10 min to remove unbroken cells and nuclei, and the supernatant was centrifuged at 280,000 ϫ g for 1 h at 4°C to collect a total membrane pellet, which was then extracted in PES buffer (2% C 12 E 9 , 0.1% SDS, 1 mM EDTA in phosphate-buffered saline, pH 7.4) for 1 h on ice with constant stirring. After centrifugation at 200,000 ϫ g for 15 min at 4°C, the supernatant was recovered, and the protein concentration was determined by a modified Lowry method (24) or the bicinchoninic acid method (Pierce protein assay kit). An equal amount of starting protein (100 -500 g) from each sample was transferred into microcentrifuge tubes, brought to a volume of 500 l with PES buffer, and used for the immunoprecipitation assay. To minimize nonspecific interaction between labeled membrane proteins and the immunoglobulins, an unrelated nonimmune rabbit serum IgG (5 g) and 50 l of protein A-Sepharose beads (50% suspension in PES buffer) were added to the extracts and incubated ("precleared") for 3 h at 4°C with constant mixing. The samples were then centrifuged at 15,000 ϫ g for 15 s to collect the protein A-Sepharose-IgG complexes, and this precleared supernatant was transferred to a new microcentrifuge tube. EAAC1 antibody (5 g of IgG) was added to the precleared extract and incubated at 4°C overnight with constant mixing. A 50-l aliquot of 50% protein A-Sepharose beads was then added and incubated for 2 h to collect the immunoprecipitates. The pellets were washed with PES extraction buffer (3 ϫ 1 min) and with PES containing 0.35 M NaCl (for a total salt concentration of 0.5 M) (4 ϫ 10 min) at 4°C with constant mixing. A series of salt washes were tested, and the data showed that binding between the antibody and the EAAC1 protein can withstand salt washing up to 1 M NaCl but that 0.5 M salt is sufficient to eliminate all of the nonspecific binding (data not shown). The immunoprecipitated proteins were eluted from the beads with the gel electrophoresis SDB containing 6 M urea and 10% ␤-mercaptoethanol for 20 min at 37°C and separated by SDS-PAGE. For fluorography, the gels were fixed at room temperature in 10% trichloroacetic acid, 40% methanol for 30 min, soaked in water for 30 min, and then incubated in 1 M sodium salicylate for 1 h before drying at 65°C under vacuum. The dried gels were exposed to autoradiographic film at Ϫ80°C with an intensifying screen, and the band intensity was quantitated in the linear range of the film on a Visage Bioscan video densitometer. A 5 g of anti-EAAC1 antibody (purified total IgG from the immune rabbit serum) was shown to be sufficient to precipitate all of the EAAC1 protein from 500 g starting material by immunoblotting EAAC1 protein in the immunoprecipitation supernatant (data not shown). Also, preliminary experiments showed that the amount of EAAC1 protein immunoprecipitated by 5 g of anti-EAAC1 antibody was proportional to the amount of starting protein from 0 -500 g (data not shown).
Endoglycosidase Digestions-To test for the endoglycosidase H (Endo H) sensitivity of the newly synthesized EAAC1, C6 cells were pulselabeled for 15 min with [ 35 S]Met-Cys and chased with medium containing 5 mM each of unlabeled methionine and cysteine for 30 -240 min, as described above. At the end of each chase period, EAAC1 was immunoprecipitated and then eluted from the protein A-Sepharose beads with 10 l of 5ϫ denaturing solution containing 2.5% SDS, 5% ␤-mercaptoethanol in water for 30 min at 37°C. After dilution to 1ϫ denaturing solution with 40 l of water, each of the eluates was collected and then divided into two microcentrifuge tubes. A 1 ⁄10 volume of 500 mM sodium citrate, pH 5.5, and 500 -1000 units (1-2 l) of Endo H (BioLabs Inc.) was added to each of the tubes. For the control tube, 1-2 l of enzyme storage buffer (20 mM Tris-HCl, pH 7.5, 50 mM NaCl, and 5 mM Na 2 EDTA) was added, instead of the enzyme. All of the samples were incubated at 37°C for 1 h and then mixed with an equal volume of 2ϫ SDB buffer before the samples were loaded onto SDS-PAGE gel for separation and autoradiographic detection.
To determine the N-linked glycosylation of both the newly synthesized forms of the EAAC1 transporter protein, protein endoglycosidase F (PNGase F) digestions were performed. Total C6 cellular membrane protein was collected from the cells, with or without pulse-chase labeling and then solubilized with PES buffer, and EAAC1 protein was immunoprecipitated. The precipitates were eluted with 10 l of 5ϫ denaturing solution (2.5% SDS, 5% ␤-mercaptoethanol) for 30 min at 37°C and then diluted to 1ϫ denaturing solution with water, as described above for the Endo H digestions. For each PNGase F digestion, 1 ⁄10 volume each of 10% Nonidet P-40 and a buffer consisting of 10% Nonidet P-40, 500 M sodium citrate, pH 7.5, as well as 500 -1000 units of PNGase F (BioLabs Inc.) were added. For the controls, the enzyme was replaced with equal volume of enzyme storage buffer. All of the samples were incubated at 37°C for 60 min and then mixed with an equal volume of 2ϫ SDB buffer before they were loaded onto a SDS-PAGE gel for separation and autoradiographic detection.
Cell Surface Protein Biotinylation-To determine the half-life of the cell surface EAAC1 protein, C6 glioma cells were washed twice with NaKRP buffer (119 mM NaCl, 5.9 mM KCl, 1.2 mM KHCO 3 , 5.6 mM glucose, 25 mM Na 2 HPO 4 , 0.5 mM CaCl 2 , 1.2 mM MgSO 4 , pH 7.5) and incubated with 0.5-1 mg/ml of sulfo-NHS-LC-biotin (Pierce) in NaKRP for 30 min to 1 h at 4°C. The specific conditions for each experiment are given in the figure legends. The biotin-containing buffer was aspirated, and the cells were rinsed twice with fresh MEM containing 50 mM glycine and then incubated in fresh MEM and 1% FBS at 37°C for specific chase times ranging from 0 to 24 h at 37°C. At the end of each chase period, the cells were washed once with ice-cold NaKRP, washed once with ice-cold SEB containing protease inhibitors (as above), and then frozen in 2 ml of SEB containing protease inhibitors at Ϫ80°C. The percentage of the biotinylation for EAAC1 was 69%, compared with a value of less than 2% for the intracellular proteins, asparagine synthetase (cytoplasmic) and GRP78 (endoplasmic reticulum) (data not shown).
To determine the transit time of newly synthesized EAAC1 transporter proteins to the plasma membrane, C6 cells were metabolically labeled with [ 35 S]Met-Cys for 15-30 min and chased in medium containing 5 mM each of nonradiolabeled methionine and cysteine for 0 -36 h. At the end of each chase time, these metabolically labeled C6 cells were washed twice with NaKRP and then cell surface-biotinylated with 0.5-1 mg/ml of sulfo-NHS-LC-biotin in NaKRP at 15°C for 30 -60 min. After aspirating the biotinylation solution, the cells were rinsed once in NaKRP and then incubated with NaKRP containing 50 mM glycine for 2 ϫ 15 min at 15°C to quench the remaining free biotin. The cells were washed once with ice-cold SEB containing protease inhibitors and frozen at Ϫ80°C until all of the samples were collected. The surfacebiotinylated C6 cells were thawed on ice and homogenized with a steel block homogenizer, and the total cellular membrane proteins were collected and solubilized with PES buffer, as described above. Equal amounts of solubilized proteins were then subjected to two successive precipitations. For the immunoprecipitation of total EAAC1 protein, the protein samples were precleared with nonimmune rabbit IgG and then immunoprecipitated with anti-EAAC1 antibody, as described above. The precipitated EAAC1 protein was eluted from the beads with 1 ml of 0.1 M glycine, pH 2.8, containing 0.5% Triton X-100 and 0.2% bovine serum albumin at room temperature for 30 min. After centrifugation at 10,000 ϫ g for 15 s to remove the beads, the eluate was removed and neutralized with 50 l of 0.1 M Tris-HCl, pH 9.5. For the selective precipitation of the biotinylated EAAC1 molecules, 25 l of packed monomeric avidin-beads was washed twice with 1 ml of PES buffer and then preincubated for 2 h with nonbiotinylated total C6 cellular membrane protein to block the nonspecific binding sites on the beads. Then the immunoprecipitated EAAC1 protein collected above was added to the pretreated monomeric avidin-beads and incubated for 18 h with constant mixing at 4°C. At the end of the incubation, the beads were washed extensively with 1 ml of PES supplemented with 350 mM NaCl for 40 min at 4°C with a total of six changes of buffer. The doubly precipitated proteins were eluted from the beads with 2ϫ SDB containing 2 mM free D-biotin and separated by gel electrophoresis followed by fluorography, as described above.

EAAC1
Localization at the Cell Surface-To demonstrate the plasma membrane localization of EAAC1 transporter in C6 cells, the accessibility of EAAC1 protein from the extracellular space was tested by cell surface biotinylation using a membrane-impermeable sulfo-NHS-LC-biotin reagent. After biotinylation, total C6 cellular membrane proteins were collected, and the biotinylated proteins were isolated using avidin precipitation and then separated by gel electrophoresis. The biotinylated EAAC1 protein was then detected using immunoblotting with anti-EAAC1 antibody. As shown in Fig. 1A, a large portion of the EAAC1 protein was readily biotinylated, demonstrating that much of the total amount of the transporter resides at the cell surface. By monitoring the percentage of total cellular EAAC1 protein that could be biotinylated, it was established that ϳ70% of EAAC1 protein was labeled by the cell surface reaction (Fig. 1B). On contrast, less than 2% of asparagine synthetase (cytoplasm) or glucose-regulated protein 78 (endoplasmic reticulum) were biotinylated. As an example of another plasma membrane protein, 89% of the NaK-ATPase was biotinylated (Fig. 1B). These data document that a majority of EAAC1 transporter protein is localized at the plasma membrane but that an intracellular pool of transporter also exists.
N-Glycosylation of EAAC1 Protein-The deduced amino acid sequence for the rat EAAC1 transporter contains four putative N-glycosylation consensus sequences, and three of these predicted N-glycosylation sites are localized within the second extracellular loop (10). It has been reported that the EAAC1 transporter is N-glycosylated and that its glycosylation pattern may vary in different host cell lines (11). To test for the Nglycosylation of EAAC1 protein in C6 cells, PNGase F digestion was used to cleave between the first GlcNAc residue and the asparagine residue (25). Without PNGase F digestion, EAAC1 monomer and oligomer bands were detected after immunoprecipitation of solubilized total membrane proteins with anti-EAAC1 antibody (Fig. 1C). Prior to enzyme treatment, the EAAC1 bands were broad, likely because of the microheterogeneity of the glycosylation or other post-translational modifications. However, a relatively sharp 57-kDa band (EAAC1 core) and, to a lesser extent, a 114-kDa band (possibly a core dimer) were detected after incubation of the immunoprecipitated EAAC1 protein with PNGase F for 15 min or more. The deglycosylated monomer band appears to represent less protein, but this apparent discrepancy may be due to the sharpness of the deglycosylated band relative to the broader smear that the glycosylated monomer for presents. These results are consistent with the data by Dowd et al. (11) and show that deglycosylation of the EAAC1 dimer does not reverse its oligomerization.
De Novo Biosynthesis Rate for EAAC1 Transporter-To determine the biosynthesis rate of EAAC1 protein in C6 glioma cells, the cells were metabolically labeled with 200 Ci/ml of [ 35 S]Met-Cys for 15-120 min and then chased for 3 h. As shown in Fig. 2A, even a 15-min pulse labeling time is sufficient to detect newly synthesized EAAC1, and all three forms (monomer, dimer, and oligomer) of EAAC1 appear to be proportional to each other, regardless of the pulse time length. These results suggest that the oligomerization is not a late step in the maturation process for the EAAC1 protein. Interestingly, the biosynthesis rate of EAAC1 was decreased when a higher number of cells were plated, indicating that EAAC1 biosynthesis is down-regulated by increased cell density (Fig. 2B). This observation is consistent with preliminary experiments showing that the total EAAC1 protein content was reduced as cells became more confluent (data not shown). Therefore, in all remaining experiments a fixed number of cells were plated (900,000/100-mm dish) and then cultured for an exact period of time (24 h) prior to labeling.
Maturation and Targeting of EAAC1 Transporter-To study the maturation process of EAAC1, C6 glioma cells were pulse-labeled with 500 Ci/ml of [ 35 S]Met-Cys for 15 min and then chased for 0 -120 min. When chased for less than 30 min, only the low molecular mass (57 kDa), immature form of EAAC1 was detected primarily (Fig. 3), but after longer chase periods, the 57-kDa form of EAAC1 matured into the 73-kDa protein.
The maturation of EAAC1 protein started after 45 min of chase time and finished after 190 min. The data in Fig. 3 show that oligomeric forms were detected not only for the mature EAAC1 protein but also for the immature EAAC1 form (e.g. chase time ϭ 0).
To address the time required for the newly synthesized EAAC1 protein to be targeted to the plasma membrane, the cells were metabolically labeled with 200 Ci/ml of [ 35 S]Met-Cys for 15 min and chased in medium containing 5 mM each of nonradiolabeled Met and Cys for 0 -360 min at 37°C (Fig. 4). The pulse time was short (15 min), and an increased amount of radioactivity was used to provide a narrow observation "window" for the plasma membrane arrival. At the end of each chase time, the cells were surface-biotinylated with the membrane-impermeable sulfo-NHS-LC-biotin to label all proteins that were accessible from the extracellular space. After the PES-soluble proteins were collected from a total cellular membrane fraction, a double precipitation procedure was performed by precipitating all of the EAAC1 protein using anti-EAAC1 antibody, eluting the transporter from the beads at low pH, and then selectively precipitating only the biotinylated EAAC1 protein using immobilized monomeric avidin-coated beads. Thus, the newly synthesized EAAC1 protein molecules that had arrived at the plasma membrane (both 35 S-and biotin-labeled), as well as those that had not yet arrived at the membrane ( 35 S-labeled and nonbiotinylated), were detected. Newly synthesized EAAC1 transporter first arrived at the cell surface at ϳ90 min (Fig. 4A). Based on data shown below (e.g. see Fig. 8), the maturation process peaks between 3 and 4 h and then FIG. 1. Cell surface biotinylation and glycosidase sensitivity of EAAC1 in C6 glioma cells. A, cells were cultured on 100-mm dishes to near confluency and cell surface-biotinylated with (ϩ) or without (Ϫ) 0.5 mg/ml of sulfo-NHS-LC-biotin for 1 h at 4°C as described under "Materials and Methods." The cells were then cultured ("chased") in MEM with 10% FBS for 0, 8, or 24 h under normal conditions to monitor the decay of the biotinylated EAAC1. At the end of each chase time, the total biotinylated proteins were precipitated with monomeric avidin-Sepharose beads and separated on SDS-PAGE, and then the biotinylated EAAC1 protein was detected by immunoblotting. B, C6 glioma cells were biotinylated with sulfo-NHS-LC-biotin for 1 h at 15°C. The cells were then homogenized and solubilized with PES buffer. To determine the total amount of protein, equal amounts of solubilized total cell protein were subjected to immunoblotting for each of the four proteins indicated (filled bars set to 100%). Equal amounts of solubilized proteins were used for monomeric avidin-Sepharose precipitation and then eluted, analyzed with SDS-PAGE, and immunoblotted with antibodies specific for each of the four proteins. The immunoblots were quantified by densitometry and expressed as a percentage of the total cellular content for that particular protein (hatched bars). For the immunoblotting, 1:2000 dilution of anti-EAAC1, 1:1000 dilution of anti-GRP78, and undiluted anti-AS and anti-NaK-ATPase antibodies were used as primary antibodies, and then 1:20,000 dilution of either goatanti-rabbit IgG (for EAAC1 and GRP78) or goat anti-mouse IgG (for AS and NaK-ATPase) were used as the secondary antibodies. C, a total C6 glioma cellular membrane fraction was isolated and solubilized with PES buffer, as described under "Materials and Methods." After immunoprecipitation with anti-EAAC1 antibody, the precipitates were eluted and divided into six aliquots. Each of those samples was then denatured and treated with (ϩ) or without (Ϫ) N-glycosidase F for 15-240 min at 37°C, as described under "Materials and Methods." All of the samples were then diluted with an equal volume of sample dilution buffer, separated on SDS-PAGE, and subjected to immunoblotting analysis with anti-EAAC1 antibody. There were no detectable differences between the 60-, 120-, and 240-min samples, so only the first 60 min are shown. The results presented are representative of several independent experiments.

FIG. 2. De novo biosynthesis of EAAC1 transporter in C6 glioma cells.
Either 3 ϫ 10 6 or 9 ϫ 10 6 C6 glioma cells were placed onto 100-mm dishes and cultured for 20 h before they were metabolically labeled with 200 Ci/ml of [ 35 S]Met-Cys for 15, 30, 60, or 120 min. After 3 h of chase in MEM medium with 10% FBS containing 5 mM each of nonradiolabeled methionine and cysteine, a total cellular membrane fraction was solubilized in PES buffer, as described under "Materials and Methods." An equal amount of starting protein was subjected to immunoprecipitation with anti-EAAC1 and then analyzed with SDS-PAGE and fluorography (A). B illustrates the densitometry data obtained from the data in A.
plateaus or increases only slightly thereafter. The densitometry analysis of the data shows that the targeting of the newly synthesized EAAC1 protein to the plasma membrane coincided with its maturation from the 57-kDa form to the mature 73-kDa monomer (Fig. 4B). Analysis of several experiments not included here and the data of Fig. 8 suggests that the apparent difference in the ratios at 2 h versus 6 h is due to experimental variability.
N-Glycosylation of EAAC1 during Maturation-To study the intracellular targeting of the newly synthesized EAAC1 protein, endoglycosidase digestions were employed. Resistance to Endo H digestion can be used as a hallmark to demonstrate that an N-glycoprotein has proceeded beyond the medial Golgi compartment (27). C6 glioma cells were pulse-labeled with 200 Ci/ml of [ 35 S]Met-Cys for 15 min and then chased in medium without radioactivity for 30 -240 min at 37°C (Fig. 5). EAAC1 protein was immunoprecipitated from a solubilized total cellular membrane fraction and subjected to Endo H digestion for 1 h at 37°C. The immature forms of EAAC1 protein were sensitive to the Endo H digestion, indicating that they are localized in the endoplasmic reticulum or cis-Golgi compartment, still contain high mannose type oligosaccharide chains, and thus are still sensitive to Endo H digestion. The basis for detecting two bands for the precursor protein in this particular series of experiments is unclear. It is interesting that both are deglycosylated and appear to be lost at the same rate during the chase period. In contrast, all of the detected mature EAAC1 monomers were resistant to Endo H digestion. This observation is consistent with the results of Fig. 4, documenting that the mature form of EAAC1 arrives at the cell surface soon after maturation from the 57-kDa to the 73-kDa form.
Determination of the Half-life and Degradation Rate for the EAAC1 Transporter-Pulse-chase labeling of C6 glioma cells was used to establish the rate of degradation of the newly synthesized EAAC1 protein in C6 glioma cells. The cells were metabolically labeled with [ 35 S]Met-Cys for 1 h and then chased in medium containing an excess of nonradiolabeled Met and Cys for 0 -60 h. As shown in Fig. 6, the amount of radiolabeled EAAC1 protein diminished as the chase time was prolonged. From Fig. 6A, it is clear that the amount of all three forms of EAAC1 are proportional to each other throughout the chase period. Therefore, the densitometry readings for the monomer were used for quantification (Fig. 6B). When the data were plotted using the common logarithm of the remaining radiolabeled EAAC1 versus the chase time, there was a lag time of about 8 h prior to initiation of the decay process (Fig.  6C). This lag period was observed consistently in all pulsechase degradation experiments. Given that it takes about 3 h for all of the pulse-labeled EAAC1 protein to arrive at the plasma membrane, these new polypeptides must be protected from decay, not only during the de novo biosynthesis and targeting process as would be expected but also during the initial 5 h after the protein reaches the plasma membrane. However, once the decay process starts, it follows first order kinetics that yields a yield a first order rate constant for degradation (K d ) of 0.12 h Ϫ1 or a half-life of about 6 h (Fig. 6C).
Degradation of the Cell Surface EAAC1 Protein-C6 plasma membrane proteins were labeled with sulfo-NHS-LC-biotin at 15°C for 1 h and then chased in normal MEM medium lacking serum for 0 -12 h at 37°C under normal culture conditions. The biotinylated EAAC1 protein remaining after each chase period was detected using precipitation with immobile monomeric avidin-Sepharose followed by immunoblotting with specific an- ti-EAAC1 antibody (Fig. 7A). The half-life of the cell surface biotinylated EAAC1 protein was 6 h (Fig. 7B), a result consistent with the pulse-chase labeling described above. In contrast to the pulse-chase studies, which determine how long it takes for the newly synthesized EAAC1 protein molecules to be degraded, cell surface biotinylation determines the turnover of the cell surface EAAC1 protein as a whole without discriminating with regard to the age of the protein. The results obtained by both approaches show that the EAAC1 protein was degraded with a half-life of about 6 h under normal conditions in C6 glioma cells.
Residence Time of the EAAC1 Transporter at the Cell Surface-The length of time that the EAAC1 protein resides at the plasma membrane and whether or not intact EAAC1 protein could be detected following removal from the plasma mem-brane were assessed using pulse-chase labeling followed by cell surface biotinylation. Fig. 8A shows the rate of disappearance of the radiolabeled and biotinylated EAAC1 protein in C6 cells. Consistent with the pulse labeling experiments shown above, a lag period of about 8 h was observed prior to the initial loss of the EAAC1 from the cell surface. The loss of the radiolabeled, biotinylated EAAC1 protein represents the disappearance of the EAAC1 from the cell surface, which can result from either protein turnover at the membrane or protein internalization. The plasma membrane-resident EAAC1 protein disappeared from the cell surface at a rate of about 11.5%/h (half-life ϭ 6 h), which is consistent with the total cellular EAAC1 protein decay rate defined by the pulse-chase studies (Figs. 6C and 8B). To confirm this observation and to compare these values within the same experiment, the disappearance rate of the newly synthesized (radiolabeled), plasma membrane-resident (biotinylated) EAAC1 protein was compared with the degradation rate for the total pool of newly synthesized EAAC1 protein (Fig.  9). The rate of EAAC1 removal from the plasma membrane was nearly identical to the rate of EAAC1 degradation. DISCUSSION The results represent a detailed study of the biosynthesis and turnover of the EAAC1 glutamate/aspartate transporter. Collectively, the data obtained indicate that the EAAC1 transporter is co-translationally modified by N-glycosylation and that the transition of the EAAC1 protein from its immature form to mature form coincides with the alteration of the attached oligosaccharide chains from the high mannose type to the complex type. These results also document that once the mature form is fully processed, it rapidly passes through the rest of the Golgi compartment and arrives at the plasma membrane.
Haugeto et al. (12) reported that GLAST1, GLT1, and EAAC1 transporters all formed homomultimers in rat brain and transfected HeLa cells. Furthermore, based on results obtained from radiation inactivation analysis, they postulated that the glutamate transporters operate as homomultimeric complexes in vivo. Consistent with that interpretation, our anti-EAAC1 antibody detected three EAAC1 bands in C6 cell extracts with estimated sizes of 73, 145, and Ͼ200 kDa, which may represent the EAAC1 monomer, dimer, and trimer, respectively. In addition, when C6 glioma cells were pulse-chase labeled, the oligomer forms for the newly synthesized immature EAAC1 protein were also detected, and no change in oligomerization was observed as the pulse or chase time was prolonged. These results suggest that oligomerization happens before EAAC1 synthesis is finished. However, that the oligomerization is an artifact of transporter isolation or manipulation cannot be ruled out. In several experiments when the total membrane fraction from C6 glioma cells was prepared without additional processing, only the EAAC1 monomer was detected. Further analysis of the oligomerization state of the transporter and the impact on function is needed.
As glycoproteins proceed through the endoplasmic reticulum and Golgi compartments, the oligosaccharide chains are modified from high mannose-to complex-type carbohydrate chains. Therefore, in pulse-chase experiments, when the chase time is short, only the immature form of the protein will be detected and, if it has not passed beyond the medial Golgi, this form will be sensitive to endoglycosidase H digestion. For EAAC1, only the immature EAAC1 protein species were detected when the chase period was 45 min or less. These results indicate that the core protein synthesis and the initial stages of glycosylation occur within this time frame. Interestingly, the completion of carbohydrate processing and the trafficking of the mature EAAC1 protein to the plasma membrane were considerably faster. Pulse-chase labeling followed by cell surface biotinylation showed that the arrival of EAAC1 transporter to the cell surface coincided with the shift of EAAC1 from its immature forms to its mature form. These results indicate that there is no significant lag time between these two processes. Furthermore, the difference between the apparent molecular mass of the mature EAAC1 monomer and its immature form is ϳ16 kDa, and no intermediates were observed during its maturation. These results suggest that during the maturation process the trimming and readdition steps for the N-glycosylation modification of each EAAC1 protein molecule occur relatively quickly. The data support a model of rapid transit of the mature EAAC1 protein from the medial Golgi to the plasma membrane.
To establish the residence time of newly synthesized EAAC1 protein at the plasma membrane, cell surface biotinylation was employed immediately after the pulse-chase labeling, followed by anti-EAAC1 immunoprecipitation. When the rate at which the EAAC1 protein disappeared from the surface was compared with that of its degradation, the majority of the EAAC1 protein appeared to be degraded reasonably soon after it left At the end of each chase period, the cells were surface-biotinylated with 0.5 mg/ml sulfo-NHS-LC-biotin in NaKRP for 1 h at 15°C, and then the free biotin was quenched with 50 mM glycine in NaKRP for 2 ϫ 15 min at 15°C. After the PES-solubilized total membrane proteins were prepared, the total EAAC1 protein was immunoprecipitated with anti-EAAC1 antibody, and then monomeric avidin-Sepharose beads were used to precipitate only the biotinylated EAAC1 protein (A). The nonbiotinylated EAAC1 protein was immunoprecipitated from the supernatant of the avidin precipitation through a second incubation with anti-EAAC1 antibody. The logarithm of the densitometry reading of the remaining radiolabeled EAAC1 monomer was plotted against the chase time (B). The data are representative of several individual experiments. At the end of each chase period, the cells were surface-biotinylated with 0.5 mg/ml sulfo-NHS-LC-biotin in NaKRP for 1 h at 15°C, and then the free biotin was quenched with 50 mM glycine in NaKRP for 2 ϫ 15 min at 15°C. As shown in A, after the PES-solubilized total membrane proteins were prepared, total EAAC1 protein was immunoprecipitated with anti-EAAC1 antibody (Total), and then monomeric avidin-Sepharose beads were used to precipitate only the biotinylated EAAC1 protein (Biotinylated). The plot of the logarithm of the densitometry values of the remaining EAAC1 monomer against the chase time is shown in B. The results shown are representative of two independent experiments. the cell surface. The disappearance rate of EAAC1 proteins from the cell surface was almost identical with the degradation rate of the total cellular EAAC1 protein pool. Our results indicate that 70% of EAAC1 resides at the plasma membrane, and for the 30% of transporters present in the intracellular pool, the synthesis and degradation rates are similar to those on the cell surface.
The present data indicate that the half-life for decay of radiolabeled EAAC1 protein is about 6 h. The decay process for the EAAC1 protein did not start as soon as it matured, even though biotinylation suggested that it was exposed at the extracellular surface of the plasma membrane. Instead, there was a lag time of about 8 h between EAAC1 synthesis and the beginning of its degradation, and at least 5 of those 8 h occurred after the protein had arrived at the plasma membrane. The newly synthesized EAAC1 protein then merges with the general EAAC1 protein pool to be degraded by a process exhibiting the expected first order kinetics. What makes these newly synthesized "protected" EAAC1 proteins distinguishable from the rest is unclear, but several possibilities exist. These newly synthesized EAAC1 protein molecules may: 1) be sequestered within specific membrane domains that are inaccessible to endocytosis; 2) lack a structural alteration or post-translational modification that is required for the recognition by an adaptor protein for endocytosis; or 3) lack the proper interaction with another regulatory protein (21) for the recognition by the degradation machinery. Collectively, these results represent the foundation on which to further investigate these possibilities as well as to consider regulation of the biosynthetic and degradative pathways as potential mechanisms for modulating EAAC1 activity under normal and disease states.
Interestingly, data from Davis et al. (19) and Sims et al. (20) indicate that when cells are treated with either protein kinase C or phosphatidylinositol 3-kinase activators, EAAC1 transporters can be recruited from an intracellular pool not accessible to an impermeant biotinylation reagent. The data in this report do not address the issue of EAAC1 recruitment or recycling. Recycling kinetics have been investigated for a number of receptors, but only a few solute transporters. Of the latter, the most thoroughly investigated is the GLUT4 glucose transporter that resides in one or more intracellular compartments and recycles between them and the plasma membrane in response to insulin (28). The current data outlining the synthesis and degradation of EAAC1 as well as documenting an intra-cellular pool of the transporter will permit more detailed studies on recruitment and recycling in the future.