Biosynthesis, Intracellular Targeting, and Degradation of the EAAC1 Glutamate/Aspartate Transporter in C6 Glioma Cells
2002; Elsevier BV; Volume: 277; Issue: 41 Linguagem: Inglês
10.1074/jbc.m202052200
ISSN1083-351X
AutoresWenbo Yang, Michael S. Kilberg,
Tópico(s)Molecular Sensors and Ion Detection
ResumoRat 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-translationallyN-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. 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-translationallyN-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. supplemented Eagle's medium sodium-free Krebs-Ringer phosphate buffer sodium-containing Krebs-Ringer phosphate buffer endoglycosidase H N-glycosidase F sulfosuccinimidyl-6-(biotinamido)hexanoate fetal bovine serum sample dilution buffer A family of Na+-dependent high affinity glutamate/aspartate transport systems, previously referred to collectively as System X−A,G, is essential for the glutamatergic transmission in the central nervous system (1Radian R. Ottersen O.P. Storm-Mathisen J. Castel M. Kanner B.I. J. Neurosci. 1990; 10: 1319-1330Crossref PubMed Google Scholar, 2Danbolt N.C. Storm-Mathisen J. Kanner B.I. Neuroscience. 1992; 51: 295-310Crossref PubMed Scopus (374) Google Scholar, 3Kanai Y. Smith C.P. Hediger M.A. Trends Neurosci. 1993; 16: 365-370Abstract Full Text PDF PubMed Scopus (201) Google Scholar), as well as for nutrition of many other cells. The human cDNAs encoding glutamate/aspartate transporter activity are designated as excitatory amino acid transporters, EAAT1–5 (4Malandro M.S. Kilberg M.S. Annu. Rev. Biochem. 1996; 65: 305-336Crossref PubMed Scopus (181) Google Scholar, 5Fairman W.A. Vandenberg R.J. Arriza J.L. Kavanaugh M.P. Amara S.G. Nature. 1995; 375: 599-603Crossref PubMed Scopus (1019) Google Scholar, 6Arriza J.L. Eliasof S. Kavanaugh M.P. Amara S.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4155-4160Crossref PubMed Scopus (808) Google Scholar), but the rodent counterparts for EAAT1–3 were originally named as GLAST (7Storck T. Schutle S. Hofmann K. Stoffel W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10955-10959Crossref PubMed Scopus (1102) Google Scholar), GLT1 (8Pines G. Danbolt N.C. Bjoras M. Zhang Y. Bendahan A. Eide L. Koepsell H. Storm-Mathisen J. Seeberg E. Kanner B.I. Nature. 1992; 360: 464-467Crossref PubMed Scopus (1141) Google Scholar), and EAAC1 (excitatory amino acidcarrier 1) (9Kanai Y. Hediger M.A. Nature. 1992; 360: 467-471Crossref PubMed Scopus (1202) Google Scholar), respectively. Rat EAAC1 cDNA encodes an integral membrane protein of 523 amino acids with a predicated nonglycosylated core molecular mass of 56.8 kDa (10Velaz-Faircloth M. Am. J. Physiol. 1996; 270: C67-C75Crossref PubMed Google Scholar), but it is N-glycosylated under normal conditions (11Dowd L.A. Coyle A.J. Rothstein J.D. Pritchett D.B. Robinson M.B. Mol. Pharmacol. 1996; 49: 465-473PubMed Google Scholar). It has been reported that GLAST, GLT1, and EAAC1 form homo-multimers, even when detected using immunoblotting under reducing conditions (12Haugeto O. Ullensvang K. Levy L.M. Chaudhry F.A. Honore T. Nielsen M. Lehre K.P. Danbolt N.C. J. Biol. Chem. 1996; 271: 27715-27722Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar). On analysis with denaturing gels, the mature monomers of these transporters each runs as a broad band between 66 and 74 kDa, which is likely due to the microheterogeneity of theirN-glycosylation sites and possibly other post-translational modifications such as phosphorylation (11Dowd L.A. Coyle A.J. Rothstein J.D. Pritchett D.B. Robinson M.B. Mol. Pharmacol. 1996; 49: 465-473PubMed Google Scholar). 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 (9Kanai Y. Hediger M.A. Nature. 1992; 360: 467-471Crossref PubMed Scopus (1202) Google Scholar, 10Velaz-Faircloth M. Am. J. Physiol. 1996; 270: C67-C75Crossref PubMed Google Scholar), and placenta (13Matthews J.C. Beveridge M.J. Malandro M.S. Rothstein J.D. Campbell-Thompson M. Verlander J.W. Kilberg M.S. Novak D.A. Am. J. Physiol. 1998; 274: C603-C614Crossref PubMed Google Scholar). 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 γ-aminobutyric acid synthesis or for other metabolic reactions in the brain (14Kanai Y. Bhide P.G. Difiglia M. Hediger M.A. Neuroreport. 1995; 6: 2357-2362Crossref PubMed Scopus (96) Google Scholar). 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 (15Bissell M.G. Rubinstein L.J. Bignami A. Herman M.M. Brain Res. 1974; 82: 77-80Crossref PubMed Scopus (138) Google Scholar). These cells express a high level of System X−A,G transport activity (16Deas J. Erecinska M. Brain Res. 1989; 483: 84-90Crossref PubMed Scopus (12) Google Scholar). Although there are reports that GLT1 is expressed in C6 cells (17Casado M. Bendahan A. Francisco Z. Niels D.C. Carmen A. Gimenez C. Kanner B.I. J. Biol. Chem. 1993; 268: 27313-27317Abstract Full Text PDF PubMed Google Scholar), most evidence indicates that C6 glioma cells express EAAC1 but not GLAST, GLT1, or EAAT4 (11Dowd L.A. Coyle A.J. Rothstein J.D. Pritchett D.B. Robinson M.B. Mol. Pharmacol. 1996; 49: 465-473PubMed Google Scholar, 18Palos T.P. Ramachandran B. Boado R. Howard B.D. Mol. Brain Res. 1996; 37: 297-303Crossref PubMed Scopus (68) Google Scholar). 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 (19Davis K.E. Straff D.J. Weinstein E.A. Bannerman P.G. Correale D.M. Rosthein J.D. Robinson M.B. J. Neurosci. 1998; 18: 2475-2485Crossref PubMed Google Scholar, 20Sims K.D. Straff D.J. Robinson M.B. J. Biol. Chem. 2000; 274: 5228-5327Abstract Full Text Full Text PDF Scopus (116) Google Scholar), suggesting the redistribution of EAAC1 from an intracellular compartment. Furthermore, Lin et al. (21Lin C.I. Orlov I. Ruggerio A.M. Dykes-Hoberg M. Lee A. Jackson M. Rothstein J.D. Nature. 2001; 410: 84-88Crossref PubMed Scopus (198) Google Scholar) 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. C6 glioma cells were obtained from the American Type Culture Collection (CCL107) and maintained in supplemented Eagle's medium (MEM)1 containing 10% FBS as a monolayer culture under a humidified atmosphere of 5% CO2, 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. Amino acid uptake by C6 glioma cells was measured using the cluster tray method (22Kilberg M.S. Methods Enzymol. 1989; 173: 564-575Crossref PubMed Scopus (47) Google Scholar). 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+, the cells were incubated at 37 °C twice for 15 min each in sodium-free Krebs-Ringers phosphate buffer (choline-KRP). To initiate transport, [3H]aspartate in 250 μl 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+-dependent transport is taken as the difference between uptake in NaKRP and choline-KRP. The data are expressed as pmol·mg−1 protein·min−1 and are presented as the averages of four assays on at least two different batches of cultured cells. Gel electrophoresis was performed in 7.5% polyacrylamide gels following the protocol originally described by Laemmli (23Laemmli U.K. Nature. 1970; 227: C1-C24Crossref PubMed Scopus (207537) Google Scholar). The protein samples were diluted with an equal volume of sample dilution buffer (SDB) consisting of 2% (w/v) SDS, 5% β-mecaptoethanol, 30 μg/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 (13Matthews J.C. Beveridge M.J. Malandro M.S. Rothstein J.D. Campbell-Thompson M. Verlander J.W. Kilberg M.S. Novak D.A. Am. J. Physiol. 1998; 274: C603-C614Crossref PubMed Google Scholar). To study the de novo biosynthesis and the intracellular targeting of the EAAC1 transporter protein in C6 cells, pulse-chase labeling withl-[35S]methionine-cysteine (ProMix; AmershamBiosciences) was used. After placing 9 × 106 cells onto each 100-mm culture dish or 2.3 × 107 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/58-cm2 surface area of methionine- and cysteine-free Dulbecco's modified Eagle's medium (Invitrogen) for 2 × 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 μCi/ml of [35S]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 mmeach 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 of the EAAC1 transporter protein was performed following the procedure outlined by Harlow and Lane (24Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar) with modifications. After pulse-chase labeling with l-[35S]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 μg/ml each of leupeptin, aprotonin, pepstatin,N-tosyl-l-phenylalanine chloromethyl ketone, andN-p-tosyl-l-lysine chloromethyl ketone) at −80 °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 × g for 10 min to remove unbroken cells and nuclei, and the supernatant was centrifuged at 280,000 × g for 1 h at 4 °C to collect a total membrane pellet, which was then extracted in PES buffer (2% C12E9, 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 × g for 15 min at 4 °C, the supernatant was recovered, and the protein concentration was determined by a modified Lowry method (24Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar) or the bicinchoninic acid method (Pierce protein assay kit). An equal amount of starting protein (100–500 μg) from each sample was transferred into microcentrifuge tubes, brought to a volume of 500 μl 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 μg) and 50 μl 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 × 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 μg of IgG) was added to the precleared extract and incubated at 4 °C overnight with constant mixing. A 50-μl 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 × 1 min) and with PES containing 0.35 mNaCl (for a total salt concentration of 0.5 m) (4 × 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% β-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 −80 °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 μg 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 μg 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 μg of anti-EAAC1 antibody was proportional to the amount of starting protein from 0–500 μg (data not shown). To test for the endoglycosidase H (Endo H) sensitivity of the newly synthesized EAAC1, C6 cells were pulse-labeled for 15 min with [35S]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 μl of 5× denaturing solution containing 2.5% SDS, 5% β-mercaptoethanol in water for 30 min at 37 °C. After dilution to 1× denaturing solution with 40 μl of water, each of the eluates was collected and then divided into two microcentrifuge tubes. A 110 volume of 500 mmsodium citrate, pH 5.5, and 500–1000 units (1–2 μl) of Endo H (BioLabs Inc.) was added to each of the tubes. For the control tube, 1–2 μl of enzyme storage buffer (20 mm Tris-HCl, pH 7.5, 50 mm NaCl, and 5 mm Na2EDTA) 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 2× 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 μl of 5× denaturing solution (2.5% SDS, 5% β-mercaptoethanol) for 30 min at 37 °C and then diluted to 1× denaturing solution with water, as described above for the Endo H digestions. For each PNGase F digestion, 110 volume each of 10% Nonidet P-40 and a buffer consisting of 10% Nonidet P-40, 500 μm 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 2× SDB buffer before they were loaded onto a SDS-PAGE gel for separation and autoradiographic detection. 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 KHCO3, 5.6 mm glucose, 25 mmNa2HPO4, 0.5 mm CaCl2, 1.2 mm MgSO4, 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 mmglycine 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 −80 °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 [35S]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 × 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 −80 °C until all of the samples were collected. The surface-biotinylated 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 0.1m 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 × g for 15 s to remove the beads, the eluate was removed and neutralized with 50 μl of 0.1m Tris-HCl, pH 9.5. For the selective precipitation of the biotinylated EAAC1 molecules, 25 μl 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 2× SDB containing 2 mm free d-biotin and separated by gel electrophoresis followed by fluorography, as described above. 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. 1 A, 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 ∼70% of EAAC1 protein was labeled by the cell surface reaction (Fig. 1 B). 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. 1 B). 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. The deduced amino acid sequence for the rat EAAC1 transporter contains four putativeN-glycosylation consensus sequences, and three of these predicted N-glycosylation sites are localized within the second extracellular loop (10Velaz-Faircloth M. Am. J. Physiol. 1996; 270: C67-C75Crossref PubMed Google Scholar). It has been reported that the EAAC1 transporter is N-glycosylated and that its glycosylation pattern may vary in different host cell lines (11Dowd L.A. Coyle A.J. Rothstein J.D. Pritchett D.B. Robinson M.B. Mol. Pharmacol. 1996; 49: 465-473PubMed Google Scholar). To test for theN-glycosylation of EAAC1 protein in C6 cells, PNGase F digestion was used to cleave between the first GlcNAc residue and the asparagine residue (25Maley F. Anal. Biochem. 1989; 180: 195-204Crossref PubMed Scopus (646) Google Scholar). Without PNGase F digestion, EAAC1 monomer and oligomer bands were detected after immunoprecipitation of solubilized total membrane proteins with anti-EAAC1 antibody (Fig. 1 C). 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. (11Dowd L.A. Coyle A.J. Rothstein J.D. Pritchett D.B. Robinson M.B. Mol. Pharmacol. 1996; 49: 465-473PubMed Google Scholar) and show that deglycosylation of the EAAC1 dimer does not reverse its oligomerization. To determine the biosynthesis rate of EAAC1 protein in C6 glioma cells, the cells were metabolically labeled with 200 μCi/ml of [35S]Met-Cys for 15–120 min and then chased for 3 h. As shown in Fig. 2 A, 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. 2 B). 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. To study the maturation process of EAAC1, C6 glioma cells were pulse-labeled with 500 μCi/ml of [35S]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 = 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 μCi/ml of [35S]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 amo
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