The Structural and Functional Units of Heteromeric Amino Acid Transporters
2006; Elsevier BV; Volume: 281; Issue: 36 Linguagem: Inglês
10.1074/jbc.m604049200
ISSN1083-351X
AutoresEsperanza Fernández, Maite Jiménez-Vidal, Marı́a Calvo, António Zorzano, Francesc Tebar, Manuel Palacı́n, Juan J. Chillarón,
Tópico(s)Photoreceptor and optogenetics research
ResumoHeteromeric amino acid transporters are composed of a catalytic light subunit and a heavy subunit linked by a disulfide bridge. We analyzed the structural and functional units of systems b0,+ and x -C, formed by the heterodimers b0,+AT-rBAT and xCT-4F2hc, respectively. Blue Native gel electrophoresis, cross-linking, and fluorescence resonance energy transfer in vivo indicate that system b0,+ is a heterotetramer [b0,+AT-rBAT]2, whereas xCT-4F2hc seems not to stably or efficiently oligomerize. However, substitution of the heavy subunit 4F2hc for rBAT was sufficient to form a heterotetrameric [xCT-rBAT]2 structure. The functional expression of concatamers of two light subunits (which differ only in their sensitivity to inactivation by a sulfhydryl reagent) suggests that a single heterodimer is the functional unit of systems b0,+ and x -C. Heteromeric amino acid transporters are composed of a catalytic light subunit and a heavy subunit linked by a disulfide bridge. We analyzed the structural and functional units of systems b0,+ and x -C, formed by the heterodimers b0,+AT-rBAT and xCT-4F2hc, respectively. Blue Native gel electrophoresis, cross-linking, and fluorescence resonance energy transfer in vivo indicate that system b0,+ is a heterotetramer [b0,+AT-rBAT]2, whereas xCT-4F2hc seems not to stably or efficiently oligomerize. However, substitution of the heavy subunit 4F2hc for rBAT was sufficient to form a heterotetrameric [xCT-rBAT]2 structure. The functional expression of concatamers of two light subunits (which differ only in their sensitivity to inactivation by a sulfhydryl reagent) suggests that a single heterodimer is the functional unit of systems b0,+ and x -C. Heteromeric amino acid transporters (HAT) 7The abbreviations used are: HAT, heteromeric amino acid transporters; DMS, dimethyl suberimidate; HSHAT, heavy subunits of HAT; LSHAT, light subunits of HAT; pCMB, p-chloromercuribenzoate; CFP, cyan fusion protein; YFP, yellow fusion protein; Wt, wild-type; FRET, fluorescence resonance energy transfer; FRETN, normalized sensitized FRET; NTA, nitrilotriacetic acid; DTT, dithiothreitol; EGFR, epidermal growth factor receptor; MTS, methanethiosulfonate; MTSEA, 2-aminoethyl methanethiosulfonate. have a unique architecture composed of disulfide-linked heavy (HSHAT) and light (LSHAT) subunits (1Palacin M. Kanai Y. Pfluegers Arch. Eur. J. Physiol. 2004; 447: 490-494Crossref PubMed Scopus (135) Google Scholar, 2François V. Closs I. Wagner A. Manuel P. Hitoshi E. Yoshikatsu K. Pfluegers Arch. Eur. J. Physiol. 2004; 447: 532-542Crossref PubMed Scopus (561) Google Scholar). HSHAT are type II membrane glycoproteins involved in trafficking the heterodimer to the plasma membrane, whereas LSHAT are polytopic membrane proteins that confer transport function and specificity (1Palacin M. Kanai Y. Pfluegers Arch. Eur. J. Physiol. 2004; 447: 490-494Crossref PubMed Scopus (135) Google Scholar, 2François V. Closs I. Wagner A. Manuel P. Hitoshi E. Yoshikatsu K. Pfluegers Arch. Eur. J. Physiol. 2004; 447: 532-542Crossref PubMed Scopus (561) Google Scholar, 3Gasol E. Jimenez-Vidal M. Chillaron J. Zorzano A. Palacin M. J. Biol. Chem. 2004; 279: 31228-31236Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Two heavy subunits are known, rBAT and 4F2hc. The former constitutes system b0,+ with the light subunit b0,+AT (4Fernandez E. Carrascal M. Rousaud F. Abian J. Zorzano A. Palacin M. Chillaron J. Am. J. Physiol. Renal Physiol. 2002; 283: 540-548Crossref PubMed Scopus (85) Google Scholar). 4F2hc dimerizes with at least six of the other LSHAT to form several transport systems (2François V. Closs I. Wagner A. Manuel P. Hitoshi E. Yoshikatsu K. Pfluegers Arch. Eur. J. Physiol. 2004; 447: 532-542Crossref PubMed Scopus (561) Google Scholar). The physiological relevance of HAT is being increasingly recognized (1Palacin M. Kanai Y. Pfluegers Arch. Eur. J. Physiol. 2004; 447: 490-494Crossref PubMed Scopus (135) Google Scholar, 2François V. Closs I. Wagner A. Manuel P. Hitoshi E. Yoshikatsu K. Pfluegers Arch. Eur. J. Physiol. 2004; 447: 532-542Crossref PubMed Scopus (561) Google Scholar). Mutations in b0,+AT-rBAT and y+LAT1 (which dimerizes with 4F2hc) cause cystinuria and lysinuric protein intolerance, respectively (5Borsani G. Bassi M.T. Sperandeo M.P. De Grandi A. Buoninconti A. Riboni M. Manzoni M. Incerti B. Pepe A. Andria G. Ballabio A. Sebastio G. Nat. 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Genet. 2005; 42: 58-68Crossref PubMed Scopus (122) Google Scholar, 9Pras E. Arber N. Aksentijevich I. Katz G. Schapiro J.M. Prosen L. Gruberg L. Harel D. Liberman U. Weissenbach J. Pras M. Kastner D.L. Nat. Genet. 1994; 6: 415-419Crossref PubMed Scopus (110) Google Scholar, 10Torrents D. Mykkanen J. Pineda M. Feliubadalo L. Estevez R. de Cid R. Sanjurjo P. Zorzano A. Nunes V. Huoponen K. Reinikainen A. Simell O. Savontaus M.L. Aula P. Palacin M. Nat. Genet. 1999; 21: 293-296Crossref PubMed Scopus (240) Google Scholar). Together with LAT2-4F2hc, these transporters cooperate in the net reabsorption of cystine and dibasic amino acids in the proximal tubule (4Fernandez E. Carrascal M. Rousaud F. Abian J. Zorzano A. Palacin M. Chillaron J. Am. J. Physiol. Renal Physiol. 2002; 283: 540-548Crossref PubMed Scopus (85) Google Scholar, 11Bauch C. Forster N. Loffing-Cueni D. Summa V. Verrey F. J. Biol. Chem. 2003; 278: 1316-1322Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 12Fernandez E. Torrents D. Chillaron J. Martin D.R. Zorzano A. Palacin M. J. Am. Soc. Nephrol. 2003; 14: 837-847Crossref PubMed Scopus (38) Google Scholar). System x -C (xCT-4F2hc) mediates cystine uptake and glutamate efflux (13Bannai S. J. Biol. Chem. 1986; 261: 2256-2263Abstract Full Text PDF PubMed Google Scholar, 14Bassi M.T. Gasol E. Manzoni M. Pineda M. Riboni M. Martin R. Zorzano A. Borsani G. Palacin M. Pfluegers Arch. Eur. J. Physiol. 2001; 442: 286-296Crossref PubMed Scopus (122) Google Scholar). Cytosolic cystine is rapidly reduced to cysteine, the limiting substrate for the synthesis of intracellular glutathione (13Bannai S. J. Biol. Chem. 1986; 261: 2256-2263Abstract Full Text PDF PubMed Google Scholar, 15Sato H. Tamba M. Ishii T. Bannai S. J. Biol. Chem. 1999; 274: 11455-11458Abstract Full Text Full Text PDF PubMed Scopus (763) Google Scholar). In vivo, system x -C is involved in cocaine relapse through the control of the basal levels of extra-synaptic glutamate (16Baker D.A. McFarland K. Lake R.W. Shen H. Tang X.C. Toda S. Kalivas P.W. Nat. Neurosci. 2003; 6: 743-749Crossref PubMed Scopus (617) Google Scholar), and it contributes to maintaining the plasma redox balance (17Sato H. Shiiya A. Kimata M. Maebara K. Tamba M. Sakakura Y. Makino N. Sugiyama F. Yagami K.i. Moriguchi T. Takahashi S. Bannai S. J. Biol. Chem. 2005; 280: 37423-37429Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar). Recent evidence indicates that LAT1-4F2hc, which transports bulky hydrophobic amino acids, may play a crucial role in carcinogenesis both in cellular growth and survival signaling, thereby making it potential target for cancer therapy (for review, see Ref. 18Fuchs B.C. Bode B.P. Semin. Cancer Biol. 2005; 15: 254-266Crossref PubMed Scopus (561) Google Scholar). Moreover, 4F2hc is an integrin-associated protein that mediates integrin-dependent signals, which promote tumorigenesis (19Feral C.C. Nishiya N. Fenczik C.A. Stuhlmann H. Slepak M. Ginsberg M.H. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 355-360Crossref PubMed Scopus (198) Google Scholar). Despite the important roles attributed to HAT, few studies have addressed the structure-function relationship of these transporters; (i) most HAT are obligate antiporters with a 1:1 stoichiometry (20Chillaron J. Roca R. Valencia A. Zorzano A. Palacin M. Am. J. Physiol. Renal Physiol. 2001; 281: 995-1018Crossref PubMed Google Scholar), (ii) light subunits appear to be necessary and sufficient for transport activity, as demonstrated for b0,+AT (21Reig N. Chillaron J. Bartoccioni P. Fernandez E. Bendahan A. Zorzano A. Kanner B. Palacin M. Bertran J. EMBO J. 2002; 21: 4906-4914Crossref PubMed Scopus (91) Google Scholar); (iii) in using xCT as a model for the light subunits, we showed a membrane topology with 12 transmembrane domains and revealed that the residues His-110 and Cys-327 are crucial for function (3Gasol E. Jimenez-Vidal M. Chillaron J. Zorzano A. Palacin M. J. Biol. Chem. 2004; 279: 31228-31236Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 22Jimenez-Vidal M. Gasol E. Zorzano A. Nunes V. Palacin M. Chillaron J. J. Biol. Chem. 2004; 279: 11214-11221Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). In non-reducing SDS-PAGE conditions 4F2hc-associated heterodimers run mainly as a 125-kDa band (23Rossier G. Meier C. Bauch C. Summa V. Sordat B. Verrey F. Kuhn L.C. J. Biol. Chem. 1999; 274: 34948-34954Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar), whereas rBAT and b0,+AT run as a band of 130 kDa (the disulfide-linked b0,+AT-rBAT) and an additional band of 250 kDa (4Fernandez E. Carrascal M. Rousaud F. Abian J. Zorzano A. Palacin M. Chillaron J. Am. J. Physiol. Renal Physiol. 2002; 283: 540-548Crossref PubMed Scopus (85) Google Scholar). However, neither the native structure nor the functional unit of the HAT is known. Here we present evidence indicating that the single heterodimer is the functional unit of systems b0,+ and x -C. 4F2hc-associated heterodimers seem not to form stable oligomers, whereas b0,+AT-rBAT is a heterotetramer in vivo. Finally, we demonstrate that rBAT promotes the oligomerization of HAT. The construction of the distinct cDNAs is described under supplemental "Experimental Procedures." Reagents and Antibodies—Reagents were obtained from Sigma if not indicated otherwise. Antibodies against human and mouse b0,+AT and rBAT are described elsewhere (4Fernandez E. Carrascal M. Rousaud F. Abian J. Zorzano A. Palacin M. Chillaron J. Am. J. Physiol. Renal Physiol. 2002; 283: 540-548Crossref PubMed Scopus (85) Google Scholar, 24Furriols M. Chillaron J. Mora C. Castello A. Bertran J. Camps M. Testar X. Vilaro S. Zorzano A. Palacin M. J. Biol. Chem. 1993; 268: 27060-27068Abstract Full Text PDF PubMed Google Scholar, 25Font M.A. Feliubadalo L. Estivill X. Nunes V. Golomb E. Kreiss Y. Pras E. Bisceglia L. d'Adamo A.P. Zelante L. Gasparini P. Bassi M.T. George Jr., A.L. Manzoni M. Riboni M. Ballabio A. Borsani G. Reig N. Fernandez E. Zorzano A. Bertran J. Palacin M. Hum. Mol. Genet. 2001; 10: 305-316Crossref PubMed Scopus (115) Google Scholar). The anti-Xpress antibody was purchased from Invitrogen, and the anti-Myc 9E10 hybridoma was from ATCC. The rabbit polyclonal antibody against mouse LAT2 was produced at Research Genetics. The antigenic peptide was PIFKPTPVKDPDSEEQP (the C-terminal 17 residues). Specificity of this antibody was tested by comparing the signal in Western blot with the preimmune antisera obtained from the same rabbit (data not shown). cRNA Synthesis, Injection, and Maintenance of Xenopus Oocytes—The synthesis of human 4F2hc cRNA has been described elsewhere (26Estevez R. Camps M. Rojas A.M. Testar X. Deves R. Hediger M.A. Zorzano A. Palacin M. FASEB J. 1998; 12: 1319-1329Crossref PubMed Scopus (72) Google Scholar). In vitro synthesis of human xCT wild type, xCT C327S, and xCT concatamers was conducted with the NotI-linearized plasmid template using the in vitro transcription protocol from AMBION (mMESSAGE mMACHINE, Ambion, Austin, TX). Mixtures of cRNAs were prepared immediately before injection with a calibrated pipette. The amount of transcribed RNA was calculated by 260-nm absorbance measurement before microinjection into Xenopus oocytes. Each of the cRNA species was synthesized at least on two occasions. In mixing experiments, oocytes were injected with 4F2hc cRNA (5 ng) together with xCT Wt (5 ng), xCT C327S (5 ng), or with a mixture of xCT Wt and xCT C327S (total amount of 5 ng of xCT cRNAs). For the concatamers, 25 ng of the corresponding cRNA together with 5 ng of 4F2hc cRNA were injected per oocyte. The procedures for injection and maintenance of oocytes have been described in detail elsewhere (27Bertran J. Werner A. Moore M.L. Stange G. Markovich D. Biber J. Testar X. Zorzano A. Palacin M. Murer H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5601-5605Crossref PubMed Scopus (198) Google Scholar). Oocytes were injected with either 50 nl of water or 50 nl of water containing the cRNAs. Oocytes were incubated in modified Barth's solution, and the experiments were performed 2-4 days after injection. Cell Culture and Transfection—Growth, maintenance, and calcium phosphate transient transfection was performed as described (25Font M.A. Feliubadalo L. Estivill X. Nunes V. Golomb E. Kreiss Y. Pras E. Bisceglia L. d'Adamo A.P. Zelante L. Gasparini P. Bassi M.T. George Jr., A.L. Manzoni M. Riboni M. Ballabio A. Borsani G. Reig N. Fernandez E. Zorzano A. Bertran J. Palacin M. Hum. Mol. Genet. 2001; 10: 305-316Crossref PubMed Scopus (115) Google Scholar). The efficiency of transfection was above 70% in all experiments. For fluorescence resonance energy transfer (FRET) analysis, the cells were plated on a 6-well plate and transiently transfected using FuGENE-6 (Roche Applied Science). This reagent (6 μl) was added to serum-free medium (100 μl) at room temperature for 5 min. This medium was incubated with plasmids encoding the CFP and YFP fusion proteins (1 μg each) for 30 min, and the mixture was added to the cells grown in culture medium. After 6 h of incubation, the cells were washed twice with phosphate-buffered saline, trypsinized, and reseeded on a 6-well plate containing one coverslip (22 mm, Electron Microscopy Science) per well. Transport Assays and Transport Reconstitution—Influx rates of 100 μm l-[3H]glutamate (ARC) or 20 μm l-[35S]cystine (Amersham Biosciences) in Xenopus oocytes or transfected HeLa cells were performed as described (22Jimenez-Vidal M. Gasol E. Zorzano A. Nunes V. Palacin M. Chillaron J. J. Biol. Chem. 2004; 279: 11214-11221Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 25Font M.A. Feliubadalo L. Estivill X. Nunes V. Golomb E. Kreiss Y. Pras E. Bisceglia L. d'Adamo A.P. Zelante L. Gasparini P. Bassi M.T. George Jr., A.L. Manzoni M. Riboni M. Ballabio A. Borsani G. Reig N. Fernandez E. Zorzano A. Bertran J. Palacin M. Hum. Mol. Genet. 2001; 10: 305-316Crossref PubMed Scopus (115) Google Scholar). The effect of pCMB and MTS reagents (Toronto Research Chemicals, Inc.) was assayed as described (3Gasol E. Jimenez-Vidal M. Chillaron J. Zorzano A. Palacin M. J. Biol. Chem. 2004; 279: 31228-31236Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 22Jimenez-Vidal M. Gasol E. Zorzano A. Nunes V. Palacin M. Chillaron J. J. Biol. Chem. 2004; 279: 11214-11221Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). 1 mm pCMB and 2.5 mm MTSEA inhibited the activity of wild-type xCT (22Jimenez-Vidal M. Gasol E. Zorzano A. Nunes V. Palacin M. Chillaron J. J. Biol. Chem. 2004; 279: 11214-11221Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) and wildtype b0,+AT, respectively. Reconstitution of wild type b0,+AT and the C321S mutant into liposomes and uptake measurements in the reconstituted system were performed as described (21Reig N. Chillaron J. Bartoccioni P. Fernandez E. Bendahan A. Zorzano A. Kanner B. Palacin M. Bertran J. EMBO J. 2002; 21: 4906-4914Crossref PubMed Scopus (91) Google Scholar). Membrane Preparation and Protein Purification by Ni2+-NTA Chromatography—Kidney brush border membranes were obtained as described (4Fernandez E. Carrascal M. Rousaud F. Abian J. Zorzano A. Palacin M. Chillaron J. Am. J. Physiol. Renal Physiol. 2002; 283: 540-548Crossref PubMed Scopus (85) Google Scholar). For the preparation of kidney total membranes, the kidney was homogenized in 25 mm Hepes, pH 7.4, 4 mm EDTA, 250 mm sucrose, and 20 mm N-ethylmaleimide with the protease inhibitors aprotinin, leupeptin, phenylmethylsulfonyl fluoride, and pepstatin on a CPCU Polytron. The homogenate was centrifuged at 10,000 × g for 10 min at 4 °C, and the supernatant was further centrifuged at 200,000 × g for 90 min at 4 °C. A similar procedure was used to obtain total membranes from HeLa cells, but the cells (∼107 cells/1 ml homogenization buffer) were homogenized by 15 passages through a 25-gauge needle. For Blue Native PAGE or cross-linking, the membranes were resuspended directly in the appropriate buffers (see below). For Ni2+-NTA chromatography, total membranes from HeLa cells were resuspended in 25 mm Tris-HCl, pH 7, 50 mm NaCl, 1% digitonin (final detergent/protein (w/w) ratio of 3.3), and solubilization proceeded for 30 min. Insoluble material was discarded by a 10,000 × g centrifugation at 4 °C for 10 min. The supernatant was diluted in the above buffer containing 15 mm imidazole and applied to the Ni2+-NTA beads (Qiagen). After 30 min of end-over-end mixing at room temperature, the beads were washed 4 times in a similar buffer containing 0.1% digitonin and 10 mm imidazole. Elution was performed by raising the imidazole concentration to 100 mm for 10 min at room temperature. The eluted material was then processed for Blue Native PAGE (see below). Blue Native and SDS-PAGE—In preliminary experiments we tested a range of detergent/protein (w/w) ratios with a fixed Coomassie Blue G/detergent ratio (w/w) of 1/2.5 (28Nicke A. Baumert H.G. Rettinger J. Eichele A. Lambrecht G. Mutschler E. Schmalzing G. EMBO J. 1998; 17: 3016-3028Crossref PubMed Scopus (487) Google Scholar). With 0.5-1% digitonin extracts and a 3.3/1 detergent/protein ratio, we observed that rBAT and b0,+AT appeared as a single band of 535 ± 18 kDa (Fig. 1A). Similar results were obtained with 0.25-0.5% dodecyl-β-d-maltoside, although a smear appeared above the ∼535-kDa band. N-Octylglucoside did not resolve any band, and Triton-X-100 increased the smearing until no distinct band could be seen (data not shown). We did not observe any additional bands. Digitonin/protein ratios below 3.3/1 increased smearing and decreased the intensity of the ∼535-kDa band, and at a ratio of 0.25/1 no band was detected; ratios up to 10/1 were similar to the 3.3/1 ratio (data not shown). Membranes were solubilized for 30 min at room temperature in 25 mm Tris-HCl, pH 7, 50 mm NaCl, 1% digitonin at a detergent/protein ratio (w/w) of 3.3 (see above). Further treatment with a range of urea concentrations, 100 mm DTT, or 2% SDS with or without 100 mm DTT was carried out at 37 °C for 30 min. After solubilization, Blue Native buffer was added, and Blue Native PAGE was performed as described (29Schagger H. von Jagow G. Anal. Biochem. 1991; 199: 223-231Crossref PubMed Scopus (1918) Google Scholar, 30Schagger H. Cramer W.A. von Jagow G. Anal. Biochem. 1994; 217: 220-230Crossref PubMed Scopus (1043) Google Scholar). Native molecular weight markers (Amersham Biosciences) were visualized by Coomassie staining. For control SDS-PAGE, SDS sample buffer (without DTT) was added to the solubilized samples, which were immediately loaded (without heating) in SDS-PAGE gels. Western blots were performed as described (31Pineda M. Wagner C.A. Broer A. Stehberger P.A. Kaltenbach S. Gelpi J.L. Martin D.R. Zorzano A. Palacin M. Lang F. Broer S. Biochem. J. 2004; 377: 665-674Crossref PubMed Scopus (43) Google Scholar). After transference of Blue Native gels, the membrane was destained in methanol 50% and acetic acid 10% to eliminate excess Coomassie Blue G (Serva) and washed with bi-distilled water before blocking. Cross-linking—Total membrane proteins from transfected HeLa cells were resuspended in phosphate-buffered saline and incubated at 2 mg/ml with the cross-linker dimethyl suberimidate (DMS, from Pierce) for 30 min at room temperature. Cross-linking was terminated by the addition of 100 mm Tris-HCl, pH 8.0, for 15 min at room temperature. FRET—For FRET analysis the concatamers CFP-b0,+AT/rBAT 8The notation LSHAT-HSHAT (i.e.b0,+AT-rBAT) indicates that the light subunit LSHAT and the heavy subunit HSHAT are disulfide-linked and expressed from distinct genes/vectors; the notation LSHAT/HSHAT (i.e. b0,+AT/rBAT) indicates that the two subunits are linked as a fusion protein (concatamer). and YFP-b0,+AT/rBAT, CFP-xCT/4F2hc and YFP-xCT/4F2hc, and the CFP-EGFR and YFP-EGFR fusion proteins were transiently expressed in HeLa cells. A Leica TCS SL laser scanning confocal spectral microscope (Leica Microsystems Heidelberg GmbH, Manheim, Germany) equipped with an argon laser, 63× oil immersion objective lens and a double dichroic filter (458/514 nm) was used. We used CFP as the donor fluorochrome paired with YFP as the acceptor fluorochrome. FRET measurements were based on the sensitized emission method previously described (32Gordon G.W. Berry G. Liang X.H. Levine B. Herman B. Biophys. J. 1998; 74: 2702-2713Abstract Full Text Full Text PDF PubMed Scopus (729) Google Scholar, 33Sorkin A. McClure M. Huang F. Carter R. Curr. Biol. 2000; 10: 1395-1398Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar) with minor modifications for the confocal microscope. In some experiments in which the sensitized emission method was performed, FRET efficiency was also calculated using the acceptor photobleaching method (34Herrick-Davis K. Grinde E. Mazurkiewicz J.E. Biochemistry. 2004; 43: 13963-13971Crossref PubMed Scopus (76) Google Scholar, 35Scholze P. Freissmuth M. Sitte H.H. J. Biol. Chem. 2002; 277: 43682-43690Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). The same set of transfected cells (but distinct dishes) was used for both methods. Sensitized Emission Method—The sensitized emission method is based on the increase of acceptor fluorescence caused by FRET during excitation. To measure FRET, three images were acquired in the same order in all experiments through 1) the CFP channel (absorbance 458 nm, emission 465-510 nm), 2) the FRET channel (absorbance 458 nm, emission 525-600 nm) 3) the YFP channel (absorbance 514 nm, emission 525-600 nm). Background was subtracted from images before performing FRET calculations. Control and experiment images were taken under the same conditions of photomultiplier gain, offset, and pinhole aperture. The FRET image must be corrected for the cross-talk of the donor emission and the direct excitation of the acceptor by the donor excitation wavelength. The crossover of the donor and acceptor fluorescence through the FRET filter is a constant proportion between the fluorescence intensity levels of donor and acceptor and their bleed-through. To correct for this spectral bleed-through of the donor and acceptor through the FRET filter, images of cells expressing only CFP-b0,+AT/rBAT, CFP-xCT/4F2hc, or CFP-EGFR and cells expressing only YFP-b0,+AT/rBAT, YFP-xCT/4F2hc, or YFP-EGFR were also taken under the same conditions as for the experiments. Corrected FRET was calculated on a pixel-by-pixel basis for the entire image by using equation,FRETC=FRET-(A×CFP)-(B×YFP) where FRET, CFP, and YFP correspond to background-subtracted images of cells expressing CFP and YFP acquired through the FRET, CFP, and YFP channels, respectively. A (equal to 0.20) and B (equal to 0.21) correspond to the cross-talk coefficient of CFP and YFP through the FRET filter set, respectively. Images of FRETC intensity were renormalized using a look-up table in which the minimum and maximum values are displayed as blue and red, respectively. Mean FRETC values were calculated from mean fluorescence intensities for each selected region of interest following Equation 1, and normalized sensitized FRET (FRETN) values for selected sub-regions of the image were calculated on the basis of the equation,FRETN=FRETC/(YFP×CFP) where FRETC, CFP, and YFP are the mean intensities of FRETC, CFP, and YFP fluorescence in the selected sub-region of the image. The negative FRETC values obtained in some experiments are due to slight over-estimation of the spectral bleed-through coefficients. Areas with unusually high or low CFP/YFP ratios (i.e. outside the 1:1 to 1:4 stoichiometric range) were excluded from analysis. All calculations were performed using the Image Processing Leica Confocal Software and Microsoft Excel. Acceptor Photobleaching Method—In the presence of FRET, bleaching of the acceptor (YFP) results in a significant increase in fluorescence of the donor (CFP). Half the cell was bleached in the YFP channel using the 514 argon laser line at 100% intensity. Before and after YFP bleaching, CFP and YFP images were collected to assess changes in donor and acceptor fluorescence. To minimize the effect of photobleaching caused by imaging, images were collected at low laser intensity. The gain of the photomultiplier tubes was adjusted to obtain the best possible dynamic range. FRET efficiency was calculated as,E=(Ipost-Ipre)×100/Ipost where Ipre is the pre-bleach CFP intensity, and Ipost is the post-bleach CFP intensity in the bleached region. As an internal negative control, an unbleached region in the same cell was measured. In Vitro Analysis of the Quaternary Structure of HAT—To analyze oligomerization of HAT, we used Blue Native PAGE (29Schagger H. von Jagow G. Anal. Biochem. 1991; 199: 223-231Crossref PubMed Scopus (1918) Google Scholar, 30Schagger H. Cramer W.A. von Jagow G. Anal. Biochem. 1994; 217: 220-230Crossref PubMed Scopus (1043) Google Scholar) followed by Western blot. We initially tested different detergents, detergent to protein ratios, and Coomassie Blue G to detergent ratios to find good experimental conditions for detection of the transporters (see "Experimental Procedures" and Refs. 29Schagger H. von Jagow G. Anal. Biochem. 1991; 199: 223-231Crossref PubMed Scopus (1918) Google Scholar and 30Schagger H. Cramer W.A. von Jagow G. Anal. Biochem. 1994; 217: 220-230Crossref PubMed Scopus (1043) Google Scholar). rBAT and b0,+AT were found as a single band of 535 ± 18 kDa (n = 12) (Fig. 1A). This band did not correspond to isolated rBAT or b0,+AT subunits because (i) it was detected in brush border membranes, where only the disulfide-linked heterodimer is found (Fig. 1A, lanes 1) and (ii) a functional, purified, His-tagged b0,+AT/rBAT concatamer (36Pfeiffer R. Loffing J. Rossier G. Bauch C. Meier C. Eggermann T. Loffing-Cueni D. Kuhn L.C. Verrey F. Mol. Biol. Cell. 1999; 10: 4135-4147Crossref PubMed Scopus (112) Google Scholar) (where "/" indicates fusion of the two subunits) had the same mobility (Fig. 1A, lanes 4). As expected, no b0,+AT appeared in membranes from the b0,+AT KO mice (37Feliubadalo L. Arbones M.L. Manas S. Chillaron J. Visa J. Rodes M. Rousaud F. Zorzano A. Palacin M. Nunes V. Hum. Mol. Genet. 2003; 12: 2097-2108Crossref PubMed Scopus (69) Google Scholar) (Fig. 1A, αb0,+AT panel, lane 2). In contrast, rBAT was still detected as the 535-kDa band because of its binding to an as yet unidentified light subunit (4Fernandez E. Carrascal M. Rousaud F. Abian J. Zorzano A. Palacin M. Chillaron J. Am. J. Physiol. Renal Physiol. 2002; 283: 540-548Crossref PubMed Scopus (85) Google Scholar, 37Feliubadalo L. Arbones M.L. Manas S. Chillaron J. Visa J. Rodes M. Rousaud F. Zorzano A. Palacin M. Nunes V. Hum. Mol. Genet. 2003; 12: 2097-2108Crossref PubMed Scopus (69) Google Scholar) (Fig. 1A, αrBAT panel, lane 2). Blue Native PAGE allows the determination of oligomerization stoichiometry with agents that partially dissociate complexes (28Nicke A. Baumert H.G. Rettinger J. Eichele A. Lambrecht G. Mutschler E. Schmalzing G. EMBO J. 1998; 17: 3016-3028Crossref PubMed Scopus (487) Google Scholar, 38Gendreau S. Voswinkel S. Torres-Salazar D. Lang N. Heidtmann H. Detro-Dassen S. Schmalzing G. Hidalgo P. Fahlke C. J. Biol. Chem. 2004; 279: 39505-39512Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Up to 5 m urea did not alter the mobility of the b0,+AT-rBAT complex (data not shown). In contrast, 2% SDS partially dissociated the complex in bands I and II, both in HeLa cells (Figs. 1, B and C) and in brush border membranes (data not shown). The relative amounts of the two bands varied slightly between experiments, but b0,+AT-rBAT never shifted completely to band II. The slower mobility of band I compared with the untreated sample could be due to partial unfolding of b0,+AT-rBAT. On the b0,+AT/rBAT concatamer, DTT may cause further unfolding that results in even a slower mobility of bands I and II (Fig. 1C). The addition of the reducing agent DTT in the presence of SDS dissociated the complex into its subunits (Fig. 1B). rBAT appeared as a single band of 162 ± 11 kDa, and b0,+AT appeared as two bands of 180 ± 14 and 84 ± 9 kDa (n = 7). SDS-PAGE followed by Western blot confirmed that DTT completely reduced the samples (data not shown). No dissociated subunits appeared with the b0,+AT/rBAT concatamer (Fig. 1C). According to a recently proposed empirical rule, the molecular weight of a polytopic transporter multip
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