COMMUNICATION
Cloning and Expression of a Plasma Membrane Cystine/Glutamate Exchange Transporter Composed of Two Distinct Proteins*

Hideyo Sato, Michiko Tamba, Tetsuro Ishii, and Shiro BannaiDagger

From the Department of Biochemistry, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8575 Japan

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

Transport system xc- found in plasma membrane of cultured mammalian cells is an exchange agency for anionic amino acids with high specificity for anionic form of cystine and glutamate. We have isolated cDNA encoding the transporter for system xc- from mouse activated macrophages by expression in Xenopus oocytes. The expression of system xc- activity in oocytes required two cDNA transcripts, and the sequence analysis revealed that one is identical with the heavy chain of 4F2 cell surface antigen (4F2hc) and the other is a novel protein of 502 amino acids with 12 putative transmembrane domains. The latter protein, named xCT, showed a significant homology with those recently reported to mediate cationic or zwitterionic amino acid transport when co-expressed with 4F2hc. Thus xCT is a new member of a family of amino acid transporters that form heteromultimeric complex with 4F2hc, with a striking difference in substrate specificity. The expression of system xc- was highly regulated, and Northern blot analysis demonstrated that the expression of both 4F2hc and xCT was enhanced in macrophages stimulated by lipopolysaccharide or an electrophilic agent. However, the expression of xCT was more directly correlated with the system xc- activity.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The transport of amino acids across the plasma membrane of mammalian cells is performed by specific systems of mediation acting on discrete groups of substrate molecules (1). We have described in cultured mammalian cells a Na+-independent anionic amino acid transport system highly specific for cystine and glutamate (2, 3). This system, designated as system xc- (4), is an exchange agency, and the anionic form of cystine is transported in exchange for glutamate (5). The exchange is obligatory with a molar ratio of 1:1. System xc- is almost ubiquitous in cultured mammalian cell lines (6), and the physiologic flows via this system are the entry of cystine and the exit of glutamate because cystine is very rare in cytosol due to a rapid reduction to cysteine, whereas the concentration of glutamate is much higher in cells than in extracellular fluid (5). Cystine taken up by the cell via system xc- is rapidly reduced to cysteine, which is incorporated into proteins and glutathione. Because cysteine is a rate-limiting precursor for glutathione synthesis, the intracellular level of glutathione is regulated by the system xc- activity (1, 7). The expression of system xc- in cultured cells seems to be highly regulated. Its activity is induced by electrophilic agents, depletion of cystine, or by oxygen (8), and this induction may be interpreted as an adaptive response because these tend to decrease glutathione. In macrophages lipopolysaccharide (LPS)1 is a potent inducer of the system xc- activity and increases glutathione (9), which may serve to protect the cells against the oxidative stress at the sites where they are activated.

Thus the physiological role of system xc- in cultured cells is obvious. However, the molecular nature of the transporter remains totally unknown. In this communication a cDNA library has been constructed from mouse peritoneal macrophages in which the system xc- activity is induced and cloning and expression of cDNA encoding the transporter protein(s) for system xc- have been performed.

    EXPERIMENTAL PROCEDURES
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INTRODUCTION
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Expression Cloning-- To isolate a cDNA encoding the cystine/glutamate transporter, we used an expression cloning approach similar to that used to clone the glucose transporter (10). Mouse peritoneal macrophages were collected from female C57BL/6N mice that had received 4 days previously an intraperitoneal injection of 2 ml of 4% thioglycollate broth. The mRNA fraction was isolated from the cells cultured for 8 h with 0.1 mM diethyl maleate and 1 ng/ml LPS (DIFCO, Salmonella typhosa 0901), and a unidirectional cDNA library was constructed using the SuperScriptTM Plasmid System (Life Technologies, Inc.). For screening, plasmids from pools of approximately 1000 clones were isolated using a purification kit (Qiagen), linearized with NotI, and transcribed in vitro in the presence of 5' 7MeGpppG 5'. Xenopus laevis oocytes were manually defolliculated and injected with 2.5-50 ng of the transcribed cRNA in 50 nl water/oocyte. Two days after injection, the rate of uptake of cystine by 3-5 oocytes was measured at 30 °C for 10 min in 125 µl of uptake medium. The uptake medium contained [14C]cystine in modified Barth's saline (10 mM HEPES, pH 7.5, 88 mM NaCl, 1 mM KCl, 0.3 mM Ca(NO3)2, 0.4 mM CaCl2, 0.8 mM MgSO4, 50 units/ml penicillin, and 50 µg/ml streptomycin). The uptake was terminated by rapidly rinsing the oocytes three times with ice-cold modified Barth's saline, and then the radioactivity in oocytes was counted. The cDNA was sequenced on both strands by dye terminator cycle sequencing method by PE Applied Biosystems.

Functional Characterization by Xenopus Oocyte Expression-- Oocytes were injected with 2.5 ng each of the cloned cRNA. Two days after injection, the rates of uptake of various 14C-labeled amino acids were measured as described above. For Na+-free uptake, NaCl was replaced by choline chloride. For efflux experiments, five oocytes were incubated in 125 µl of modified Barth's saline for 10 min at 30 °C in the presence or absence of 0.1 mM L-cystine. Then the saline was removed and analyzed by a JEOL JLC 300 amino acid analyzer.

Northern Blot Analysis-- The cDNA probes for clones Dr4 and Cm30 were PstI restriction fragments from 365 up to 1211 base pairs and from 102 up to 947, respectively. Both probes were labeled using [alpha -32P]dCTP and RediprimeTM II random prime labeling system (Amersham Pharmacia Biotech). RNA was electrophoresed on a 1% agarose gel in the presence of 2.2 M formaldehyde, transferred to Hybond N+ membrane (Amersham Pharmacia Biotech), and hybridized in a solution containing 50% formamide for 16 h at 42 °C. The membranes were washed twice for 15 min at room temperature with 1 × SSC, 0.1% SDS and then washed twice for 15 min at 68 °C with 0.25 or 0.1 × SSC, 0.1% SDS.

In Vitro Translation-- In vitro translation of cRNA was performed using the rabbit reticulocyte lysate system with or without canine pancreatic membrane (Promega). The experiments were done according to the manufacturer's protocol.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

To clone system xc- transporter, cRNA was prepared from pools of about 1000 clones and tested using the expression of cystine uptake activity in Xenopus oocytes. One of the pools was positive, and it was divided into 11 groups (A through K), which were tested subsequently. However, none of these groups were positive. Because the mixture of cRNAs prepared from these 11 groups was positive, we hypothesized that the transporter is composed of heteromultimeric complex. Thus the mixture of cRNAs from 10 groups, i.e. cRNAs from those lacking one specified group (-A through -K) was tested, and the results showed that -C and -D were negative, whereas the other nine were positive (Fig. 1). This indicates that both cRNAs from group C and those from group D are required for the expression of the cystine transport activity. Then we found that the combination of cRNAs from group C and those from group D were sufficient for the expression. Group C was further subdivided and tested in the presence of total cRNAs from group D until a single clone, Cm30, was isolated. Similarly the group D was subdivided and tested in the presence of cRNA from Cm30, and a single clone Dr4 was isolated. Sequence analysis of the insert of Cm30 proved its identity with the cDNA of 4F2 heavy chain (4F2hc) cell surface antigen (two nucleotides of 1828 were different with no change in amino acid sequence). Clone Dr4 insert was composed of 2250 base pairs and contained a single open reading frame that encodes a putative protein designated xCT (system xc-transporter-related protein).


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Fig. 1.   The rate of uptake of cystine in Xenopus oocytes injected with cRNA from the plasmids of various groups divided from the positive pool. The pool of about 1000 clones that showed the positive signal with respect to cystine uptake was divided into the 11 groups (A through K), and 10 of the 11 groups, i.e. lacking one specified group, were put together, making combinations of all groups except for A (-A), all groups except for B (-B), and so on. cRNAs were prepared from these combined groups (labeled by -A through -K) or from the undivided pool (labeled by All). The cRNA was also prepared from the mixture of groups C and D (C+D). They were injected at 50 ng/oocyte, and 2 days after injection, the rates of uptake of L-[14C]cystine at 50 µM were measured for 10 min at 30 °C. Data represent the means ± S.D. of three independent experiments.

cRNAs of 4F2hc and xCT were about 2 kb, and when they were injected into Xenopus oocytes in equal amounts, the uptake of cystine and glutamate was increased greatly and the uptake of aspartate was increased to a much lesser extent (Fig. 2). The uptake of arginine, leucine, serine, and alanine remained unchanged or slightly increased. cRNA of 4F2hc or xCT alone did not enhance the uptake of cystine and glutamate at all. However, 4F2hc cRNA significantly enhanced the uptake of arginine and leucine, probably because 4F2hc induces system y+L-like activity in oocytes (11, 12). The uptake of cystine and glutamate co-expressed by 4F2hc and xCT was mainly (83 ± 5%) Na+-independent, with Km of 0.081 mM for cystine and 0.16 mM for glutamate. To determine the specificity of this uptake, the ability of various amino acids to inhibit the uptake of cystine or glutamate was investigated. The cystine or glutamate uptake exhibited very similar inhibitor specificity to each other, and the uptake of cystine was potently inhibited by glutamate and vice versa (Fig. 3). System xc- mediates an obligatory exchange with 1:1 stoichiometry. Because the free glutamate pool in Xenopus oocytes is very large, whereas cystine/cysteine content is trivial (13), we measured the influx (uptake) of cystine and the efflux of glutamate simultaneously by using [14C]cystine for the influx measurement and by the direct determination of glutamate effluxed from the oocytes into the medium by amino acid analyzer. Results are summarized in Table I. The efflux of glutamate was dependent on the presence of cystine in the medium, and its rate was nearly equal to that of cystine influx, indicating that cystine/glutamate exchange occurred at a 1:1 molar ratio. Analysis of amino acids in the saline in which oocytes were incubated showed that the efflux of amino acids other than glutamate was trivial regardless of the presence of cystine. All these properties of the cystine/glutamate transport elicited in oocytes by the co-expression of 4F2hc and xCT are consistent with those of system xc- in mouse peritoneal macrophages and human fibroblasts (2, 5, 14).


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Fig. 2.   The rate of uptake of amino acids in Xenopus oocytes injected with cRNAs of 4F2hc and/or xCT. Oocytes were injected with water, 2.5 ng each of 4F2hc cRNA (labeled as 4F2hc), xCT cRNA (labeled as xCT), or both (labeled as 4F2hc + xCT). Two days after injection, the rates of uptake of the indicated L-[14C]amino acids at 50 µM were measured for 10 min at 30 °C. Data represent the means ± S.D. of five to six independent experiments.


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Fig. 3.   Comparison of the inhibitory potential of various amino acids on the uptake of L-cystine and L-glutamate. Oocytes were injected with 2.5 ng each of 4F2hc cRNA and xCT cRNA, and 2 days after injection the rates of uptake of 20 µM L-[14C]cystine (A) or L-[14C]glutamate (B) were measured in the absence (Ctl) or presence of the various L-amino acids indicated. Inhibitor amino acids were added at 2 mM except for cystine (at 0.5 mM because of its solubility). Data represent the means ± S.D. of five to six independent experiments and is expressed as a percentage of the control uptake. HC, L-homocysteate; mAIB, 2-methylaminoisobutyrate; BCH, 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid; Cyss, L-cystine.

                              
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Table I
Efflux of L-glutamate and uptake of L-cystine
Oocytes previously injected with water or 2.5 ng each of 4F2hc cRNA and xCT cRNA were incubated in the absence (L-cystine = 0 mM) or presence of 0.1 mM L-cystine for 10 min at 30 °C, and glutamate effluxed from the oocytes was determined. Uptake of L-[14C]cystine at 0.1 mM was measured independently using the oocytes simultaneously prepared. The amino acid pool of oocytes just before the efflux and uptake measurements (i.e., oocytes defolliculated, injected, and incubated for 2 days) was determined by an amino acid analyzer. L-Glutamate content was 2560 ± 120 pmol/oocyte, about 5.6 mM in concentration assuming that water content was 460 nl/oocyte (13). Data represent the means ± S.D. of four independent experiments.

cDNA for xCT encoded a putative protein of 502 amino acids and a relative molecular mass of 55.5 kDa (Fig. 4A). Assignment of the first ATG as the translation initiation site is based on its resemblance to a consensus sequence (GCCATGG). In vitro translation showed a band of an approximately 40-kDa protein, which was not glycosylated by canine pancreatic microsomes (data not shown). Comparison of the sequence of this predicted protein (xCT) against protein data bases revealed that the protein is novel and has a significant homology (44-47% identity) with the recently identified system L-like amino acid transporters from human (15) and rat (16) and system y+L-like amino acid transporters from human (17). All of these proteins induce transport activity only when co-expressed with 4F2hc. Analysis of the amino acid sequence according to the algorithm of Kyte and Doolittle (18) predicts an extremely hydrophobic protein (Fig. 4B) that may contain as many as 12 membrane-spanning domains.


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Fig. 4.   A, the predicted amino acid sequence of xCT. The cysteine residue conserved in L-like and y+L-like amino acid transporters is labeled with an asterisk. B, hydropathy plot of xCT (Kyte-Doolittle (18) hydropathy analysis using a window of 17 amino acids). The abscissa indicates the amino acid number.

Northern blot analysis showed three xCT transcripts (12, 3.5, and 2.5 kb) in macrophages cultured for 8 h with LPS and/or diethyl maleate (Fig. 5A). These multiple bands remained after high stringency washing and may represent alternative splicing, alternative polyadenylation sites, or a combination of both. xCT-specific signals are not visible in RNA from freshly prepared macrophages and are very faint in RNA from macrophages cultured for 8 h. The pattern of expression of xCT transcript is consistent with the system xc- transport activity of the macrophages previously reported (9). Fig. 5B shows that 4F2hc message was expressed at a very low level in freshly prepared macrophages and at a much higher level in macrophages cultured for 8 h with or without LPS and diethyl maleate. Expression of xCT in major organs was investigated (Fig. 5D), and the 12-kb message was expressed in brain but not in other organs tested.


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Fig. 5.   Northern blot analysis of xCT and 4F2hc. Total RNA was isolated from the macrophages freshly prepared (lane 1), cultured for 8 h (lane 2), cultured for 8 h with 0.1 mM diethyl maleate (lane 3), cultured for 8 h with 1 ng/ml LPS (lane 4), and cultured for 8 h with 0.1 mM diethyl maleate and 1 ng/ml LPS (lane 5). Ten µg each of total RNA was loaded per lane. The hybridization was performed with 32P-labeled cDNAs of xCT (A), 4F2hc (B), and beta -actin (C). D, poly(A)+ RNA was purified from mouse heart (lane 1), lung (lane 2), liver (lane 3), kidney (lane 4), and brain (lane 5), and 4 µg of each was loaded. The hybridization was performed with 32P-labeled cDNA of xCT.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Recently evidence has been presented to indicate a heterodimeric protein structure of some amino acid transporters in mammals (15-17). The common component is 4F2hc, which has been implicated in system L-like and system y+L-like transport activity. The counterpart component has been cloned on the assumption that the transport activity is induced when it is co-expressed with 4F2hc. Cell surface glycoprotein 4F2hc is less hydrophobic and contains a single transmembrane domain, whereas the counterpart proteins are much more hydrophobic with 12 predicted transmembrane domains. Experiments using sulfhydryl-blocking agents or cysteine-to-serine mutagenesis in 4F2hc suggested that the heterodimeric complex is linked by a disulfide bridge involving Cys109 of 4F2hc (17, 19). System xc- is an anionic amino acid transport system, and the present study clearly indicated that the transporter for system xc- belongs to the group requiring 4F2hc as an essential component (provisionally designated as 4F2 group). In xCT the conserved cysteine residue, which is a putative site for a disulfide bond with Cys109 of 4F2hc, was located at position 158 (Fig. 4A). Thus the membrane topology of (4F2hc + xCT) transporter is probably very similar to other 4F2 group transporters, although the substrate specificity is markedly different.

The present study, together with those on 4F2 group transporters, suggests that one of the most intrinsic features of the 4F2 group transporters is an exchange of the substrate amino acids. System L- and y+L-like transport activity expressed in oocytes seem to show an obligatory exchange, although the molar ratio of exchange is not determined (15, 16, 20). System L and y+L naturally expressed in cells show marked transstimulation (1, 21), that is, the substrate amino acids in one side of the membrane stimulate the exodus of the substrate amino acids from the opposite side of the membrane. However, it has not been proved yet that the naturally occurring system L or y+L is locked into exchange, and the possibility that the obligatory exchange mechanism of an L- or y+L-like system is an oocyte artifact has not been ruled out. In contrast the transport by system xc- naturally occurring in cells such as fibroblasts has been established to be an obligatory exchange with a molar ratio of 1:1, consistent with the transport expressed by (4F2hc + xCT) in oocytes. Although further study is required, it is likely that the Na+ independence and obligatory exchange are characteristics of 4F2 group transporters.

Although the significance of system xc- is evident in cultured cells, little is known about the physiological relevance of system xc- in vivo. System xc- might affect plasma cystine/cysteine level as predicted from the cell culture model (7). The plasma cystine level is suggested to be a physiological regulator of immune responses (22), particularly in HIV infection where glutathione plays an important role in HIV suppression and T cell function (23). Isolation of cDNA clone for system xc- will enable us to investigate a physiological role of this system xc- transporter in vivo.

    ACKNOWLEDGEMENTS

We thank Drs. K. Sakamoto and H. Kitayama for helpful discussions.

    FOOTNOTES

* This work was supported in part by grants from the Tokyo Biochemical Research Foundation and the Ministry of Education, Science, and Culture in Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB022345.

Dagger To whom correspondence should be addressed. Tel.: 81-298-53-3066; Fax: 81-298-53-3039.

    ABBREVIATIONS

The abbreviations used are: LPS, lipopolysaccharide; 4F2hc, 4F2 heavy chain; kb, kilobase(s); HIV, human immunodeficiency virus.

    REFERENCES
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ABSTRACT
INTRODUCTION
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REFERENCES
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