©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Evidence That the Rat Hepatic Mitochondrial Carrier Is Distinct from the Sinusoidal and Canalicular Transporters for Reduced Glutathione
EXPRESSION STUDIES IN XENOPUSLAEVIS OOCYTES (*)

Carmen Garca-Ruiz (§) , Albert Morales (¶) , Anna Colell (§) , Joan Rodés , Jiau-R. Yi (1), Neil Kaplowitz (1), José C. Fernández-Checa (**)

From the (1)Liver Unit, Servicio de Bioqumica, Department of Medicine, Hospital Clinic i Provincial, Universidad de Barcelona, 08036 Barcelona, Spain, Instituto Mixto de Investigaciones Biomédicas August Pi i Suer (UB-CSIC), 08036 Barcelona, Spain, and Division of Gastrointestinal and Liver Diseases, Department of Medicine, University of Southern California (USC) and the USC Center for Liver Diseases, School of Medicine, Los Angeles, California 90033

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Mitochondrial GSH derives from a mitochondrial transport system (RmGshT), which translocates cytosol GSH into the mitochondrial matrix. Mitochondria of oocytes, isolated 3-4 days after microinjection of total liver mRNA, expressed a RmGshT compared with water-injected oocytes. The expressed RmGshT exhibited similar functional features as reported in isolated mitochondria of rat liver such as ATP stimulation, inhibition by glutamate, and insensitivity to inhibition by sulfobromophthalein-glutathione (BSP-GSH) and S-(2,4-dinitrophenyl)glutathione (DNP-GSH). The expressed RmGshT is localized in the inner mitochondrial membrane since expression is still observed in mitoplasts prepared from total liver mRNA-injected oocytes. Fractionation of poly(A) RNA identified a single mRNA species of 3-3.5 kilobases encoding for the RmGshT, which was stimulated by ATP and inhibited by glutamate but not by BSP-GSH or DNP-GSH. Microinjection of this fraction did not lead to expression of plasma membrane GSH transport in intact oocytes, and conversely, oocytes microinjected with cRNA for rat liver sinusoidal GSH transporter (RsGshT) or rat liver canalicular GSH transporter (RcGshT) did not express mitochondrial GSH transport activity. Thus, our results show the successful expression of the rat hepatic mitochondrial GSH carrier, which is different from RsGshT and RcGshT, and provide the strategic basis for the cloning of this important carrier.


INTRODUCTION

GSH participates in multiple cell functions, and therefore intracellular reduced GSH is of major importance in protecting cells against damage by toxic xenobiotics, reactive oxygen compounds, and radiation(1, 2, 3) . There is increasing evidence that GSH within mitochondria is essential to guarantee a normal functioning organelle that supplies the energy needed to maintain normal cell functions (4-8). Furthermore, it has been shown in several cell types and organs that depletion of mitochondrial GSH results in cell injury and death, which is prevented by the selective increase of the mitochondrial pool of GSH using GSH derivatives such as GSH ethyl ester(4, 5, 6, 7) . Mitochondria do not synthesize GSH de novo; thus the mitochondrial pool of GSH is derived from the activity of a mitochondrial transporter (RmGshT)()that translocates GSH from cytosol into the mitochondrial matrix(4, 9, 10, 11) . The exact mechanism of this transport system is presently unknown and remains controversial, since recent studies have suggested diffusion of GSH into mitochondria through a channel (11) and transport via a high affinity system stimulated by ATP (10) or subject to a rapid exchange between cytosol and mitochondria(4) .

Despite playing a critical role in cell function, very little is known about the molecular properties of this critical transporter since there have been no reports, to our knowledge, to purify or express this transporter to initiate the identification of its molecular properties. Furthermore, functional characterization of this transporter, especially in relationship to the functional features of other plasma membrane GSH carriers, has not been examined(10, 12, 13, 14) . Therefore, the purpose of the present study was first to determine the feasibility to functionally express the hepatic mitochondrial GSH transporter as the initial stage toward the molecular cloning of this mitochondrial membrane carrier using Xenopus laevis oocytes and second to functionally characterize the expressed GSH mitochondrial transporter in relation to known features of the sinusoidal (RsGshT) and canalicular (RcGshT) GSH plasma membrane carriers that have been recently cloned(13, 14) .


EXPERIMENTAL PROCEDURES

Materials

GSH, GSSG, acivicin, L-methionine, L-cystathionine, actinomycin D, collagenase type IA, emetine, chloramphenicol, and sucrose were obtained from Sigma. ATP and dithiothreitol were from Boehringer Mannheim. 145 Ci/mmol [S]GSH was purchased from DuPont NEN. S-(2,4-dinitrophenyl)glutathione (DNP-GSH) and sulfobromophthalein-glutathione (BSP-GSH) were enzymatically synthesized and purified as described previously(15) . The conjugates were collected, lyophilized, and stored at -20 °C until use.

Animals

Male Sprague-Dawley rats weighing 180-220 g were obtained from PanLab (Barcelona, Spain) and fed adlibitum until used. Mature X. laevis females were purchased from CRBM (CNRS, Montpelier, France) and kept under standard conditions(12, 16) .

Isolation of Rat Liver Poly(A)RNA, Oocyte, and Microinjection of mRNA

mRNA isolation and fractionation on sucrose density gradient (30-10%) were performed as described previously(17) . Stage 5 and 6 defolliculated oocytes were selected and maintained in modified Barth's solution at room temperature with daily changes of medium(12, 13, 16) . Mature stage 6 oocytes were injected (33-66 nl/oocyte) with mRNA (0.1-3 ng/nl) using a computerized pressure-controlled microinjector (Atto Instruments, Potomac, MD).

Isolation of Mitochondria and Mitoplast from Oocytes

Water or total mRNA-microinjected oocytes were fractionated to isolate the mitochondrial fraction(18) . Batches of 150-200 microinjected oocytes were washed 3 times with Barth's medium and transferred to 190 mM mannitol, 1 mM CaCl, 0.5 mM EDTA, and 10 mM TES, pH 7.4, and homogenized in a loose-fitting glass homogenizer with 10 strokes at 4 °C. The crude mitochondrial pellet was separated from the pigment and yolk on a sucrose density gradient as described(19) . The mitochondrial fraction was collected and washed in 195 mM mannitol, 1 mM EDTA, 5 mM TES, pH 7.4, and kept on ice until use for GSH transport.

Mitoplasts were prepared by fractionation of whole mitochondria with digitonin (0.15 mg/mg of mitochondrial protein) as described previously (20, 21).

cRNA Synthesis from RsGshT and RcGshT Clones

cDNA clones from RsGshT and RcGshT were described previously(13, 14) . cDNA were used for in vitro transcription with T7 RNA polymerase in the presence of mG(5`)ppp(5`)G (Pharmacia Biotech, Barcelona, Spain), using a protocol supplied with the Riboprobe transcription system (Promega Biotec, Madison, WI). Complementary RNA (cRNA) from each clone was injected into oocytes and kept at room temperature for 3 days before transport experiments. Plasma membrane transport of [S]GSH was performed as described previously(12, 13, 14) .

Mitochondrial GSH Transport

To check the expression of mitochondrial GSH transport, initial rates of GSH uptake into mitochondria isolated from total or size-fractionated mRNA-injected oocytes was determined in uptake buffer containing 5 mM Hepes, pH 7.2, 220 mM mannitol, 70 mM sucrose, 0.1 mM EDTA, 0.1% bovine serum albumin (fatty acid-free), 5 mM succinate, 1 mM potassium phosphate, and 1 µCi of [S]GSH (0.025-50 mM) in a final volume of 250 µl. Uptake was initiated by the addition of mitochondrial suspension (0.5-0.7 mg/ml, final concentration) and after 15 s terminated by addition of 1.0 ml of ice-cold uptake buffer followed by vacuum filtering (Millipore, 0.45 µm) to separate medium from mitochondria. Retained mitochondria on filter were washed twice with 2.5 ml of ice-cold buffer(15) . In some cases, to check for the molecular form of GSH associated with the mitochondria, filters with retained mitochondria were placed in borosilicate glass tubes with 1 ml of distilled water to release intramitochondrial contents retained on filters. Samples were treated with 10% trichloroacetic acid and spun 13,000 g for 2 min, and the supernatants were analyzed for HPLC as described(22) . To study the ATP-dependent uptake of mitochondrial GSH, ATP (2 mM) was added to the uptake medium and transport experiments for 15 s and was terminated as described above. For a longer period of transport, ATP and an ATP-regenerating system (10 mM creatine phosphate and 100 µg/ml creatine kinase) were included in the incubation.


RESULTS AND DISCUSSION

Expression of GSH Transport in Mitochondria of Oocytes Injected with Total mRNA from Rat Liver

Xenopus oocytes contain about 10 times as many mitochondria as a typical somatic cell(19) . The final mitochondrial band was enriched in succinic dehydrogenase activity (4-5-fold) relative to homogenate. Electron microscopy examination revealed sealed mitochondria uncontaminated with other intracellular structures. For purposes of comparison of the basal status of mitochondrial GSH with that of hepatocytes, we expressed our results as per 8 oocytes, since their volume is equivalent to that of 10 hepatocytes(12) . Most of the GSH content, which was comprised of >95% reduced GSH, was found in cytosol (11-13 nmol/8 oocytes), which is 3-4 times lower relative to hepatocytes (30-40 nmol/10 cells). Mitochondria of basal oocytes were found to have also a lower GSH content (<1.0 nmol/8 oocytes) than hepatic mitochondria (5-7 nmol/10 cells). These values of basal GSH content in total mRNA-injected oocytes did not differ from water-injected oocytes. Therefore, to discern if injection of total rat liver-mRNA leads to expression of GSH transport in mitochondria, we loaded cytosol of oocytes with GSH to provide substrate for the operation of the mitochondrial transporter. As shown in , mitochondria isolated from oocytes 3 days after mRNA injection and preloaded with GSH 2 h before mitochondria isolation showed a much greater level of GSH (2.0-2.5 nmol/8 oocytes (3-4-fold increase)) compared with mRNA-injected oocytes not loaded with GSH or water-injected GSH-loaded oocytes. These results strongly suggest that injection of total rat liver mRNA leads to expression of the mitochondrial transport system leading to an increased steady-state level of mitochondrial GSH when cytosol GSH was available as a substrate.

To examine transport characteristics of RmGshT, we next studied its properties in isolated mitochondria from oocytes expressing rat liver poly(A) RNA. Three days after microinjection of total mRNA, isolated mitochondria exhibited a time-dependent uptake of [S]GSH, with an initial linear rate up to 1 min, compared with mitochondria from water-injected control oocytes (not shown). The molecular form of [S]GSH associated with retained mitochondria on filters was checked by HPLC and confirmed as >90% reduced GSH. However, when the uptake of labeled GSH was examined in the presence of added ATP, the initial rate of transport increased significantly compared with mitochondria from mRNA-injected oocytes without addition of ATP (not shown). This ATP stimulation of GSH uptake into mitochondria from mRNA-injected oocytes was a unique feature of mitochondrial GSH transport since neither RsGshT nor RcGshT displays ATP stimulation(15) . The small endogenous uptake of GSH in control mitochondria from water-injected oocytes was not stimulated by ATP (not shown). Mitoplasts from mRNA-injected oocytes exhibited the same magnitude of transport compared with whole mitochondria, suggesting that the expressed mitochondrial GSH carrier is located in the inner mitochondrial membrane (Fig. 1). In addition, our studies indicated that for the stimulation of transport observed by ATP, this molecule has to be hydrolyzed since the presence of ADP or non-hydrolyzable ATP analogues did not result in stimulation of GSH uptake (not shown). Further studies revealed that the expression of GSH transport in mitochondria from oocytes following injection of total mRNA from rat liver was dependent on time of culture of oocytes injected with total rat liver mRNA, taking at least 3 days to process the delivered mRNA into a functional protein inserted in mitochondria, without much further increase after 5 days of culture of microinjected oocytes (not shown). In addition, increasing the amount of total mRNA (10-100 µg) from liver injected into the oocyte led to a proportional increase in the expression of activity of transport in mitochondria compared with control water-injected oocytes (not shown). Expression of the mitochondrial GSH carrier in oocytes microinjected with total rat liver mRNA was unaffected by pretreatment of oocytes with actinomycin D and was abrogated by treatment with emetine, a specific inhibitor of the cytoplasmic translation process, indicating that the expression of the mitochondrial GSH transporter was not the result of activation of an endogenous Xenopus gene but rather the consequence of processing and translation of the rat liver mRNA (not shown).


Figure 1: Expression of transport of GSH in mitoplast from oocytes microinjected with total mRNA. Mitochondria were prepared and fractionated with digitonin to obtain a matrix plus inner membrane structure (as described under ``Experimental Procedures'') checking monoamine oxidase activity to monitor disappearance of outer membrane. Mitochondria or mitoplasts were incubated with [S]GSH (5 mM) to determine expression of GSH transport activity. When present, ATP was added at 2.5 mM in the medium.



Inhibitor Specificity and Identification of the mRNA Size Fraction Encoding the Mitochondrial GSH Transporter

We further characterized the functional features of the expressed mitochondrial GSH transport activity by examining cis-competition with known inhibitors of the RsGshT, RcGshT, and preferred substrates of the ATP-driven multispecific organic anion transporter(15, 23) . We examined the cis-effects on 5 mM [S]GSH uptake of BSP-GSH, DNP-GSH, BSP, methionine, and cystathionine, previously shown to interact with and inhibit plasma membrane GSH carriers to determine substrate specificity of the expressed mitochondrial GSH carrier(12, 13, 14, 15, 24, 25) . As seen in Fig. 2, GSH thioether adducts, BSP-GSH and DNP-GSH, did not lead to inhibition of mitochondrial GSH transport in total mRNA-injected oocytes with or without ATP. The lack of effect by DNP-GSH contradicts the possibility that the expressed GSH transporter in mitochondria from oocytes is due to expression and mitochondrial insertion of RcGshT or multispecific organic anion carrier. BSP and GSSG, similar to DNP-GSH, did not inhibit mitochondrial GSH transport (not shown). On the other hand, results with BSP-GSH, a specific inhibitor of RsGshT and the high affinity canalicular GSH carrier(24) , indicated that the GSH transport into mitochondria is not mediated by expression of these carriers. In addition, other specific inhibitors of RsGshT, such as methionine and cystathionine, which do not interact with the high affinity canalicular GSH carrier(26) , did not inhibit the transport of GSH into mitochondria (not shown). However, as shown in Fig. 2, glutamate inhibited the activity of basal and ATP-stimulated mitochondrial GSH transport expressed in total mRNA-injected oocytes, confirming its effects on rat liver mitochondria(10) . Endogenous GSH uptake by mitochondria from water-injected oocytes was not inhibited by glutamate (not shown). In contrast, RsGshT and RcGshT are not affected by glutamate (24, 25) (Fig. 3). Other GSH S-conjugates including S-methylglutathione, S-butylglutathione, and S-octylglutathione (1-5 mM) did not inhibit the basal or the ATP-stimulated transport in mitochondria from mRNA-injected oocytes (not shown).


Figure 2: Expression of mitochondrial GSH transport following injection of size-fractionated rat liver mRNA. Total mRNA (80-90 µg) was fractionated by continuous sucrose gradient centrifugation. Each size fraction was injected into oocytes (3-5 ng/oocyte) and incubated in modified Barth's medium at room temperature. Mitochondria were then isolated and incubated with [S]GSH (5 mM (A) or 0.1 mM (B)) for 15 s to determine the initial rate of GSH uptake. For the ATP dependence, ATP was included in the incubation medium at 2.5 mM. For cis-competition studies, mitochondria were incubated in the presence of labeled GSH plus BSP-GSH, DNP-GSH (1 mM each), or glutamate (10 mM) with and without ATP. The inset of B shows the effect of inhibitors on the expression of GSH transport encoded by fraction 5. The molecular form taken up into mitochondria was checked by HPLC and was >95% reduced GSH. Results are mean ± S.D. of n = 3 for A and n = 2 for B.




Figure 3: GSH uptake by intact oocytes and isolated mitochondria. Oocytes were injected with full-length cRNA prepared from cloned RsGshT or RcGshT (0.5 ng/oocyte) or 5 ng of mRNA size fraction 5 that expressed mitochondrial GSH transport activity as determined in Fig. 2. 3 days later, cell membrane transport of [S]GSH (10 mM) was determined (A) in intact oocytes (cell uptake) in the presence of BSP-GSH (10 mM), ATP (2.5 mM), or glutamate (10 mM). Mitochondria were isolated from other oocytes from the same preparations of injected oocytes, and the initial rate of transport of [S]GSH was determined (B) in the absence or presence of ATP (2.5 mM) or glutamate (10 mM). Results are mean ± S.D. of n = 3 oocyte preparations.



In order to enrich the mRNA encoding selectively for the mitochondrial GSH transport system total mRNA was size-fractionated in a continuous sucrose density gradient. Of all fractions tested, only one (fraction 5) resulted in transport of GSH in mitochondria, which was 8-10 times over that shown by oocytes injected with water using low (0.1 mM) and high (5 mM) GSH (Fig. 2). These two concentrations were employed to ensure not missing high and low affinity components since both have been reported(10) . The presence of ATP in the medium resulted in a further increase of transport of GSH in mitochondria from oocytes expressing fraction 5, which was greater at lower GSH concentrations. Similar to the transport activity expressed in oocytes after injection of total mRNA, fraction 5 mediated expression of mitochondrial GSH transport, which was insensitive to the presence of BSP-GSH, DNP-GSH, and other GSH S-conjugates (1-5 mMS-methylglutathione, S-butylglutathione, and S-octylglutathione) and thioether amino acids (methionine and cystathionine). However, the enriched activity expressed by fraction 5 was inhibited by glutamate (5 mM). Despite the difference of amount of mRNA injected (33 ng for total mRNA versus 3-5 ng of fraction 5) there was a 2-3-fold increase in the activity of transport of GSH in mitochondria expressing fraction 5, indicating an overall enrichment of 20-30-fold compared with total mRNA. Based on agarose gel electrophoresis (data not shown) fraction 5 corresponded to a mRNA size class between 3 and 3.5 kb; this differs from RcGshT (4 kb) and RsGshT (2.7 kb). The expressed GSH transport system in mitochondria was unaffected by the presence of aspartate (10 mM, not shown), suggesting that GSH transport expressed in mitochondria is not mediated via the glutamate/aspartate antiporter.

Hepatic Mitochondrial GSH Transporter Is Distinct from RsGshT and RcGshT

To determine directly the relationship between plasma membrane and mitochondrial GSH transporters, we examined expression of both activities (cell uptake and isolated mitochondrial uptake) in oocytes injected with either fraction 5 mRNA or cRNA prepared from the two recently cloned plasma membrane transporters, RcGshT and RsGshT (13, 14). As shown on Fig. 3, injection of RsGshT and RcGshT resulted in expression of GSH uptake in intact oocytes that was unaffected by ATP and glutamate. However, the presence of BSP-GSH in the medium resulted in inhibition of GSH uptake only when the sinusoidal clone was injected into oocytes. In addition, methionine and cystathionine also inhibited transport by RsGshT (not shown). Oocytes injected with RcGshT displayed sensitivity to inhibition of transport by DNP-GSH (Fig. 3), BSP, and di-BSP (not shown). However, injection of mRNA fraction 5 did not lead to expression of GSH transport in intact oocytes. Moreover, injection of the cRNA from RsGshT and RcGshT did not result in expression of GSH transport in isolated mitochondria (Fig. 3B), whereas the injection of the mRNA size fraction 5 resulted in expression of GSH transport in mitochondria sensitive to inhibition by glutamate and stimulated by ATP. Furthermore, since previous studies have identified only one transcript when rat liver RNA is hybridized with full-length cDNA probes for RsGshT (2.7 kb) and RcGshT (4.0 kb), it is unlikely that alternate processing of a transcript of a gene common to one of these transporters could account for RmGshT. Thus, on the basis of ATP stimulation, inhibitor specificity, and mRNA size and expression in oocytes, it is likely that the mitochondrial GSH transporter is encoded by a distinct gene. In addition, the studies reported here provide the basis for a cloning strategy for this transporter.

  
Table: Expression of mitochondrial GSH transport in oocytes microinjected with mRNA from rat liver

Oocytes were fractionated into cytosol and mitochondria 3 days after microinjection of water (33 nl) or total rat liver mRNA (33 ng) followed by a second injection for GSH loading (66 nl of a stock solution of 50 mM GSH) or water (66 nl) 2 h before isolation of mitochondria. Oocytes were washed three times with Barth's medium after GSH loading to remove excess GSH. Results are the mean ± S.D. of n = 3 oocyte preparations.



FOOTNOTES

*
This work was supported by National Institute on Alcohol Abuse and Alcoholism Grant AA09526 (to N. K. and J. C. F-C.), Dirección General Poltica Cientfica y Técnica (DIGICYT) Grant PB92-1110, Fondo Investigaciones Sanitarias (FISS) Grant 94-0046/01, and by a grant from Europharma (to J. C. F-C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Partially supported by a grant from Europharma.

Fellow from the FISS.

**
To whom all correspondence and reprint requests should be addressed: Liver Unit, Hospital Clinic i Provincial, Villarroel, 170, 08036 Barcelona, Spain. Fax: 34-3-451-5272. E-mail: checa@clinic.med.ub.es.

The abbreviations used are: RmGshT, rat liver mitochondrial GSH transporter; RcGshT, rat liver canalicular GSH transporter; RsGshT, rat liver sinusoidal GSH transporter; DNP-GSH, S-(2,4-dinitrophenyl)glutathione; BSP-GSH, sulfobromophthalein-glutathione; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; HPLC, high pressure liquid chromatography; kb, kilobase(s).


ACKNOWLEDGEMENTS

We thank Dr. J. A. Bomb for assistance with the electron microscopy studies.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.