Abstract
The dicarboxylate carrier (DCC) is one of two carriers responsible for glutathione (GSH) transport into rat kidney mitochondria. The central hypothesis of the present study was that overexpression of this carrier in renal proximal tubular cells increases content of mitochondrial GSH, which in turn can protect these cells from chemical-induced injury. We first cloned the carrier protein and verified its properties. This was accomplished by reverse transcribing total rat kidney RNA and polymerase chain reaction amplification with primers based on the complete cDNA sequence for the mitochondrial DCC protein. DCC was expressed as a His6-tagged protein, purified from Escherichia coli inclusion bodies, and reconstituted into proteoliposomes for transport assays. Time- and concentration-dependent uptake of bothl-[3H-glycyl]GSH and [2-14C]malonate was observed with kinetics, substrate specificity, and inhibitor sensitivities similar to those observed in rat kidney proximal tubules. We next transiently transfected NRK-52E cells with the cDNA for rat kidney DCC to overexpress the protein. The presence of the recombinant DCC-His6 protein was confirmed by immunoblots. Transport of both GSH and malonate into the mitochondrial fraction of transfected cells was enhanced 2.45- to 11.3-fold, compared with that in wild-type cells. Transfected cells exhibited markedly less apoptosis from tert-butyl hydroperoxide orS-(1,2-dichlorovinyl)-l-cysteine than did wild-type cells, validating the central hypothesis and providing us with a valuable and novel tool with which to further study GSH and thiol redox status in renal mitochondria, and the function of GSH transport in regulation of processes such as apoptosis and oxidative phosphorylation.
The tripeptide GSH plays numerous critical roles in mammalian cells, including maintenance and regulation of cellular thiol and redox status and serving as a substrate in various xenobiotic metabolism reactions (Reed, 1990). Within renal proximal tubular cells, there are at least two major pools of GSH, one in the cytoplasm and one in the mitochondrial matrix (Schnellmann et al., 1988). Cellular fractionation studies showed that the mitochondrial pool in rabbit and rat renal proximal tubule comprises approximately 15 to 30% of total cellular GSH (Schnellmann et al., 1988; Lash et al., 1998), which equates with the approximate volume fraction of mitochondria in the proximal tubules. GSH is the predominant nonprotein thiol in mitochondria, and its presence in this organelle at millimolar levels is necessary to maintain redox status, and hence activity of numerous sulfhydryl-dependent processes (Beatrice et al., 1984; Yagi and Hatefi, 1984; Lê-Quôc and Lê-Quôc, 1985, 1989). Accordingly, selective depletion of the mitochondrial pool of GSH has been associated with enhanced susceptibility to several types of chemical-induced toxicity, such as oxidative stress (Martensson and Meister, 1989; Reed, 1990; Shan et al., 1993; Fernandez-Checa et al., 1997). Furthermore, decreased concentrations of mitochondrial GSH are associated withS-(1,2-dichlorovinyl)-l-cysteine (DCVC)-induced acute toxicity (Lash and Anders, 1987) and apoptosis (Chen et al., 2001) in renal mitochondria and renal proximal tubular cells, respectively; apoptosis in general (Kroemer et al., 1998); and chronic ethanol exposure and alcoholic liver disease in liver mitochondria (Fernandez-Checa et al., 1991, 1993, 1997).
The predominant, if not exclusive, localization of enzymes catalyzing de novo synthesis of GSH is the cytoplasm (Griffith and Meister, 1985;Lash et al., 1998), indicating that transport of the intact tripeptide from the cytoplasm across the mitochondrial inner membrane must occur to supply the mitochondria with its pool of GSH. GSH does not diffuse passively across the mitochondrial inner membrane. Rather, because mitochondria possess a membrane potential with the matrix space negative relative to the cytoplasm and because GSH is a negatively charged molecule at physiological pH, GSH must be transported actively or in exchange for another anion (Smith et al., 1996).
Eight known anion carriers are present on the mitochondrial inner membrane that could conceivably play a role in the mitochondrial uptake of GSH from the cytoplasm. In previous studies (McKernan et al., 1991;Chen and Lash, 1998; Chen et al., 2000), we examined the role of these carriers by assessing substrate specificity and patterns of inhibition, and determined that two carriers, the dicarboxylate carrier (DCC) and the oxoglutarate carrier (OGC), are responsible for at least 80% of the total uptake of cytoplasmic GSH into renal cortical mitochondria. These two carriers, along with the mono- and tricarboxylate carriers, share a common 30-kDa molecular mass subunit and are believed to belong to a carrier “superfamily” (Palmieri et al., 1996). Function of each of these carriers is electroneutral, catalyzing the exchange of anions across the inner membrane without any net transfer of charge.
Both the DCC and OGC proteins have been purified to homogeneity and reconstituted into proteoliposomes (Bisaccia et al., 1985, 1988; Kaplan and Pedersen, 1985; Lançar-Benba et al., 1994). Two difficulties with studying the purified DCC protein from mammalian tissues relates to its low abundance in the inner mitochondrial membrane and its instability in its purified form, due to autoxidation and inactivation (Kaplan and Pedersen, 1985; Bisaccia et al., 1988). To circumvent this limitation, the cDNA for the DCC from Norway rat was isolated using related expressed sequence tags homologous to genes inCaenorhabditis elegans and yeast (Kakhniashvili et al., 1997). The rat DCC was then expressed in Escherichia coliand functionally reconstituted into proteoliposomes (Fiermonte et al., 1998).
In this communication, we significantly expand on this earlier report (Fiermonte et al., 1998) and our prior studies of GSH transport in mitochondria from rat kidney proximal tubules (Chen and Lash, 1998;Lash et al., 1998), by expressing recombinant rat DCC (as the N-His6 fusion protein). By reconstituting purified DCC into proteoliposomes and transiently expressing DCC in a cell line derived from rat kidney proximal tubules (NRK-52E), we show that DCC transports GSH and malonate with properties similar to those observed in intact rat kidney mitochondria. The recombinant DCC-His6 in transfected NRK-52E cells localizes in mitochondria where it mediates GSH uptake, thereby demonstrating function of the carrier in an intact renal cell. Furthermore, overexpression of the DCC in NRK-52E cells markedly protected against apoptosis induced by either DCVC or tert-butyl hydroperoxide (tBH), suggesting the toxicological significance of the mitochondrial GSH transport process.
Materials and Methods
Materials.
[3H-glycyl]GSH (44.8 Ci/mmol) was purchased from PerkinElmer Life Sciences (Boston, MA). [2-14C]Malonate (56 mCi/mmol) was purchased from ICN Pharmaceuticals (Costa Mesa, CA). γ-Glutamyl amino acids, S-alkyl GSH derivatives, and ophthalmic acid were purchased from Sigma-Aldrich (St. Louis, MO), Bachem California (Torrance, CA), or Roche Applied Science (Indianapolis, IN). Restriction enzymes for PCR, other enzymes (e.g., DNA polymerases and T7 RNA polymerase), and plasmids were purchased from New England Biolabs (Beverly, MA), Promega (Madison, WI), and Invitrogen (Carlsbad, CA). PCR primers were custom synthesized by Integrated DNA Technology, Inc. (Coralville, IA). Cloning vectors (pGEM-T Easy, pRSET, and pcDNA3.1/V5-His-TOPO) were purchased from Promega and Invitrogen. Dowex-1, Triton X-100, Percoll, and phospholipids were purchased from Sigma-Aldrich. Materials for gel electrophoresis (SDS, acrylamide, agarose, and buffers) were purchased from Bio-Rad (Hercules, CA) or Sigma-Aldrich. ProBond nickel-chelating resin was purchased from Invitrogen. Millicell polycarbonate filter inserts (0.4-μm pore size, 25 mm in diameter) were purchased from Millipore Corporation (Bedford, MA). NRK-52E cells (catalog number CRL-1571), cell culture medium (catalog number 30-2002; Dulbecco's modified Eagle's medium with 4 mMl-glutamine adjusted to contain 1.5 g/l sodium bicarbonate, 4.5 g/l glucose, and 1.0 mM sodium pyruvate), and 10% (w/v) bovine calf serum were purchased from American Type Culture Collection (Manassas, VA). Antibodies to the His6fusion proteins were purchased from Invitrogen. Double-distilled, deionized water was used for all experiments. All other chemicals and reagents were purchased from commercial vendors and were of the highest purity available.
Amplification of Rat Kidney Mitochondrial DCC cDNA by reverse transcription-PCR and Bacterial Expression.
Total RNA was previously isolated from renal cortical homogenates of male Fischer-344 rats (200–300 g b.wt.; Charles River Laboratories, Inc., Wilmington, MA) by acid phenol extraction using the TRIzol extraction kit (Invitrogen) and was stored at −80°C until needed. Total rat kidney RNA was reverse transcribed with Superscript II reverse transcriptase and amplified with forward and reverse primers based on the complete cDNA sequence (1946 base pairs) for the mitochondrial DCC protein from the Norway rat (Rattus norvegicus; GenBank accession numberAJ223355). PCR primers were 5′-CGG GCC AGG TCG CTG CTG CTC T-3′ (sense, positions 8–29) and 5′-GCC AAG GCA GGG TCG GAA GGC GTA G-3′ (antisense, positions 1039–1063), where the coding region is from positions 34 to 894; PCR conditions were 30 cycles of 94°C × 3 min for denaturation and 68°C × 2 min for annealing/extension. The 1056-base pair product was ligated into a T-A cloning vector (pGEM-T Easy) for transformation. Confirmation of the PCR product was by automated DNA sequencing.
The full-length cDNA for rat kidney DCC was subcloned into the pRSET T7 expression vector for high-level expression in E. coli as an N-terminal polyhistidine (His6) fusion product. Bacterial cells containing the overexpressed protein were harvested by centrifugation (5000g × 5 min at 4°C), supernatants were discarded, and pellets were resuspended in 20 ml of TE-1 buffer (50 mM Tris-HCl and 2 mM EDTA, pH 8.0). Cells were then lysed by incubation for 15 min at 30°C and occasional mixing with 100 μg of lysozyme/ml (freshly prepared in TE-1 buffer) and 0.1% (v/v) Triton X-100. Bacterial DNA in the cell lysate was sheared by sonication, the suspension centrifuged at 12,000g × 15 min, and the pellet resuspended in 2 ml of TE-2 buffer (10 mM Tris-HCl, 0.1 mM EDTA, and 1 mM dithioerythritol, pH 7.0) and recentrifuged. The final pellet was resuspended in 2 ml of TE-2 buffer and was solubilized with 1.2% (w/v) sarkosyl dissolved in TE-2 buffer. The inclusion body fraction was isolated by centrifugation of the resuspended pellet at 131,000g × 4.5 h through a step sucrose gradient [12.4 ml of 40% (w/v) sucrose and 18.6 ml of 53% (w/v) sucrose (sucrose solutions prepared in TE-2 buffer)]. The inclusion body pellet was resuspended in 30 ml of TE-2 buffer and centrifuged at 12,000g × 15 min. The DCC protein was then extracted by resuspension of the inclusion body pellet in 2 ml of 1.2% (w/v) sarkosyl dissolved in TE-2 buffer and centrifugation at 314,000g × 30 min.
Purity of the DCC-His6 fusion protein obtained from the inclusion body fraction was enhanced by fractionation of the inclusion body pellet (2 ml, approximately 25 mg of protein) on a column (1.0 × 6.8 cm) with 4 ml of nickel-chelating resin and equilibrated with ProBond buffer (20 mM potassium phosphate, pH 7.8, containing 500 mM NaCl). The column was then washed with ProBond buffer adjusted to pH 6.0 (25 ml) and finally with 25 ml each of ProBond buffer, pH 6.0, containing 100 mM and then 300 mM imidazole.
Preparation of Proteoliposomes for Reconstitution of Purified DCC.
Liposomes and proteoliposomes were prepared essentially as described previously (Chen et al., 2000). Briefly, liposomes were prepared by transferring 700 mg of phosphatidylcholine into a 30-ml polypropylene tube and drying under a stream of nitrogen. After redissolving the dried lipid in diethyl ether and removal of solvent under nitrogen, liposome buffer (7 ml; 120 mM HEPES, 50 mM KCl, and 1 mM EDTA, pH 7.0) was added to the dried lipid, the tube was flushed with nitrogen, sealed, and vigorously mixed on a vortex mixer. Lipid was then dispersed in a bath sonicator for approximately 60 min and was stored at −80°C for up to 3 months. Proteoliposomes were prepared by mixing purified carrier protein (200 μl, 50 μg of protein) or liposome buffer (for a blank control) with 0.5 ml of liposomes and 50 μl of substrate solution (buffer containing 20 mM phosphate and 20 mM malate) in a final volume of 1 ml. After mixing on a vortex mixer, the proteoliposomes were rapidly frozen in liquid nitrogen and stored at −80°C for up to 1 month until use. Immediately before assay, the proteoliposome suspension was thawed, sonicated with a bath sonicator at room temperature, placed on ice for 1 min, and then passed through an anion exchange column (Dowex-1, 100–200 mesh, equilibrated with liposome buffer and prepared in a 9-inch Pasteur pipette) to remove external substrate by elution with liposome buffer. The opalescent fraction (approximately 1 ml) contains the proteoliposomes and was collected. The proteoliposomes were equilibrated at room temperature for approximately 5 min before being used in transport assays. This freeze-thaw cycle and sonication method give rise to a heterogeneous population of large, primarily unilamellar proteoliposomes that are suitable for transport studies (Kaplan and Pedersen, 1985).
Transport Assays in Reconstituted Proteoliposomes.
Transport of substrates (i.e., GSH and malonate) into proteoliposomes was measured by first preincubating 200 μl of proteoliposomes (preloaded with 1 mM phosphate and 1 mM malate) for 1 min with 12 μl of “reconstitution buffer” (120 mM HEPES, 50 mM KCl, and 1 mM EDTA, pH 7.4). Competitive inhibitors were added simultaneously with substrates. Transport was initiated by addition of 12 μl of 20× radiolabeled substrate. At various times (30–240 s), 30-μl aliquots were transferred to a microcentrifuge tube containing 70 μl of reconstitution buffer + 40 mM of an irreversible transport inhibitor (i.e., pyridoxal 5′-phosphate), and tubes were mixed and placed on ice until the next processing step. After completion of time courses, samples were loaded onto Sephacryl S-100 mini-columns, which were prepared in 5.75-inch Pasteur pipettes. These columns were placed in 16 × 125-mm glass test tubes and were centrifuged at 2300 rpm × 2 min in a tabletop clinical centrifuge. Supernatant from each tube was transferred into a 5-ml scintillation vial, and each tube was washed with 200 μl of scintillation fluid, which was then added to scintillation vials.
Uptake rate was calculated by linear curve fitting as described previously (Chen and Lash, 1998; Chen et al., 2000), plotting ln [Ptotal/(Ptotal− Pt )] versus time according toHalestrap (1975). Ptotal represents the total uptake of substrate at equilibrium (estimated by exponential decay curve fitting of time course data), andPt represents substrate uptake at timet (0 to 5 min). The initial rate of uptake was then determined from the first order rate equation v =k(Ptotal).
As a control, liposomes were pseudo-reconstituted with buffer and no protein. These control liposomes exhibited less than 5% of the measured uptake of either GSH or malonate (data not shown), similar to previous reconstitution studies (Chen et al., 2000), indicating that nearly all of the measured substrate transport was due to the function of the reconstituted carrier protein.
Culture of NRK-52E Cells.
NRK-52E cells were cultured on collagen-coated, polystyrene T-25 or T-175 culture flasks with Dulbecco's modified Eagle's medium containing 4% (w/v) glutamine, 1.5 g/l sodium bicarbonate, 4.5 g of glucose/l, 1 mM sodium pyruvate, and 10% (v/v) bovine calf serum in an atmosphere of 5% CO2/95% air at 37°C. On reaching confluence (5–9 days), subcultures were prepared by a 15-min treatment with 0.02% (w/v) EDTA, 0.05% (w/v) trypsin solution and replating the cells at a density of 4 × 104cells/cm2.
Transfection of NRK-52E Cells.
NRK-52E cells were transiently transfected with rat kidney DCC cDNA to overexpress mitochondrial GSH transport activity. Plasmid DNA was purified following amplification in E. coli cells, using the Promega Wizard Prep purification kit. cDNA encoding the rat kidney DCC was subcloned into the pcDNA3.1/V5-His-TOPO vector and transfected into the NRK-52E cells by either the calcium phosphate method (Current Protocols in Molecular Biology, 1999) or with FuGENE 6 from Roche Applied Science. For the calcium phosphate method, purified plasmid containing 200 mM CaCl2 in HEPES buffer, pH 7.4, was added dropwise to subconfluent T-175 flasks to a final concentration of 20 μg/10 ml of Dulbecco's modified Eagle's medium. After 24 h, medium was removed and the cells were washed twice with phosphate-buffered saline (PBS). After 48 h, cells were harvested by incubating with Cellstripper (Mediatech, Herndon, VA) for 5 min and gentle scraping. These harvested cells were then used for isolation of mitochondria for transport measurements and Western blot analyses (see below). For the FuGENE 6 method, transient transfections were carried out as described by the manufacturer using a 3:1 ratio of FuGENE 6 reagent to plasmid DNA. After 24 h, cells were harvested as described above.
Isolation of Mitochondria from NRK-52E Cells and Transport Measurements.
Mitochondria were prepared from seven confluent T-175 flasks, which contain approximately 70 to 85 × 106 NRK-52E cells, by differential centrifugation as described previously (McKernan et al., 1991). Yield of mitochondrial protein was 6 to 10.5 mg. Purity of mitochondria was determined by measurement of activities of marker enzymes for mitochondria (succinate dehydrogenase and glutamate dehydrogenase), plasma membrane (alkaline phosphatase, (Na+ + K+)-stimulated ATPase), cytoplasm (lactate dehydrogenase), and endoplasmic reticulum (glucose 6-phosphatase) in whole cells and the mitochondrial fraction. Most of the total cellular activity of the mitochondrial marker enzymes (≥75%) was recovered in the mitochondrial fraction, whereas minimal-to-modest amounts of total cellular activity of markers for the other subcellular organelles were recovered in this fraction. Specifically, approximately 5% of total cellular activity of markers for the plasma membrane and endoplasmic reticulum and <2% of total activity of the cytoplasmic marker enzyme were recovered in the mitochondrial fraction. This indicates that the mitochondrial fraction obtained from the NRK-52E cells is of high purity and has minimal contamination with other subcellular organelles.
For transport measurements, mitochondria were suspended in 3 ml of sucrose-triethanolamine buffer in 25-ml Erlenmeyer flasks. Radiolabeled malonate (1 mM final concentration, containing 0.8 μCi; 38.5 cpm/nmol) or GSH (5 mM final concentration, containing 0.8 μCi; 23.4 cpm/nmol) was added to initiate transport. Aliquots (0.5 ml) were removed at 1, 2, 5, and 10 min and were added to 1.5-ml microcentrifuge tubes, which were centrifuged at 13,000g × 30 s. The mitochondrial pellets were washed twice with buffer, suspended in 0.5 ml of buffer, and transferred to a scintillation vial and radioactivity determined. No differences were observed if measurements were performed in mitochondria treated with antimycin A to inhibit metabolism, as found previously (Chen and Lash, 1998).
SDS-Polyacrylamide Gel Electrophoresis and Western Blotting.
For gel electrophoresis, 10 or 20 μg of protein was loaded per well onto a 10% (w/v) or a 12% (w/v) polyacrylamide gel (Bio-Rad), and separation was achieved according to the method of Laemmli (1970). Total protein was visualized by staining with Coomassie Brilliant Blue G. For Western blot analysis, protein was transferred by electroblotting to a nitrocellulose membrane (MSI, Westborough, MA), blocked with 3% (w/v) bovine serum albumin (Promega), washed with Tris-buffered saline containing Tween 20, probed with a monoclonal anti-RGSHHHH tag antibody (QIAGEN, Valencia, CA), washed with Tris-buffered saline containing Tween 20, and then probed with an anti-mouse IgG antibody conjugated to alkaline phosphatase (Jackson Immunoresearch Laboratories, West Grove, PA). Immunoreactive bands were visualized after incubation with a solution containing 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium (Promega).
Flow Cytometry Analysis of Cell Cycle and Quantitation of Apoptosis.
Cell cultures were washed twice with sample buffer (PBS plus 1 g of glucose/l filtered through a 0.22-μm filter), dislodged by trypsin/EDTA incubation, centrifuged at 400g × 10 min at 22°C, and resuspended in sample buffer. Cell concentrations were adjusted to 1 to 3 × 106 cells/ml with sample buffer and 1 ml of the cell suspension was centrifuged at 400g × 10 min at 22°C. All of the supernatant except 0.1 ml/106cells was removed and the remaining cells were mixed on a vortex mixer in the remaining fluid for 10 s. Next, 1 ml of ice-cold ethanol (70%, v/v) was added to the sample in a dropwise manner, with samples being mixed for 10 s between drops. Tubes were capped and fixed in ethanol at 4°C. After fixation, cells were stained with propidium iodide (50 μg/ml) containing RNase A (100 U/ml). Samples were then mixed, centrifuged at 1000g × 5 min at 22°C, and all the ethanol except 0.1 ml was removed. Cells were mixed in the residual ethanol and 1 ml of the propidium iodide staining solution was added to each tube. After mixing again, cells were incubated at room temperature for at least 30 min. Samples were analyzed within 24 h by flow cytometry using an FACSCalibur flow cytometer (BD Biosciences, San Jose, CA), which is a core facility of the National Institute of Environmental Health Sciences Center for Molecular Toxicology with Human Applications at Wayne State University. Analyses were performed with 20,000 events per sample using the ModFit LT version 2 for Macintosh data acquisition software package (Verity Software House, Inc., Topsham, ME; distributed by BD Immunocytochemistry System BDIS). Propidium iodide was detected by the FL-2 photomultiplier tube. Fractions of apoptotic cells were quantified by analysis of the sub-G1 (subdiploid) peak with ModFit cell cycle analysis. The percentages of distribution of cells in the various stages of the cell cycle (G0/G1, S, and G2/M) were also calculated. Cell aggregates were discarded in the flow cytometry analysis by postfixation aggregate discrimination.
Data Analysis.
Data were normalized to protein content, which was determined by the bicinchoninic acid protein assay from Pierce Chemical (Rockford, IL). All measurements were performed three to five times. Significant differences between means were first assessed by a one-way or two-way analysis of variance, depending on the comparisons being tested. When significant “F values” were obtained, the Fisher's protected least significant differencet test was performed to determine which means were significantly different from one another, using a two-tail probability,P < 0.05, as the criteria for significance.
Results
Bacterial Expression of the Rat Kidney Mitochondrial DCC.
The full-length cDNA for the rat DCC was prepared from rat kidney total RNA by RT-PCR. The cDNA was subcloned into the pRSET-B expression vector for expression of DCC protein as an N-terminal His6 fusion protein. Expression of DCC in one clone (15) was readily induced with IPTG (Fig.1A); the arrow shows the time-dependent increase in expression of a 35.5-kDa protein, the expected molecular mass of the DCC-His6 product. This was confirmed by Western analysis (Fig. 1B). Figure 1A also shows that DCC-His6 could be solubilized from inclusion bodies. The detergent-solubilized extract from inclusion bodies was fractionated on a Ni2+-affinity column. The purified DCC-His6 was approximately 90% pure by Coomassie staining (Fig. 2). The overall yield of DCC-His6 from a 1-liter culture of bacteria was typically 20 to 25 mg of protein.
Reconstitution of Recombinant Rat Kidney DCC-His6Protein.
The partially purified DCC fraction was reconstituted into proteoliposomes to assess its transport activity with GSH and dicarboxylate substrates. Time courses for the uptake of GSH (0.05–10 mM; Fig. 3, A and B) and malonate (0.01–5 mM; Fig. 3, C and D) were determined in phosphate- and malate-containing proteoliposomes. In preloaded proteoliposomes, transport exhibited a characteristic spike, reflecting a heteroexchange of the trans- for cis-substrates. Thereafter, intravesicular radioactive substrate modestly decreased. Reconstituted DCC exhibited time- and concentration-dependent uptake of both GSH and malonate. For both substrates at nearly all concentrations tested, maximal intravesicular content of transported substrate was achieved by 2 min. Initial rates (after 2 min) and maximal intravesicular contents of malonate were approximately 5- to 6-fold higher than those for GSH.
Analysis of the kinetics of GSH transport in proteoliposomes reconstituted with the purified recombinant DCC showed Michaelis-Menten kinetics with obvious saturation (Fig.4A). Eadie-Hofstee analysis showed an excellent correlation coefficient (r2= 0.912) with Vmax (in nanomoles per minute per milligram of protein) andKm (in millimolar concentration) values of 263 and 8.39, respectively (Fig. 4B). Malonate also exhibited Michaelis-Menten kinetics with saturation, although the shape of the calculated curve for the V versus S plot differed somewhat from that of GSH transport (Fig. 4C). Eadie-Hofstee analysis showed a reasonably good fit (r2 = 0.676) withVmax (in nanomoles per minute per milligram of protein) and Km (in millimolar concentration) values of 473 and 0.59, respectively (Fig.4D).
Transport of GSH and malonate by reconstituted DCC-His6 was characterized further by studying the effect of potential competitive and noncompetitive inhibitors. Uptake of 1 mM GSH into proteoliposomes was significantly inhibited by several competitive inhibitors [i.e., butylmalonate (Kaplan and Pedersen, 1985; Chen et al., 2000),l-γ-glutamyl-l-glutamate (McKernan et al., 1991; Chen and Lash, 1998; Chen et al., 2000), and the GSH analog ophthalmic acid (Chen et al., 2000)], each present at a concentration of 10 mM, in amounts ranging from 56 to 75% (Table1). Additionally, uptake of GSH was also significantly inhibited by pyridoxal 5′-phosphate and two mercurials (mersalyl and p-mercuribenzoate), which are noncompetitive inhibitors (Kaplan and Pedersen, 1985). Inhibition by these three compounds and by butylmalonate is consistent with the known properties of the DCC (Kaplan and Pedersen, 1985; Lançar-Benba et al., 1994;Kakhniashvili et al., 1997; Fiermonte et al., 1998).
Uptake of 1 mM malonate into proteoliposomes containing DCC-His6 was also significantly inhibited by butylmalonate, pyridoxal 5′-phosphate, and mersalyl (Table2). Additionally, 10 mM GSH inhibited uptake of 1 mM malonate by approximately 40%, which is consistent with the relative affinities of the carrier protein for the two substrates as determined in Fig. 4. Although glutamate is a dicarboxylate, it did not inhibit malonate transport, which agrees with previous findings in isolated renal cortical mitochondria (Chen and Lash, 1998) and a partially purified and reconstituted preparation of mitochondrial inner membrane transporters (Chen et al., 2000).
Mitochondrial GSH Transport Activity in Transfected NRK-52E Cells.
To better explore the role of the DCC in GSH transport in an intact renal cell and to develop a strategy to increase mitochondrial GSH levels and protect cells from various forms of chemical-induced injury, NRK-52E cells were transiently transfected with the cDNA for the rat kidney mitochondrial DCC in pcDNA3.1/V5-His-TOPO. Mitochondrial fractions were prepared from three clones and were tested for the presence of the DCC-His6 fusion protein by immunoblot analysis with antibody directed to the His6 tag (Fig.5). Whereas no reactivity was detected in the wild-type cells, a single, prominent band at approximately 35.5 kDa was detected in mitochondrial extracts from each of the three clones, indicating expression of the recombinant protein. The relative expression of the recombinant protein varied 1.72-fold, as judged by densitometry.
The functional effects of transfecting DCC-His6into NRK-52E cells were determined by measuring uptake of 5 mM GSH and 1 mM malonate in suspensions of mitochondria prepared from both wild-type and transfected cells (Fig. 6). Transport of both GSH and malonate was markedly increased in the transfected cells. The stimulation of uptake (at 2 min) in the different populations of transfected cells ranged from 2.45- to 9.60-fold for GSH and from 4.18- to 11.3-fold for malonate. Moreover, the amount of stimulation of uptake seemed to correlate qualitatively with the intensity of the bands on immunoblots, with clone 3 exhibiting the most stimulation of transport and the highest relative expression of the three clones (Figs. 5 and 6).
Influence of Overexpression of rDCC-His6 on Sensitivity to Chemical-Induced Apoptosis.
The influence of overexpression of the DCC protein on sensitivity of the NRK-52E cells to chemical-induced apoptosis was next examined. Both wild-type cells (NRK-52E-WT) and transfectants overexpressing rDCC-His6(NRK-52E-rDCC+) were incubated with 1 mM GSH for 24 h, followed by incubation for 1, 2, or 4 h without treatment, or with tBH (10 or 50 μM) or DCVC (50 or 200 μM), in the presence of 20 μM GSH. The 20 μM GSH was chosen to mimic the concentration of GSH in the extracellular or periplasmic space and is that to which renal cells are normally exposed (Lash and Jones, 1985).
Induction of apoptosis by tBH or DCVC was assessed by FACS analyses and compared with controls (treated with GSH but no tBH or DCVC) (Figs.7 and8). Results clearly illustrate both the time dependence of the induction of apoptosis in response to these treatments and the significant protection afforded the NRK-52E cells upon transfection with the rDCC-His6 protein. For both the wild-type and DCC-His6-transfected cells, incubation with only media (i.e., controls) produced <1% apoptosis after 1, 2, or 4 h (Fig. 7, A–C). In contrast, wild-type cells incubated with 10 μM tBH exhibited 13.1, 15.1, or 32.9% apoptosis after 1, 2, or 4 h, respectively (Fig. 7, D–F), and wild-type cells incubated with 50 μM DCVC exhibited 2.28, 7.74, or 25.9% apoptosis after 1, 2, or 4 h, respectively (Fig. 7, G–I). Transfectants exhibited markedly less apoptosis: Transfectants incubated with 10 μM tBH exhibited a maximum of 3.89% apoptosis after 4 h (Fig. 8F) and those incubated with 50 μM DCVC exhibited a maximum of 16.4% apoptosis after 4 h (Fig. 8I).
Protection of NRK-52E cells by overexpression of rDCC-His6 is more completely illustrated in Fig. 9: The percentage of apoptotic cells, as indicated by FACS analyses, is plotted against time for cells incubated with either media, 10 or 50 μM tBH, or 50 or 200 μM DCVC in wild-type cells (Fig. 9A) and transfectants (Fig. 9B). Markedly lower amounts of apoptosis for cells incubated with either concentration of tBH or DCVC were observed at all time points for transfected cells.
Discussion
Mitochondrial GSH status is a critical determinant of cellular energetics and function and plays a key role in determining susceptibility of cells to various forms of chemical or pathological injury. The ability to manipulate GSH status in this organelle can provide a greater understanding of precisely how mitochondrial GSH functions in various processes, including oxidative phosphorylation and apoptosis, but also a tool with which to modulate cellular injury upon exposure to various toxicants. The present communication describes one such approach in which one of the major mechanisms by which cells compartmentalize GSH is genetically altered to produce protection from two model toxicants.
Over the past several years, we have been studying the transport of GSH into renal proximal tubular mitochondria (McKernan et al., 1991; Chen and Lash, 1998; Chen et al., 2000). Transport of GSH from the cytoplasm into the mitochondrial matrix is the primary, if not sole, source of GSH for this subcellular compartment because of the absence of significant synthesis of GSH from its precursors within the mitochondrial matrix (Griffith and Meister, 1985; McKernan et al., 1991; Smith et al., 1996). Two electroneutral anion carriers, the DCC and OGC, were identified as being responsible for most, if not all, of the transport of GSH into renal cortical mitochondria (Chen and Lash, 1998; Chen et al., 2000). This conclusion was based on examination of substrate specificity and inhibitor studies. Although a partially purified preparation of mitochondrial anion carriers was reconstituted and used by others to study the transport of various dicarboxylates (Kaplan and Pedersen, 1985; Saint-Macary and Foucher, 1985; Bisaccia et al., 1988) and by us to study GSH transport (Chen et al., 2000), there are inherent problems or limitations with this experimental approach. First, the carrier proteins are present in the mitochondrial inner membrane at relatively low abundance, making it difficult to obtain sufficient amounts of material for experimentation. Second, of the various anion carriers of the inner membrane, the DCC protein, in particular, is relatively unstable in a purified form (Lançar-Benba et al., 1994), making it difficult to obtain sufficient amounts of pure protein suitable for functional assays or protein chemistry. The availability of the complete cDNA sequence from the rat (Fiermonte et al., 1998) affords us the opportunity to express the DCC protein in relatively large amounts, to study its catalytic function in greater detail and manipulate its molecular regulation to probe its physiological and toxicological function.
We showed previously that GSH transport into renal cortical mitochondria was insensitive to membrane potential (McKernan et al., 1991; Chen and Lash, 1998), which is consistent with the function of the electroneutral DCC and OGC. This suggests that GSH is transported by these carriers as a dianion. As discussed previously (Chen and Lash, 1998), the relatively alkaline pH of the mitochondrial matrix and the suggested presence of a microenvironment near the active site of the carrier proteins effectively lowers the pKa of the thiol group so that a larger proportion of GSH molecules may be in the thiolate form.
In the present study, we expressed the full-length DCC cDNA from rat kidney mitochondria in bacteria as an N-terminal His6 fusion protein to facilitate its isolation and detection with His6-specific antibodies. DCC synthesis was induced with IPTG in a time-dependent manner and was extracted from inclusion bodies for fractionation on a Ni2+-Sepharose affinity column. Our results demonstrate that the partially purified DCC-His6, reconstituted in lipid membranes, was functional. Function was shown by measurements of time and concentration dependence of GSH and malonate uptake. As we found in isolated mitochondria from rat kidneys (Chen and Lash, 1998) and in the enriched and reconstituted anion carrier preparation (Chen et al., 2000), transport of both substrates exhibits Michaelis-Menten kinetics and the rate of uptake and maximal accumulation of malonate are at least 5-fold greater than those for GSH. Nonetheless, the kinetics of GSH uptake calculated from the reconstituted, bacterially expressed DCC protein are reasonable compared with those observed previously in other in vitro model systems. The shape of the time courses for uptake of GSH and malonate (Fig. 3), which exhibited maxima at 2-min time points and slight decreases thereafter, is consistent with that which we and others typically observe in artificial proteoliposome vesicles in which the transport process under investigation involves an exchange of substrates on opposite sides of the membrane; the slight overshoot is likely due to trans-stimulation of uptake.
Sensitivity of GSH and malonate uptake to various competitive and noncompetitive inhibitors provided additional evidence that recombinant DCC-His6 was functionally identical to the protein detected in partially purified preparations from rat or rabbit kidney. This includes inhibition by butylmalonate, pyridoxal 5′-phosphate, and mercurials. Furthermore, GSH inhibited malonate uptake. With respect to the specificity of GSH uptake, γ-glutamyl amino acids and ophthalmic acid were good inhibitors, consistent with the properties we described previously for GSH transport into renal cortical mitochondria (McKernan et al., 1991; Chen and Lash, 1998). The properties that apply to dicarboxylate transport are also comparable with those previously reported in the literature (Bisaccia et al., 1985, 1988; Saint-Macary and Foucher, 1985; Kaplan and Pedersen, 1985;Fiermonte et al., 1993, 1998; Lançar-Benba et al., 1994; Kaplan et al., 1995; Iacobazzi et al., 1996; Palmieri et al., 1996;Kakhniashvili et al., 1997).
An important goal for the present study was to establish transport function upon transfection of the His-tagged DCC construct into an intact renal cell line, NRK-52E cells, derived from normal rat kidney proximal tubules. Unlike some other commonly used renal epithelial cell lines, basic cellular energetics and redox status in NRK-52E cells have not been characterized extensively. In a separate study (Lash et al., 2002), we provided baseline data on mitochondrial function and GSH transport and metabolism. With the exception of very low activity of brush-border membrane enzymes, including γ-glutamyltransferase, GSH metabolism and plasma membrane transport in NRK-52E cells were comparable with what are commonly observed in freshly isolated or primary cultures or rat renal proximal tubular cells. Notably, the NRK-52E cells have a low content of mitochondria, as judged by use of a mitochondria-specific fluorescent dye and confocal microscopy, and low activities of several mitochondrial dehydrogenases, of oxidative phosphorylation, and of GSH and malonate transport across the inner membrane, compared with those in freshly isolated or primary cultures of rat renal proximal tubular cells. This finding is certainly not uncommon in an immortalized, epithelial cell line. This “deficiency” in the NRK-52E cells in comparison with the in vivo renal proximal tubule may actually facilitate our studies because extracellular degradation of GSH, which can cause experimental artifacts in transport studies, is minimal in these cells.
Immunoblot analysis using an antibody directed against the C-terminal His6 tag of our expressed protein confirmed successful transfection of the NRK-52E cells. More importantly, mitochondria isolated from transfectants exhibited significantly increased rates of uptake and higher maximal accumulation of both GSH and malonate than in those isolated from wild-type cells. The amount of stimulation of substrate uptake seemed to approximately correlate with the level of expression of the recombinant protein in the transfected NRK-52E cells. These observations were carried further by demonstrating that transfectants exhibited much less apoptosis compared with wild-type cells when incubated with tBH or DCVC, which are both oxidants and mitochondrial toxicants (Lash and Anders, 1987; Hagen et al., 1988; Lash and Tokarz, 1990; McKernan et al., 1991; Lash et al., 2002). Hence, overexpression of the DCC from rat kidney in a stable cell line derived from rat proximal tubule provides marked protection from oxidant-induced apoptosis. This experimental approach allows us to manipulate the mitochondrial thiol/disulfide redox and GSH status, thereby providing a novel tool with which to probe mitochondrial function and modulate cellular susceptibility to oxidants and other mitochondrial toxicants.
In summary, although we have confirmed that the bacterially expressed and reconstituted DCC protein exhibits identical function for transport of GSH and dicarboxylates as determined previously, the novelty of the present results is that we demonstrate function of a cloned and bacterially expressed mitochondrial transporter with GSH, which provides the framework for studies on the molecular regulation of and more controlled manipulation of mitochondrial GSH status. Furthermore, we show that NRK-52E cells exhibited increased rates of mitochondrial uptake of GSH and dicarboxylates when these cells are transiently transfected to overexpress the DCC protein and that these cells are relatively resistant to chemical-induced apoptosis.
Footnotes
-
This work was funded by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK40725 (to L.H.L. and L.H.M.). Core facilities funded by the National Institute of Environmental Health Sciences Center for Molecular Toxicology with Human Applications (Grant P30-ES06639) at Wayne State University were used for some of these studies.
-
DOI: 10.1124/jpet.102.040220
- Abbreviations:
- GSH
- glutathione
- DCVC
- S-(1,2-dichlorovinyl)-l-cysteine
- DCC
- dicarboxylate carrier
- OGC
- oxoglutarate carrier
- NRK
- normal rat kidney
- tBH
- tert-butyl hydroperoxide
- PCR
- polymerase chain reaction
- PBS
- phosphate-buffered saline
- FACS
- fluorescence-activated cell sorting
- IPTG
- isopropyl β-d-thiogalactoside
- Received June 11, 2002.
- Accepted July 9, 2002.
- The American Society for Pharmacology and Experimental Therapeutics