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Role of Voltage-Dependent Anion Channels in Glutathione Transport into Yeast Mitochondria
Biochemical and Biophysical Research Communications 276, 940 –944 (2000) doi:10.1006/bbrc.2000.3572, available online at http://www.idealibrary.com on
Role of Voltage-Dependent Anion Channels in Glutathione Transport into Yeast Mitochondria Brian S. Cummings, 1 Rowena Angeles, Roy B. McCauley, and Lawrence H. Lash 2 Department of Pharmacology, School of Medicine, Wayne State University, Detroit, Michigan 48201
Received August 31, 2000
Glutathione (GSH) is imported into mitochondria from the extra-mitochondrial cytoplasm. Translocation across the inner membrane of mitochondria is thought to occur via the dicarboxylate and 2-oxoglutarate carriers; however, the means by which GSH passes through the outer membrane is unknown. Disruption of the outer membrane of yeast mitochondria using either digitonin or osmotic shock did not alter GSH accumulation as compared with accumulation in intact mitochondria. These results suggested that passage across the outer membrane was not the rate-limiting step in GSH accumulation. Mitochondria isolated from yeast strains with a disruption in the major pore-forming protein of the outer membrane, VDAC1, accumulated GSH to a greater extent than mitochondria isolated from a wild-type strain. Disruption of the gene for VDAC2 did not affect GSH import. Thus, neither VDAC form is essential for GSH translocation into mitochondria, and the participation of another outer membrane channel in GSH import is possible. © 2000 Academic Press Key Words: glutathione; mitochondria; porin; voltage-gated anion channel; outer membrane; yeast.
Thiol-disulfide status, which is predominantly regulated by glutathione (GSH), is critical to the maintenance of numerous functions in mitochondria, including membrane structure and integrity (1, 2), ion homeostasis (3), redox status (4), and activities of numerous sulfhydryl-dependent enzymes (1). Distinct pools of GSH are found in the mitochondria of liver and Abbreviations used: GSH, glutathione; GSSG, glutathione disulfide; HPLC, high-performance liquid chromatography; VDAC, voltage-dependent anion channel. 1 Present address: Division of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, 4301 Markham Street, Little Rock, AR 72205. 2 To whom correspondence should be addressed at Department of Pharmacology, Wayne State University School of Medicine, 540 East Canfield Avenue, Detroit, MI 48201. Fax: (313) 577-6739. E-mail: [email protected]. 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
kidney (5, 6), and the toxicological importance of this pool is highlighted by the numerous studies that have demonstrated a specific role for depletion of the mitochondrial, rather than the cytoplasmic, pool of GSH in various forms of chemical or pathological injury (7–10). Regulation of this mitochondrial pool of GSH has been the subject of recent investigation. Inasmuch as all available data indicate that most, if not all, GSH synthesis in mammalian cells occurs in the cytoplasm (11, 12), one must conclude that the mitochondrial pool of GSH derives from transport of cytoplasmic GSH into the mitochondria (13). Because GSH is a charged molecule at physiological pH, transport of GSH into mitochondria is energetically unfavorable due to the prevailing membrane potential and pH gradient across the mitochondrial inner membrane. We have recently demonstrated that GSH transport across the inner membrane of mitochondria of epithelial cells from rat kidney cortex occurs by action of the dicarboxylate and 2-oxoglutarate carriers (14 –17). These two carriers function to exchange dicarboxylates with inorganic phosphate or 2-oxoglutarate with other dicarboxylates, respectively, and belong to a “superfamily” of anion carriers from the mitochondrial inner membrane (18). The means by which GSH traverses the outer membrane is not known; however, the voltage dependent anion channel of the outer membrane (VDAC1) is a likely candidate for playing a role in GSH transport into mitochondria. VDAC1 regulates adenine nucleotide flux across the outer membrane (19), and disruption of the gene for VDAC1 in yeast causes a slow growth phenotype (20). More recently, VDAC1 has been shown to control the release of cytochrome c into the extramitochondrial cytoplasm via interactions with members of the Bcl-2 gene family (21). VDAC1 apparently regulates fluxes across the outer membrane in apoptotic cell death as well as during normal growth. The outer membrane of yeast mitochondria also contain an isoform of VDAC1 called VDAC2. Although it has not been possible to demonstrate channel forming
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Disruption of the mitochondrial outer membrane. Mitoplasts were prepared by osmotic shock and treatment with digitonin (23 and 25, respectively). The method used for immunoblotting has been described recently (26).
TABLE 1
Strains of Saccharomyces cerevisiae Used in This Study Nickname wt dp1 dp2
Strain
Statistical analyses. Significant differences between means were first evaluated by a one-way analysis of variance. When significant F values were obtained, the Fisher’s protected least significant difference t test was performed to determine which means were significantly different from each other with two-tailed P values ⬍ 0.05 considered significant.
JHRY20-2Ca: MATa, GAL⫹, leu2, his3-⌬200 & ura3-52 JHRY20-Ca: MATa, GAL⫹, yvdacl⬋LEU2, his3-⌬200 & ura3-52 JHRY20-Ca: MATa, GAL⫹, yvdac2⬋TRP1, his3-⌬200 & ura3-52
RESULTS AND DISCUSSION properties for VDAC2, its participation in GSH translocations has not been ruled out (22). In the present study, we used mitoplasts and mitochondria from yeast strains that have disruptions in either VDAC1 or VDAC2 to investigate the role of these channels in the translocation of GSH into mitochondria. The results suggest that the outer membrane is sufficiently permeable to GSH that the carriers allowing passage across the inner membrane can be saturated, and that neither VDAC1 (the major poreforming protein in the outer membrane) nor its isoform, VDAC2, is essential for GSH translocation. MATERIALS AND METHODS Measurement of GSH uptake with radiolabeled GSH. Yeast mitochondria were prepared as described previously (23) and used immediately. Incubations were processed essentially as described previously (15). Yeast mitochondria (0.2 ml, 0.25 to 0.5 mg protein) were incubated at 25°C with 0.033 ml of GSH (0.05 Ci of [ 3Hglycyl]-GSH, 44.8 Ci/mmol [New England Nuclear, Boston, MA], and unlabeled GSH to a final concentration of 5 mM) and 0.1 ml of mitochondrial buffer (20 mM triethanolamine/HCl, pH 7.4, 225 mM sucrose, 3 mM potassium phosphate, 5 mM MgCl 2, 20 mM KCl, and 0.1 mM phenylmethylsulfonyl fluoride to inhibit proteolysis). At indicated times, incubations were stopped by centrifugation for 30 s at 10,000g. Supernatants were removed and saved and pellets were resuspended in mitochondrial buffer and centrifuged again at 10,000g for 30 s. Supernatants were removed and combined with the first supernatants and the pellets were resuspended in 0.25 ml of mitochondrial buffer and saved for liquid scintillation counting. Measurement of GSH uptake by HPLC. GSH and glutathione disulfide (GSSG) content in yeast mitochondria were also determined by ion-exchange high-performance liquid chromatography (HPLC) with an amine column and a methanol-acetate solvent system after derivatization with iodoacetate and 1-fluoro-2,4dinitrobenzene, as described previously (15, 24). In some experiments, fractions eluting from the HPLC column were collected and radioactivity was quantified by scintillation counting, with peaks of radioactivity compared with those for GSH, GSSG, and GSH degradation products on the chromatogram. This confirmed that ⬎90% of the radiolabeled material transported into the yeast mitochondria was GSH and was neither oxidation nor degradation products. Measurement of GSH efflux from mitochondria. Yeast mitochondria were preloaded with 5 mM [ 3H-glycyl]-GSH for 4 min, incubation mixtures were then centrifuged to remove extramitochondrial GSH, and mitochondria were resuspended in GSH-free, mitochondrial buffer. Aliquots were then incubated at 25°C for another 4 min, and GSH content in both the pellet and supernatant was measured as described above.
To evaluate the importance of the outer membrane in GSH transport, mitoplasts were prepared from mitochondria isolated from the Saccharomyces cerevisiae strain JHRY20-2Ca (wt, see Table 1 for a description of this and other strains used in these experiments). Mitoplasts were prepared using both osmotic shock and extraction with digitonin. As can be seen in Fig. 1, mitoplasts that were prepared by osmotic rupture of the outer membrane were depleted of the intermembrane space protein, cytochrome b 2; however, these mitoplasts retained markers for the outer membrane (VDAC1), matrix (ATP11p), and inner membrane (beta subunit of the F 0F 1 ATPase). Mitoplasts also were prepared by extraction with 0.32% (w/v) digitonin. Lower concentrations of digitonin did not fully release cytochrome b 2 while higher concentrations partially depleted ATP11p, suggesting that the inner membrane may have been compromised (Fig. 1). Extraction with 0.32% (v/v) digitonin only partially depleted VDAC1, indicating that the outer membrane was incompletely removed. Both types of mitoplasts were compared with intact mitochondria for their ability to accumulate radiolabeled GSH. As can be seen in Table 2, rupture of the outer membrane by either method apparently had no
FIG. 1. Protein composition of control, osmotically shocked, and digitonin-fractionated yeast mitochondria. The protein composition of yeast mitochondria (25 g protein per lane) was compared by immunoblotting equivalent amounts of mitochondria that had their outer membrane disrupted by osmotic shock (23) or the indicated concentrations of digitonin (25). Cytochrome b 2 (b 2) is a marker for the intermembrane space while ATP11p and the beta subunit of the F 0F 1-ATPase (F 1) are markers for the matrix and inner membrane, respectively.
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 2
Import of Radiolabeled GSH into Mitochondria and Mitoplasts Treatment
1-min Incubation
4-min Incubation
None Osmotic rupture 0.32% (w/v) Digitonin
1.1 ⫾ 0.1 1.1 ⫾ 0.2 0.7 ⫾ 0.3
1.7 ⫾ 0.2 1.3 ⫾ 0.7 1.5 ⫾ 0.7
Note. Accumulation of GSH in mitochondria or mitoplasts from wt yeast strain was determined by scintillation counting after incubation with tracer of [ 3H-glycyl]-GSH and cold GSH to a total concentration of 5 mM. Values (nmol/mg protein) are means ⫾ SD from four separate mitochondrial preparations.
effect on the translocation of GSH. There were two interpretations for this experiment. The outer membrane was not rate-limiting for GSH import (perhaps, because the outer membrane channels were present in abundance), or GSH translocation was limited by specifically located channels (perhaps at sites of contact between the inner and outer membrane (25)) that were not removed by either method of preparation of mitoplasts. The most likely channel for GSH translocation through the outer membrane is the voltage dependent anion channel, VDAC1. VDAC1 is involved in the passage of adenine nucleotides across the outer membrane (19). Yeast mitochondria contain a second VDAC form (VDAC2); however, there is no evidence that VDAC2 can form channels (22). To determine whether these proteins were involved in GSH import, mitochondria were isolated from yeast strains that had a genetic disruption in one of the VDAC genes. These mitochondria were compared with mitochondria isolated from wild-type (wt) yeast for their ability to accumulate radiolabeled GSH (Fig. 2). As might have been expected, mitochondria isolated from the yeast strain with a disruption in the VDAC2 gene, dp2, were able to accumulate GSH as well as mitochondria isolated from wt. On the other hand, mitochondria isolated from the strain with a VDAC1 disruption accumulated about twice as much GSH as did those isolated from wt. Clearly, neither VDAC was essential for GSH translocation. However, the enhanced accumulation of GSH in the cells with the VDAC1 disruption suggests that alterations in outer membrane structure and/or permeability can modulate GSH translocation into the matrix. It seemed improbable that VDAC1 depletion would directly cause an increase in the mitochondrial accumulation of GSH. Instead, it seemed more likely that the VDAC1 disruption might have caused a decrease in the efflux of GSH from the mitochondria. To test this possibility, mitochondria isolated from wt, dp1 and dp2 were loaded with radioactive GSH. These mitochondria were incubated in a medium that did not contain GSH
and the mitochondrial content of labeled GSH was measured at the times indicated in Fig. 3. Indeed, depletion of VDAC1 impaired efflux of GSH; however, the rate of GSH efflux from mitochondria isolated from wt and dp2 was so slight that it is unlikely that an altered rate of efflux could have resulted in the differences in GSH content seen in Fig. 2. In agreement with the higher total accumulation of GSH in mitochondria from the dp1 strain (cf. Fig. 2), these mitochondria had significantly higher initial values of GSH than either of the other two strains after the preloading period. The GSH used in these analyses was commercially prepared using radiolabeled glycine, and it is possible that the labeled GSH was degraded more rapidly in mitochondria isolated from dp1. That is, the increased accumulation might be radiolabeled glycine rather than GSH. This is unlikely for several reasons. First, HPLC analysis of the mitochondrial content of radioactive substances did not indicate that labeled glycine accumulated significantly in mitochondria from any of the strains (data not shown). In fact, virtually all the radioactivity migrated with the same retention time as GSH. Also, import experiments were performed using unlabeled GSH, and the accumulation was estimated using HPLC. Under these conditions (Table 3), mitochondria isolated from dp1 still accumulated about twice as much GSH as those isolated from wt. Finally, the activity of ␥-glutamyltransferase, which degrades GSH, was indistinguishable among the three types of mitochondria (data not shown). It is also unlikely that mitochondria isolated from dp1 oxidized GSH more rapidly. The accumulation of GSSG (estimated by HPLC) after 4 min of incubation
FIG. 2. Import of GSH into yeast mitochondria. Total mitochondrial accumulation of GSH in mitochondria from wild-type (wt) and two mutant strains (dp1, dp2) of yeast was measured at the indicated times with radiolabeled GSH (final GSH concentration, 5 mM) and scintillation counting. Results are means ⫾ SD of measurements from three separate mitochondrial preparations. Values in the dp1 strain were significantly (P ⬍ 0.05) greater than those in either the wt or dp2 strains at all time points after the zero time point.
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was 0.7, 0.9 and 0.7 nmol per mg mitochondrial protein for mitochondria isolated from wt, dp1 and dp2, respectively. Therefore, it is unlikely that the increase in GSH accumulation by mitochondria isolated from dp1 was a consequence of increased retention of GSSG. Yeast vacuolar membrane vesicles have been used recently as a model system to study plasma membrane transport of GSH (27–29). However, it is unlikely that the transport properties we observed are due to contaminating vacuolar fragments. The yeast vacuolar system, YCF1, is an orthologue of the mammalian multidrug resistance associated proteins (28, 29). Unlike the translocations measured here, this system is ATPdependent (27–29). There has been no other work published on GSH transport in yeast. Transport of organic anions, in particular citric acid cycle intermediates, has been studied in yeast (30 –32), and the characteristics of these carriers appear to be very similar to those in mammalian mitochondria. Hence, yeast mitochondria appear to serve as a useful model in which to study mitochondrial metabolite transport. The experiments described here show that neither VDAC1 nor VDAC2 was essential for translocation of GSH into the mitochondrial matrix. In fact, depletion of VDAC1 caused an increase in GSH accumulation. A possible explanation is that mitochondria in the dp1 strain are swollen; however, electron microscopy does not support this hypothesis (33). The reason for this increase is unclear; however, the mitochondrial effects of VDAC1 disruption are pleiotropic (20). It may be that the effects of this disruption on GSH accumulation may be secondary to another effect on the mitochondria.
TABLE 3
Import of GSH into Mitochondria Isolated from wt, dp1 and dp2 Cells Strain
0-min Incubation
4-min Incubation
wt dp1 dp2
0.6 ⫾ 0.2 0.5 ⫾ 0.3 1.0 ⫾ 0.1
3.2 ⫾ 0.9 8.1 ⫾ 2.7* 4.5 ⫾ 2.0
Note. Intramitochondrial accumulation of GSH was measured by HPLC analysis with derivatization after 0- or 4-min incubation of mitochondria from the three yeast strains with unlabeled 5 mM GSH. Values (nmol/mg protein) are means ⫾ SD from four separate mitochondrial preparations. * Significantly different (P ⬍ 0.05) from accumulation in wt.
The more important question is: If GSH does not traverse the outer membrane through VDAC1, how does it? There are two plausible explanations. The first is that GSH passage may occur throughout the outer membrane but via another channel. If so, the rate of flux through this channel must exceed the rate of translocation across the inner membrane since disruption of the outer membrane with either a detergent or osmotic shock does not improve the rate of flux. Alternatively, translocation may occur by a means other than VDAC1 or VDAC2 at discrete sites that are not as affected by either digitonin treatment or osmotic shock (e.g., contact sites between the inner and outer membranes). In either case, neither genetic disruption of VDAC1 nor breaching the outer membrane would be expected to affect the rate of flux. ACKNOWLEDGMENTS This research was supported in part by NIDDK Grant R01DK40725 to L.H.L. and by NIMH Grant 47181 and NIEHS Grant 5P30 ESO6639 to R.M. The contributions of Drs. M. Forte, Vollum Institute, Portland, Oregon (yeast strains), S. Ackerman, Wayne State University, Detroit, Michigan (antibodies to ATP11p and the beta subunit of the F 0F 1 ATPase), and G. Schatz, Biozentrum, Basel, Switzerland (antibodies to cytochrome b 2) are acknowledged.
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FIG. 3. Efflux of GSH from yeast mitochondria. Total mitochondrial content of GSH from wild-type (wt) and two mutant strains (dp1, dp2) of yeast was measured at the indicated times after preloading mitochondria with 5 mM [ 3H-glycyl]-GSH for 4 min. GSH content was measured by scintillation counting. Results are means ⫾ SD of measurements from three separate mitochondrial preparations. 943
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Role of Voltage-Dependent Anion Channels in Glutathione Transport into Yeast Mitochondria Brian S. Cummings, 1 Rowena Angeles, Roy B. McCauley, and Lawrence H. Lash 2 Department of Pharmacology, School of Medicine, Wayne State University, Detroit, Michigan 48201
Received August 31, 2000
Glutathione (GSH) is imported into mitochondria from the extra-mitochondrial cytoplasm. Translocation across the inner membrane of mitochondria is thought to occur via the dicarboxylate and 2-oxoglutarate carriers; however, the means by which GSH passes through the outer membrane is unknown. Disruption of the outer membrane of yeast mitochondria using either digitonin or osmotic shock did not alter GSH accumulation as compared with accumulation in intact mitochondria. These results suggested that passage across the outer membrane was not the rate-limiting step in GSH accumulation. Mitochondria isolated from yeast strains with a disruption in the major pore-forming protein of the outer membrane, VDAC1, accumulated GSH to a greater extent than mitochondria isolated from a wild-type strain. Disruption of the gene for VDAC2 did not affect GSH import. Thus, neither VDAC form is essential for GSH translocation into mitochondria, and the participation of another outer membrane channel in GSH import is possible. © 2000 Academic Press Key Words: glutathione; mitochondria; porin; voltage-gated anion channel; outer membrane; yeast.
Thiol-disulfide status, which is predominantly regulated by glutathione (GSH), is critical to the maintenance of numerous functions in mitochondria, including membrane structure and integrity (1, 2), ion homeostasis (3), redox status (4), and activities of numerous sulfhydryl-dependent enzymes (1). Distinct pools of GSH are found in the mitochondria of liver and Abbreviations used: GSH, glutathione; GSSG, glutathione disulfide; HPLC, high-performance liquid chromatography; VDAC, voltage-dependent anion channel. 1 Present address: Division of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, 4301 Markham Street, Little Rock, AR 72205. 2 To whom correspondence should be addressed at Department of Pharmacology, Wayne State University School of Medicine, 540 East Canfield Avenue, Detroit, MI 48201. Fax: (313) 577-6739. E-mail: [email protected]. 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
kidney (5, 6), and the toxicological importance of this pool is highlighted by the numerous studies that have demonstrated a specific role for depletion of the mitochondrial, rather than the cytoplasmic, pool of GSH in various forms of chemical or pathological injury (7–10). Regulation of this mitochondrial pool of GSH has been the subject of recent investigation. Inasmuch as all available data indicate that most, if not all, GSH synthesis in mammalian cells occurs in the cytoplasm (11, 12), one must conclude that the mitochondrial pool of GSH derives from transport of cytoplasmic GSH into the mitochondria (13). Because GSH is a charged molecule at physiological pH, transport of GSH into mitochondria is energetically unfavorable due to the prevailing membrane potential and pH gradient across the mitochondrial inner membrane. We have recently demonstrated that GSH transport across the inner membrane of mitochondria of epithelial cells from rat kidney cortex occurs by action of the dicarboxylate and 2-oxoglutarate carriers (14 –17). These two carriers function to exchange dicarboxylates with inorganic phosphate or 2-oxoglutarate with other dicarboxylates, respectively, and belong to a “superfamily” of anion carriers from the mitochondrial inner membrane (18). The means by which GSH traverses the outer membrane is not known; however, the voltage dependent anion channel of the outer membrane (VDAC1) is a likely candidate for playing a role in GSH transport into mitochondria. VDAC1 regulates adenine nucleotide flux across the outer membrane (19), and disruption of the gene for VDAC1 in yeast causes a slow growth phenotype (20). More recently, VDAC1 has been shown to control the release of cytochrome c into the extramitochondrial cytoplasm via interactions with members of the Bcl-2 gene family (21). VDAC1 apparently regulates fluxes across the outer membrane in apoptotic cell death as well as during normal growth. The outer membrane of yeast mitochondria also contain an isoform of VDAC1 called VDAC2. Although it has not been possible to demonstrate channel forming
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Vol. 276, No. 3, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Disruption of the mitochondrial outer membrane. Mitoplasts were prepared by osmotic shock and treatment with digitonin (23 and 25, respectively). The method used for immunoblotting has been described recently (26).
TABLE 1
Strains of Saccharomyces cerevisiae Used in This Study Nickname wt dp1 dp2
Strain
Statistical analyses. Significant differences between means were first evaluated by a one-way analysis of variance. When significant F values were obtained, the Fisher’s protected least significant difference t test was performed to determine which means were significantly different from each other with two-tailed P values ⬍ 0.05 considered significant.
JHRY20-2Ca: MATa, GAL⫹, leu2, his3-⌬200 & ura3-52 JHRY20-Ca: MATa, GAL⫹, yvdacl⬋LEU2, his3-⌬200 & ura3-52 JHRY20-Ca: MATa, GAL⫹, yvdac2⬋TRP1, his3-⌬200 & ura3-52
RESULTS AND DISCUSSION properties for VDAC2, its participation in GSH translocations has not been ruled out (22). In the present study, we used mitoplasts and mitochondria from yeast strains that have disruptions in either VDAC1 or VDAC2 to investigate the role of these channels in the translocation of GSH into mitochondria. The results suggest that the outer membrane is sufficiently permeable to GSH that the carriers allowing passage across the inner membrane can be saturated, and that neither VDAC1 (the major poreforming protein in the outer membrane) nor its isoform, VDAC2, is essential for GSH translocation. MATERIALS AND METHODS Measurement of GSH uptake with radiolabeled GSH. Yeast mitochondria were prepared as described previously (23) and used immediately. Incubations were processed essentially as described previously (15). Yeast mitochondria (0.2 ml, 0.25 to 0.5 mg protein) were incubated at 25°C with 0.033 ml of GSH (0.05 Ci of [ 3Hglycyl]-GSH, 44.8 Ci/mmol [New England Nuclear, Boston, MA], and unlabeled GSH to a final concentration of 5 mM) and 0.1 ml of mitochondrial buffer (20 mM triethanolamine/HCl, pH 7.4, 225 mM sucrose, 3 mM potassium phosphate, 5 mM MgCl 2, 20 mM KCl, and 0.1 mM phenylmethylsulfonyl fluoride to inhibit proteolysis). At indicated times, incubations were stopped by centrifugation for 30 s at 10,000g. Supernatants were removed and saved and pellets were resuspended in mitochondrial buffer and centrifuged again at 10,000g for 30 s. Supernatants were removed and combined with the first supernatants and the pellets were resuspended in 0.25 ml of mitochondrial buffer and saved for liquid scintillation counting. Measurement of GSH uptake by HPLC. GSH and glutathione disulfide (GSSG) content in yeast mitochondria were also determined by ion-exchange high-performance liquid chromatography (HPLC) with an amine column and a methanol-acetate solvent system after derivatization with iodoacetate and 1-fluoro-2,4dinitrobenzene, as described previously (15, 24). In some experiments, fractions eluting from the HPLC column were collected and radioactivity was quantified by scintillation counting, with peaks of radioactivity compared with those for GSH, GSSG, and GSH degradation products on the chromatogram. This confirmed that ⬎90% of the radiolabeled material transported into the yeast mitochondria was GSH and was neither oxidation nor degradation products. Measurement of GSH efflux from mitochondria. Yeast mitochondria were preloaded with 5 mM [ 3H-glycyl]-GSH for 4 min, incubation mixtures were then centrifuged to remove extramitochondrial GSH, and mitochondria were resuspended in GSH-free, mitochondrial buffer. Aliquots were then incubated at 25°C for another 4 min, and GSH content in both the pellet and supernatant was measured as described above.
To evaluate the importance of the outer membrane in GSH transport, mitoplasts were prepared from mitochondria isolated from the Saccharomyces cerevisiae strain JHRY20-2Ca (wt, see Table 1 for a description of this and other strains used in these experiments). Mitoplasts were prepared using both osmotic shock and extraction with digitonin. As can be seen in Fig. 1, mitoplasts that were prepared by osmotic rupture of the outer membrane were depleted of the intermembrane space protein, cytochrome b 2; however, these mitoplasts retained markers for the outer membrane (VDAC1), matrix (ATP11p), and inner membrane (beta subunit of the F 0F 1 ATPase). Mitoplasts also were prepared by extraction with 0.32% (w/v) digitonin. Lower concentrations of digitonin did not fully release cytochrome b 2 while higher concentrations partially depleted ATP11p, suggesting that the inner membrane may have been compromised (Fig. 1). Extraction with 0.32% (v/v) digitonin only partially depleted VDAC1, indicating that the outer membrane was incompletely removed. Both types of mitoplasts were compared with intact mitochondria for their ability to accumulate radiolabeled GSH. As can be seen in Table 2, rupture of the outer membrane by either method apparently had no
FIG. 1. Protein composition of control, osmotically shocked, and digitonin-fractionated yeast mitochondria. The protein composition of yeast mitochondria (25 g protein per lane) was compared by immunoblotting equivalent amounts of mitochondria that had their outer membrane disrupted by osmotic shock (23) or the indicated concentrations of digitonin (25). Cytochrome b 2 (b 2) is a marker for the intermembrane space while ATP11p and the beta subunit of the F 0F 1-ATPase (F 1) are markers for the matrix and inner membrane, respectively.
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Import of Radiolabeled GSH into Mitochondria and Mitoplasts Treatment
1-min Incubation
4-min Incubation
None Osmotic rupture 0.32% (w/v) Digitonin
1.1 ⫾ 0.1 1.1 ⫾ 0.2 0.7 ⫾ 0.3
1.7 ⫾ 0.2 1.3 ⫾ 0.7 1.5 ⫾ 0.7
Note. Accumulation of GSH in mitochondria or mitoplasts from wt yeast strain was determined by scintillation counting after incubation with tracer of [ 3H-glycyl]-GSH and cold GSH to a total concentration of 5 mM. Values (nmol/mg protein) are means ⫾ SD from four separate mitochondrial preparations.
effect on the translocation of GSH. There were two interpretations for this experiment. The outer membrane was not rate-limiting for GSH import (perhaps, because the outer membrane channels were present in abundance), or GSH translocation was limited by specifically located channels (perhaps at sites of contact between the inner and outer membrane (25)) that were not removed by either method of preparation of mitoplasts. The most likely channel for GSH translocation through the outer membrane is the voltage dependent anion channel, VDAC1. VDAC1 is involved in the passage of adenine nucleotides across the outer membrane (19). Yeast mitochondria contain a second VDAC form (VDAC2); however, there is no evidence that VDAC2 can form channels (22). To determine whether these proteins were involved in GSH import, mitochondria were isolated from yeast strains that had a genetic disruption in one of the VDAC genes. These mitochondria were compared with mitochondria isolated from wild-type (wt) yeast for their ability to accumulate radiolabeled GSH (Fig. 2). As might have been expected, mitochondria isolated from the yeast strain with a disruption in the VDAC2 gene, dp2, were able to accumulate GSH as well as mitochondria isolated from wt. On the other hand, mitochondria isolated from the strain with a VDAC1 disruption accumulated about twice as much GSH as did those isolated from wt. Clearly, neither VDAC was essential for GSH translocation. However, the enhanced accumulation of GSH in the cells with the VDAC1 disruption suggests that alterations in outer membrane structure and/or permeability can modulate GSH translocation into the matrix. It seemed improbable that VDAC1 depletion would directly cause an increase in the mitochondrial accumulation of GSH. Instead, it seemed more likely that the VDAC1 disruption might have caused a decrease in the efflux of GSH from the mitochondria. To test this possibility, mitochondria isolated from wt, dp1 and dp2 were loaded with radioactive GSH. These mitochondria were incubated in a medium that did not contain GSH
and the mitochondrial content of labeled GSH was measured at the times indicated in Fig. 3. Indeed, depletion of VDAC1 impaired efflux of GSH; however, the rate of GSH efflux from mitochondria isolated from wt and dp2 was so slight that it is unlikely that an altered rate of efflux could have resulted in the differences in GSH content seen in Fig. 2. In agreement with the higher total accumulation of GSH in mitochondria from the dp1 strain (cf. Fig. 2), these mitochondria had significantly higher initial values of GSH than either of the other two strains after the preloading period. The GSH used in these analyses was commercially prepared using radiolabeled glycine, and it is possible that the labeled GSH was degraded more rapidly in mitochondria isolated from dp1. That is, the increased accumulation might be radiolabeled glycine rather than GSH. This is unlikely for several reasons. First, HPLC analysis of the mitochondrial content of radioactive substances did not indicate that labeled glycine accumulated significantly in mitochondria from any of the strains (data not shown). In fact, virtually all the radioactivity migrated with the same retention time as GSH. Also, import experiments were performed using unlabeled GSH, and the accumulation was estimated using HPLC. Under these conditions (Table 3), mitochondria isolated from dp1 still accumulated about twice as much GSH as those isolated from wt. Finally, the activity of ␥-glutamyltransferase, which degrades GSH, was indistinguishable among the three types of mitochondria (data not shown). It is also unlikely that mitochondria isolated from dp1 oxidized GSH more rapidly. The accumulation of GSSG (estimated by HPLC) after 4 min of incubation
FIG. 2. Import of GSH into yeast mitochondria. Total mitochondrial accumulation of GSH in mitochondria from wild-type (wt) and two mutant strains (dp1, dp2) of yeast was measured at the indicated times with radiolabeled GSH (final GSH concentration, 5 mM) and scintillation counting. Results are means ⫾ SD of measurements from three separate mitochondrial preparations. Values in the dp1 strain were significantly (P ⬍ 0.05) greater than those in either the wt or dp2 strains at all time points after the zero time point.
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was 0.7, 0.9 and 0.7 nmol per mg mitochondrial protein for mitochondria isolated from wt, dp1 and dp2, respectively. Therefore, it is unlikely that the increase in GSH accumulation by mitochondria isolated from dp1 was a consequence of increased retention of GSSG. Yeast vacuolar membrane vesicles have been used recently as a model system to study plasma membrane transport of GSH (27–29). However, it is unlikely that the transport properties we observed are due to contaminating vacuolar fragments. The yeast vacuolar system, YCF1, is an orthologue of the mammalian multidrug resistance associated proteins (28, 29). Unlike the translocations measured here, this system is ATPdependent (27–29). There has been no other work published on GSH transport in yeast. Transport of organic anions, in particular citric acid cycle intermediates, has been studied in yeast (30 –32), and the characteristics of these carriers appear to be very similar to those in mammalian mitochondria. Hence, yeast mitochondria appear to serve as a useful model in which to study mitochondrial metabolite transport. The experiments described here show that neither VDAC1 nor VDAC2 was essential for translocation of GSH into the mitochondrial matrix. In fact, depletion of VDAC1 caused an increase in GSH accumulation. A possible explanation is that mitochondria in the dp1 strain are swollen; however, electron microscopy does not support this hypothesis (33). The reason for this increase is unclear; however, the mitochondrial effects of VDAC1 disruption are pleiotropic (20). It may be that the effects of this disruption on GSH accumulation may be secondary to another effect on the mitochondria.
TABLE 3
Import of GSH into Mitochondria Isolated from wt, dp1 and dp2 Cells Strain
0-min Incubation
4-min Incubation
wt dp1 dp2
0.6 ⫾ 0.2 0.5 ⫾ 0.3 1.0 ⫾ 0.1
3.2 ⫾ 0.9 8.1 ⫾ 2.7* 4.5 ⫾ 2.0
Note. Intramitochondrial accumulation of GSH was measured by HPLC analysis with derivatization after 0- or 4-min incubation of mitochondria from the three yeast strains with unlabeled 5 mM GSH. Values (nmol/mg protein) are means ⫾ SD from four separate mitochondrial preparations. * Significantly different (P ⬍ 0.05) from accumulation in wt.
The more important question is: If GSH does not traverse the outer membrane through VDAC1, how does it? There are two plausible explanations. The first is that GSH passage may occur throughout the outer membrane but via another channel. If so, the rate of flux through this channel must exceed the rate of translocation across the inner membrane since disruption of the outer membrane with either a detergent or osmotic shock does not improve the rate of flux. Alternatively, translocation may occur by a means other than VDAC1 or VDAC2 at discrete sites that are not as affected by either digitonin treatment or osmotic shock (e.g., contact sites between the inner and outer membranes). In either case, neither genetic disruption of VDAC1 nor breaching the outer membrane would be expected to affect the rate of flux. ACKNOWLEDGMENTS This research was supported in part by NIDDK Grant R01DK40725 to L.H.L. and by NIMH Grant 47181 and NIEHS Grant 5P30 ESO6639 to R.M. The contributions of Drs. M. Forte, Vollum Institute, Portland, Oregon (yeast strains), S. Ackerman, Wayne State University, Detroit, Michigan (antibodies to ATP11p and the beta subunit of the F 0F 1 ATPase), and G. Schatz, Biozentrum, Basel, Switzerland (antibodies to cytochrome b 2) are acknowledged.
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FIG. 3. Efflux of GSH from yeast mitochondria. Total mitochondrial content of GSH from wild-type (wt) and two mutant strains (dp1, dp2) of yeast was measured at the indicated times after preloading mitochondria with 5 mM [ 3H-glycyl]-GSH for 4 min. GSH content was measured by scintillation counting. Results are means ⫾ SD of measurements from three separate mitochondrial preparations. 943
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