Ceramide Channels Increase the Permeability of the Mitochondrial Outer Membrane to Small Proteins

Artigo Acesso aberto Revisado por pares

Ceramide Channels Increase the Permeability of the Mitochondrial Outer Membrane to Small Proteins

2002; Elsevier BV; Volume: 277; Issue: 30 Linguagem: Inglês

10.1074/jbc.m200754200

ISSN

1083-351X

Autores

Leah J. Siskind, Richard Kolesnick, Marco Colombini,

Tópico(s)

Neuroscience and Neuropharmacology Research

Resumo

Ceramides are known to play a major regulatory role in apoptosis by inducing cytochrome c release from mitochondria. We have previously reported that C2- and C16-ceramide, but not dihydroceramide, form large channels in planar membranes (Siskind, L. J., and Colombini, M. (2001)J. Biol. Chem. 275, 38640–38644). Here we show that ceramides do not trigger a cytochrome c secretion or release mechanism, but simply raise the permeability of the mitochondrial outer membrane, via ceramide channel formation, to include small proteins. Exogenously added reduced cytochromec was able to freely permeate the mitochondrial outer membrane with entry to and exit from the intermembrane space facilitated by ceramides in a dose- and time-dependent manner. The permeability pathways were eliminated upon removal of C2-ceramide by bovine serum albumin, thus ruling out a detergent-like effect of C2-ceramide on membranes. Ceramide channels were not specific to cytochrome c, as ceramides induced release of adenylate kinase, but not fumerase from isolated mitochondria, showing some specificity of these channels for the outer mitochondrial membrane. SDS-PAGE results show that ceramides allow release of intermembrane space proteins with a molecular weight cut-off of about 60,000. These results indicate that the ceramide-induced membrane permeability increases in isolated mitochondria are via ceramide channel formation and not a release mechanism, as the channels that allow cytochrome c to freely permeate are reversible, and are not specific to cytochromec. Ceramides are known to play a major regulatory role in apoptosis by inducing cytochrome c release from mitochondria. We have previously reported that C2- and C16-ceramide, but not dihydroceramide, form large channels in planar membranes (Siskind, L. J., and Colombini, M. (2001)J. Biol. Chem. 275, 38640–38644). Here we show that ceramides do not trigger a cytochrome c secretion or release mechanism, but simply raise the permeability of the mitochondrial outer membrane, via ceramide channel formation, to include small proteins. Exogenously added reduced cytochromec was able to freely permeate the mitochondrial outer membrane with entry to and exit from the intermembrane space facilitated by ceramides in a dose- and time-dependent manner. The permeability pathways were eliminated upon removal of C2-ceramide by bovine serum albumin, thus ruling out a detergent-like effect of C2-ceramide on membranes. Ceramide channels were not specific to cytochrome c, as ceramides induced release of adenylate kinase, but not fumerase from isolated mitochondria, showing some specificity of these channels for the outer mitochondrial membrane. SDS-PAGE results show that ceramides allow release of intermembrane space proteins with a molecular weight cut-off of about 60,000. These results indicate that the ceramide-induced membrane permeability increases in isolated mitochondria are via ceramide channel formation and not a release mechanism, as the channels that allow cytochrome c to freely permeate are reversible, and are not specific to cytochromec. N-acetyl-d-erythro-sphingosine N-hexanoyl-d-erythro-sphingosine N-acetyl-d-erythro-sphinganine N-hexadecyl-d-erythro-sphingosine N-octadecyl-d-erythro-sphinganine inner mitochondrial membrane potential bovine serum albumin 2,4-dinitrophenol 1,4-piperazinediethanesulfonic acid Ceramide is a sphingosine-based lipid involved in the regulation of several cellular processes, including differentiation, growth suppression, cell senescence, and apoptosis (1Radin N.S. Eur. J. Biochem. 2001; 268: 193-204Crossref PubMed Scopus (110) Google Scholar, 2Kolesnick R.N. Krönke M. Annu. Rev. Physiol. 1998; 60: 643-665Crossref PubMed Scopus (733) Google Scholar, 3Ariga T. Jarvis W.D. Yu R.K. J. 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Biol. Chem. 2002; 277: 11788-11794Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Dihydroceramide, despite the fact that it differs from ceramide only by the reduction of one double bond, does not induce apoptosis nor form channels (34Siskind L.J. Colombini M. J. Biol. Chem. 2000; 275: 38640-38644Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). The ceramide channels are composed of many ceramide monomers and thus their formation is exceedingly sensitive to the free ceramide concentration. Altering the activity of local enzymes that synthesize and catabolize ceramide would cause channels to assemble or disassemble thus regulating the permeability of the outer membrane to small proteins. Ceramides have been reported to have other effects on mitochondria including enhanced generation of reactive oxygen species (20Zamzami N. Marchetti P. Castedo M. Decaudin D. Macho A. Hirsch T. Susin S.A. Petit P.X. Mignotte B. Kroemer G. J. Exp. 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Med. 1995; 182: 367-377Crossref PubMed Scopus (1443) Google Scholar, 27Arora A.S. Jones B.J. Patel T.C. Bronk S.F. Gores G.J. Hepatology. 1997; 25: 958-963Crossref PubMed Scopus (152) Google Scholar, 28Di Paola M. Cocco T. Lorusso M. Biochemistry. 2000; 39: 6620-6628Crossref Scopus (215) Google Scholar, 29Ghafourifar P. Klein S.D. Schucht O. Schenk U. Pruschy M. Rocha S. Richter C. J. Biol. Chem. 1999; 274: 6080-6084Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar), and inhibition and/or activation of the activities of various components of the mitochondrial electron transport chain (28Di Paola M. Cocco T. Lorusso M. Biochemistry. 2000; 39: 6620-6628Crossref Scopus (215) Google Scholar, 43Gudz T.I. Tserng K.-Y. Hoppel C.L. J. Biol. Chem. 1997; 272: 24154-24158Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar). It is therefore unclear whether ceramide acts directly or indirectly on cytochrome c release. Here we show that treatment of rat liver mitochondria with either C2- or C16-ceramide causes the outer membrane to be freely permeable to cytochrome c, not just cytochromec release, and allows the release of proteins of up to about 60 kDa from the intermembrane space. The permeability increase induced by C2-ceramide is largely reversed by treatment with fatty acid-depleted bovine serum albumin (BSA). These results bolster the hypothesis that ceramide-induced cytochrome c release from mitochondria is via the formation of ceramide channels in dynamic equilibrium with ceramide in other structural states. The following reagents were purchased from Avanti Polar Lipids (Alabaster, AL): C2-ceramide, C2-dihydroceramide, C16-ceramide, C18-dihydroceramide, and asolectin (polar extract of soybean phospholipids). Antimycin A, 2,4-dinitrophenol (DNP), fatty acid-depleted BSA, horse heart cytochrome c, and sodium ascorbate were purchased from Sigma. Planar membranes were formed by the monolayer method (44Montal M. Mueller P. Proc. Natl. Acad. Sci. U. S. A. 1972; 69: 3561-3566Crossref PubMed Scopus (1647) Google Scholar), as modified (45Colombini M. Methods Enzymol. 1987; 148: 465-475Crossref PubMed Scopus (54) Google Scholar), across a 100-μm diameter hole in a Saran partition using 1% (w/v) asolectin (soybean phospholipids), 0.2% (w/v) cholesterol in hexane solution. The aqueous solution contained 1.0 m KCl, 1 mmMgCl2, and 5 mm HEPES or 5 mm PIPES (pH 7.0). The voltage was clamped (trans-side was ground) and the current was recorded. C2-ceramide was stirred into the water phase from a Me2SO solution, whereas C16-ceramide was first dissolved in ethanol at 37 °C prior to addition. In both cases, the final concentration of the vehicle was no more than 0.5%. Fatty acid-depleted BSA was prepared as a stock solution (0.5 mm) in the same aqueous solution bathing the membrane with 0.5% azide (for storage purposes). Rat liver mitochondria were isolated by differential centrifugation of tissue homogenate as previously described (46Parsons D.F. Williams G.R. Chance B. Ann. N. Y. Acad. Sci. 1966; 137: 643-666Crossref PubMed Scopus (238) Google Scholar). Briefly, livers from male Sprague-Dawley rats (fasted overnight with water ad labitum) were quickly excised, cut, washed repeatedly in cold isolation medium (70 mm sucrose, 210 mm mannitol, 0.1 mmEGTA, 1.0 mm Tris-Cl, pH 7.4), and minced. Homogenization and differential centrifugation were performed with isolation medium supplemented with 0.5% (w/v) BSA. BSA was removed by a final 9000 × g centrifugation and subsequently mitochondria were suspended in BSA-free medium. 1 mm horse heart cytochrome c was reduced by an excess of ascorbate (20 mm), 0.2 m Tris-Cl (pH 7.5). The reduced cytochrome c was separated from the ascorbate using a Sephadex G-10 gel filtration column. The concentration of reduced cytochrome c was determined spectrophotometrically (ε550 nm(Red.-Ox.) = 18.5 mm−1cm−1). Mitochondria were diluted 20–40-fold in isolation buffer to achieve an appropriate rate of cytochrome coxidation after hypotonic shock and kept on ice. 20 μl of diluted mitochondria were added to 750 μl of isolation buffer supplemented with 0.5 mm DNP and 5 μm antimycin A (final concentration of 0.3–1.2 mg of mitochondrial protein/ml) and allowed to reach room temperature. Ceramide was added in such a way that the vehicle was no more than 1% of the total volume. Me2SO was the vehicle for C2-ceramide and C2-dihydroceramide. C16-ceramide and C18-ceramide were dissolved in 100% ethanol at 37 °C as described in Ref. 28Di Paola M. Cocco T. Lorusso M. Biochemistry. 2000; 39: 6620-6628Crossref Scopus (215) Google Scholar and added to mitochondria at room temperature. After the indicated incubation period, 20 μl of reduced cytochromec (25–35 μm final concentration in the cuvette) was added and the initial rate of oxidation was assayed spectrophotometrically as a decrease in absorbance at 550 nm (ΔεRed.-Ox. = 18.5 mm−1cm−1) or the difference in absorbance at 550 and 536 nm (ε550 nm = 27.7 mm−1cm−1; ε536 nm = 7.7 mm−1 cm−1). All initial rates were expressed as nanomoles of cytochrome c oxidized per s/mg of mitochondrial protein. KCN was used to inhibit cytochromec oxidase and hence stop the reaction. Equal volumes of vehicle and/or dihydroceramide were used as controls. The intermembrane space enzyme adenylate kinase (47Sotocassa G.L. Kuylenstierna B. Ernster L. Bergstrand A. Methods Enzymol. 1967; 10: 448-463Crossref Scopus (223) Google Scholar) and the matrix enzyme fumarase (48Hill R.L. Bradshaw R.A. Methods Enzymol. 1969; 13: 91-99Crossref Scopus (209) Google Scholar) were assayed by standard methods. To increase the concentration of any released enzyme in the medium (for detection purposes) following exposure to ceramide, it was necessary to use higher mitochondrial concentrations than those used to measure cytochrome c permeation through the outer membrane. To achieve comparable conditions in both types of experiments, the ceramide to mitochondria ratio was kept constant rather than the total concentration of added ceramide. This can be justified by recalling that ceramide exerts its effect on the membranes and that the critical concentration should be the concentration of ceramide in the membrane; a constant ratio of ceramide to mitochondrial protein should result in a constant level of ceramide in the mitochondrial outer membrane. The literature supports this notion. Muriel et al.(42Muriel M.-P. Lamberg N. Darios F. Michel P.P. Hirsch E.C. Agid Y. Ruberg M. J. Comp. Neurol. 2000; 426: 297-315Crossref PubMed Scopus (41) Google Scholar) found that the concentration of C2-ceramide that induced the death of 50% of the PC12 cells after 24 h depended on the cell density used. Similarly, Simon and Gear (35Simon C.G., Jr. Gear A.R.L. Biochemistry. 1998; 37: 2059-2069Crossref PubMed Scopus (55) Google Scholar) found that C2-ceramide inhibition of platelet aggregation and its ability to induce 6-carboxyfluorescein release from vesicles was dependent on the ratio of ceramide to total lipid, as opposed to the absolute ceramide concentration. Thus, we scaled the amount of ceramide to the amount of mitochondria used (i.e. moles of ceramide to milligrams of mitochondrial protein). The mitochondrial preparation was diluted 5-fold in isolation buffer without antimycin A or DNP. A 250-μl aliquot (5–10 mg of protein/ml) was incubated for 10 min with C2- or C16-ceramide at either a high or low molar ratio, 5 and 20 μm equivalent ratios of ceramide to milligrams of mitochondrial protein as in the accompanying cytochrome coxidation experiments. The mitochondria were then pelleted at 12,000 rpm for 5 min and 200 μl of supernatant was then added to 700 μl of either adenylate kinase reaction mixture (50 mm Tris-HCl, pH 7.5, 5 mm MgSO4, 10 mm glucose, 5 mm ADP, 0.2 mm NADP, 10 units of hexokinase, and 10 units of glucose-6-phosphate dehydrogenase) or fumarase reaction mixture (50 mm sodium phosphate and 50 mml-malate, pH 7.3). Adenylate kinase was detected as an increase in absorbance at 340 nm. Fumarase was detected as an increase in absorbance at 250 nm. Untreated mitochondria and mitochondria with lysed outer (adenylate kinase) or lysed outer and inner (fumarase) membranes served as negative and positive controls, respectively. In the case of the adenylate kinase assay, the outer mitochondrial membrane was lysed hypotonically. In the fumarase assay, the outer and inner mitochondrial membranes were lysed via sonication under severe hypo-osmotic conditions in the presence of 1 mm EDTA as described in Ref. 49Holden M.J. Colombini M. Biochim. Biophys. Acta. 1993; 1144: 393-402Crossref Scopus (54) Google Scholar. Equal volumes of vehicle and dihydroceramide were used as controls. Five milliliters of mitochondria that was diluted 5-fold (5–10 mg/ml) with isolation buffer (without antimycin A or DNP) was allowed to reach room temperature and then incubated for 10 min with C2- or C16-ceramide at 20 μm equivalent ratios of moles of ceramide to mitochondrial protein. Untreated mitochondria, dihydroceramide, and vehicles served as controls. The mitochondria were then spun at 35,000 rpm (50Ti rotor) for 30 min at 4 °C. The supernatant was removed and treated with 10% trichloroacetic acid overnight at 4 °C to pellet the proteins. The pellet was washed repeatedly with 1:1 ethanol:ether (v/v) to remove the trichloroacetic acid. The pellets were redissolved by adding Tris-OH, β-mercaptoethanol, and SDS in amounts equivalent to those used in the SDS-PAGE sample buffer and heated to near boiling for 10 min. After returning to room temperature, bromphenol blue was added and then HCl was added just until the color changed. 8 m urea was added to help stabilize the solution and provide for the added density (instead of glycerol). Proteins from the hypotonically lysed mitochondria were diluted 4-, 8-, and 16-fold. Samples were separated on a 15% acrylamide SDS-PAGE supplemented with 4 m urea and the bands stained with GelCode Blue stain (Pierce). Mitochondrial protein was measured using the BCA method (Pierce). Bovine serum albumin was the standard. It has already been reported that addition of either C2- or C16-ceramide to isolated mitochondria results in cytochrome c release (27Arora A.S. Jones B.J. Patel T.C. Bronk S.F. Gores G.J. Hepatology. 1997; 25: 958-963Crossref PubMed Scopus (152) Google Scholar, 28Di Paola M. Cocco T. Lorusso M. Biochemistry. 2000; 39: 6620-6628Crossref Scopus (215) Google Scholar, 29Ghafourifar P. Klein S.D. Schucht O. Schenk U. Pruschy M. Rocha S. Richter C. J. Biol. Chem. 1999; 274: 6080-6084Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). However, from the literature it is not clear how this release might come about. We previously demonstrated that ceramides form large channels in phospholipid membranes (34Siskind L.J. Colombini M. J. Biol. Chem. 2000; 275: 38640-38644Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar), whereas the biologically inactive C2- and C18-dihydroceramides do not (34Siskind L.J. Colombini M. J. Biol. Chem. 2000; 275: 38640-38644Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). The addition of either C16- or C2-ceramide to the aqueous phase on either one or both sides of a planar phospholipid membrane results in pore formation as indicated by discrete stepwise current increases (Fig.1). Discontinuous changes in current are diagnostic of channel formation. Ceramide channels have a wide distribution of discrete conductance increases (34Siskind L.J. Colombini M. J. Biol. Chem. 2000; 275: 38640-38644Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar) (Fig. 1), ranging from 1 nanoSiemens to more than 200 nanosiemens. Small channels are seen early on when the total membrane conductance is low. With time, larger discrete conductance increases are evident reflecting the formation of larger structures. Eventually the conductance grows with fluctuations of current overlapping into current noise. Depicted in Fig. 1 are representative examples of some of these small and large channels observed with C2- and C16-ceramide. According to the bulk properties of water, pores with conductance ranging from 1 to 200 nanosiemens would have estimated diameters ranging from 0.8 to 11 nm, respectively. We hypothesize that similar channels form in the outer mitochondrial membrane, channels large enough to allow cytochrome c to exit. In this hypothesis, ceramide does not trigger a cytochromec secretion or release mechanism, but simply raises the permeability of the outer mitochondrial membrane, via ceramide channel formation, to include small proteins. The hypothesis predicts the following: 1) cytochrome c should freely permeate and thus both entry to and exit from the intermembrane space should be facilitated; 2) the permeability pathways should be eliminated by removal of ceramide; 3) the release should not be specific to cytochrome c. 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Ceramide Channels Increase the Permeability of the Mitochondrial Outer Membrane to Small Proteins