Bax Is Present as a High Molecular Weight Oligomer/Complex in the Mitochondrial Membrane of Apoptotic Cells
2001; Elsevier BV; Volume: 276; Issue: 15 Linguagem: Inglês
10.1074/jbc.m010810200
ISSN1083-351X
AutoresBruno Antonsson, Sylvie Montessuit, Belén G. Sánchez, Jean‐Claude Martinou,
Tópico(s)ATP Synthase and ATPases Research
ResumoBax is a Bcl-2 family protein with proapoptotic activity, which has been shown to trigger cytochrome crelease from mitochondria both in vitro and in vivo. In control HeLa cells, Bax is present in the cytosol and weakly associated with mitochondria as a monomer with an apparent molecular mass of 20,000 Da. After treatment of the HeLa cells with the apoptosis inducer staurosporine or UV irradiation, Bax associated with mitochondria is present as two large molecular weight oligomers/complexes of 96,000 and 260,000 Da, which are integrated into the mitochondrial membrane. Bcl-2 prevents Bax oligomerization and insertion into the mitochondrial membrane. The outer mitochondrial membrane protein voltage-dependent anion channel and the inner mitochondrial membrane protein adenosine nucleotide translocator do not coelute with the large molecular weight Bax oligomers/complexes on gel filtration. Bax oligomerization appears to be required for its proapoptotic activity, and the Bax oligomer/complex might constitute the structural entirety of the cytochromec-conducting channel in the outer mitochondrial membrane. Bax is a Bcl-2 family protein with proapoptotic activity, which has been shown to trigger cytochrome crelease from mitochondria both in vitro and in vivo. In control HeLa cells, Bax is present in the cytosol and weakly associated with mitochondria as a monomer with an apparent molecular mass of 20,000 Da. After treatment of the HeLa cells with the apoptosis inducer staurosporine or UV irradiation, Bax associated with mitochondria is present as two large molecular weight oligomers/complexes of 96,000 and 260,000 Da, which are integrated into the mitochondrial membrane. Bcl-2 prevents Bax oligomerization and insertion into the mitochondrial membrane. The outer mitochondrial membrane protein voltage-dependent anion channel and the inner mitochondrial membrane protein adenosine nucleotide translocator do not coelute with the large molecular weight Bax oligomers/complexes on gel filtration. Bax oligomerization appears to be required for its proapoptotic activity, and the Bax oligomer/complex might constitute the structural entirety of the cytochromec-conducting channel in the outer mitochondrial membrane. Apoptosis is mediated through two major pathways, the death receptor pathway and the mitochondrial pathway (1Hengartner M.O. Nature. 2000; 407: 770-776Crossref PubMed Scopus (6296) Google Scholar). The mitochondrial pathway is controlled and regulated by the Bcl-2 family of proteins (2Yang E. Korsmeyer S.J. Blood. 1996; 88: 386-401Crossref PubMed Google Scholar, 3Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar, 4Kelekar A. Thompson C.B. Trends Cell Biol. 1998; 8: 324-330Abstract Full Text Full Text PDF PubMed Scopus (539) Google Scholar). This protein family can be divided into antiapoptotic (Bcl-2, Bcl-XL, Bcl-w, Mcl-1, A1) and proapoptotic (Bax, Bak, Bok/Mtd, Bcl-Xs, Bid, Bad, Bik/Nbk, Bim, Blk) members (5Kroemer G. Nat. Med. 1997; 3: 614-620Crossref PubMed Scopus (1717) Google Scholar, 6Jacobson M.D. Curr. Biol. 1997; 7: R277-R281Abstract Full Text Full Text PDF PubMed Google Scholar, 7Reed J.C. Nature. 1997; 387: 773-776Crossref PubMed Scopus (1391) Google Scholar). Although the overall amino acid sequence homology between the family members is relatively low, they contain highly conserved domains, referred to as Bcl-2 homology domains (BH1 to -4). The activity of these proteins appears to be regulated, at least partly, by formation of homo- and heterocomplexes (8Oltvai Z.N. Milliman C.L. Korsmeyer S.J. Cell. 1993; 74: 609-619Abstract Full Text PDF PubMed Scopus (5878) Google Scholar, 9Knudson C.M. Korsmeyer S.J. Nat. Genet. 1997; 16: 358-363Crossref PubMed Scopus (350) Google Scholar, 10Yin X.-M. Oltvai Z.N. Korsmeyer S.J. Nature. 1994; 369: 321-323Crossref PubMed Scopus (1221) Google Scholar, 11Borner C. Martinou I. Mattmann C. Irmler M. Schaerer E. Martinou J.-C. Tschopp J. J. Cell Biol. 1994; 126: 1059-1068Crossref PubMed Scopus (175) Google Scholar, 12Sedlak T.W. Oltvai Z.N. Yang E. Wang K. Boise L.H. Thompson C.B. Korsmeyer S.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7834-7838Crossref PubMed Scopus (784) Google Scholar, 13Hsu Y.-T. Youle R.J. J. Biol. Chem. 1997; 272: 13829-13834Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar). The conserved BH domains are involved in these interactions. In the proapoptotic proteins, Bax and Bak, the BH3 domain is essential for complex formation as well as for their "killing" effect (4Kelekar A. Thompson C.B. Trends Cell Biol. 1998; 8: 324-330Abstract Full Text Full Text PDF PubMed Scopus (539) Google Scholar, 14Chittenden T. Flemington C. Houghton A.B. Ebb R.G. Gallo G.J. Elangovan B. Chinnadurai G. Lutz R.J. EMBO J. 1995; 14: 733-736Crossref Scopus (438) Google Scholar, 15Hunter J.J. Parslow T.G. J. Biol. Chem. 1996; 271: 8521-8524Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 16Wang K. Yin X.M. Chao D.T. Milliman C.L. Korsmeyer S.J. Genes Dev. 1996; 10: 2859-2869Crossref PubMed Scopus (808) Google Scholar, 17Zha J. Harada H. Osipov K. Jockel J. Waksman G. Korsmeyer S.J. J. Biol. Chem. 1997; 272: 24101-24104Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). The hydrophobic C-terminal domain present in some of the proteins has been implicated in targeting the proteins to intracellular membranes (11Borner C. Martinou I. Mattmann C. Irmler M. Schaerer E. Martinou J.-C. Tschopp J. J. Cell Biol. 1994; 126: 1059-1068Crossref PubMed Scopus (175) Google Scholar, 18Krajewski S. Tanaka S. Takayama S. Schibler M.J. Fenton W. Reed J.C. Cancer Res. 1993; 53: 4701-4714PubMed Google Scholar). Thus, many members of the family including Bcl-2, Bcl-XL, Bak, and Bax are acting at the level of mitochondria. It now appears clear that mitochondria have an important function in at least some apoptotic signaling cascades (3, 19– 20). Following a death stimulus, many proteins from the mitochondrial intramembrane space, including cytochrome c, adenylate kinase, Smac/DIABLO, procaspases, and apoptosis-inducing factor, have been reported to be released into the cytosol (21Bossy-Wetzel E. Newmeyer D.D. Green D.R. EMBO J. 1998; 17: 37-49Crossref PubMed Scopus (1107) Google Scholar, 22Single B. Leist M. Nicotera P. Cell Death Diff. 1998; 5: 1001-1003Crossref PubMed Scopus (97) Google Scholar, 23Kohler C. Gahm A. Noma T. Nakazawa A. Orrenius S. Zhivotovsky B. FEBS letter. 1999; 447: 10-12Crossref PubMed Scopus (97) Google Scholar, 24Lorenzo H.K. Susin S.A. Penninger J. Kroemer G. Cell Death Diff. 1999; 6: 516-524Crossref PubMed Scopus (426) Google Scholar, 25Du C. Fang M. Li Y. Li L. Wang X. Cell. 2000; 102: 33-42Abstract Full Text Full Text PDF PubMed Scopus (2941) Google Scholar, 26Verhagen A.M. Ekert P.G. Pakusch M. Silke J. Connolly L.M. Reid G.E. Moritz R.L. Simpson R.J. Vaux D.L. Cell. 2000; 102: 43-53Abstract Full Text Full Text PDF PubMed Scopus (1985) Google Scholar). The release of cytochrome c has been shown to be a fast process, depleting the mitochondria of cytochrome c within a few minutes (27Goldstein J.C. Waterhouse N.J. Juin P. Evan G.I. Green D.R. Nat. Cell Biol. 2000; 2: 156-162Crossref PubMed Scopus (886) Google Scholar). In the cytosol, cytochrome c forms a complex with Apaf-1, dATP, and procaspase 9 (28Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6261) Google Scholar). The complex formation leads to caspase 9 activation followed by downstream activation of other caspases, ultimately leading to cell death. Mounting evidence points to Bax and other proapoptotic family members as the central regulators of the release of proteins from the mitochondrial intramembrane space. Overexpression of Bax in cells or the addition of purified recombinant Bax directly to isolated mitochondria triggers the release of cytochrome c (29Rosse T. Olivier R. Monney L. Rager M. Conus S. Fellay I. Jansen B. Borner C. Nature. 1998; 391: 496-499Crossref PubMed Scopus (799) Google Scholar, 30Eskes R. Antonsson B. Osen-Sand A. Montessuit S. Richter C. Sadoul R. Mazzei G. Nichols A. Martinou J.-C. J. Cell Biol. 1998; 143: 217-224Crossref PubMed Scopus (586) Google Scholar, 31Jurgensmeier J.M. Xie Z. Deveraux Q. Ellerby L. Bredesen D. Reed J.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4997-5002Crossref PubMed Scopus (1375) Google Scholar, 32Finucane D.M. Bossy-Wetzel E. Waterhouse N.J. Cotter T.G. Green D.R. J. Biol. Chem. 1999; 274: 2225-2233Abstract Full Text Full Text PDF PubMed Scopus (653) Google Scholar). However, the mechanism through which Bax triggers the permeability of the outer mitochondrial membrane is unclear. The three-dimensional solution structure of the full-length Bax protein has recently been solved by NMR (33Suzuki M. Youle R.J. Tjandra N. Cell. 2000; 103: 645-654Abstract Full Text Full Text PDF PubMed Scopus (909) Google Scholar). The Bax structure shows a high similarity to the overall conformation of the two other Bcl-2 family proteins for which structural information is available, the antiapoptotic protein Bcl-XL and the BH3 domain-only protein Bid (34Muchmore S.W. Sattler M. Liang H. Meadows R.P. Harlan J.E. Yoon H.S. Nettesheim D. Chang B.S. Thompson C.B. Wong S.-L. Ng S.-C. Fesik S.W. Nature. 1996; 381: 335-341Crossref PubMed Scopus (1289) Google Scholar, 35Chou J.J. Li H. Salvesen G.S. Yuan J. Wagner G. Cell. 1999; 96: 615-624Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar, 36McDonnell J.M. Fushman D. Milliman C.L. Korsmeyer S.J. Cowburn D. Cell. 1999; 96: 625-634Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar). The proteins contain central hydrophobic helices (α5 and α6) surrounded by amphipathic helices. Of the three proteins, the Bax structure is the only one that contains the C-terminal hydrophobic domain. This domain forms a helix (α9) that protects the BH3-containing hydrophobic cleft on the protein. The structures of the Bcl-2 proteins are reminiscent of diphtheria toxin and the colicins A and E1. These toxins are pore-forming proteins that function as membrane channels that allow passage of ions or small polypeptides. Bax and other Bcl-2 family proteins have been shown to possess channel-forming activity in artificial membranes (37Minn A.J. Velez P. Schendoe S.L. Liang H. Muchmore S.W. Fesik S.W. Fill M. Thompson C.B. Nature. 1997; 385: 353-357Crossref PubMed Scopus (723) Google Scholar, 38Schendel S.L. Xie Z. Montal M.O. Matsuyama S. Montal M. Reed J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5113-5118Crossref PubMed Scopus (548) Google Scholar, 39Antonsson B. Conti F. Ciavatta A.M. Montessuit S. Lewis S. Martinou I. Bernasconi L. Bernard A. Mermod J.-C. Mazzei G. Maundrell K. Gambale F. Sadoul R. Martinou J.-C. Science. 1997; 277: 370-372Crossref PubMed Scopus (932) Google Scholar, 40Schlesinger P.H. Gross A. Yin X.-M. Yamamoto K. Saito M. Waksman G. Korsmeyer S.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11357-11362Crossref PubMed Scopus (443) Google Scholar). We have shown that in contrast to oligomeric Bax, monomeric recombinant Bax cannot form channels in liposomes nor trigger cytochromec release from isolated mitochondria (41Antonsson B. Montessuit S. Lauper S. Eskes R. Martinou J.-C. Biochem. J. 2000; 345: 271-278Crossref PubMed Scopus (563) Google Scholar). Recently, Bax tetramers were shown to form a channel large enough to allow the release of cytochrome c from liposomes (42Saito M. Korsmeyer S.J. Schlesinger P.H. Nat. Cell Biol. 2000; 2: 553-555Crossref PubMed Scopus (411) Google Scholar). Here we show that, in cultured cells exposed to the apoptosis inducer staurosporine or UV irradiation, Bax forms oligomers, which possibly form complexes with yet unidentified mitochondrial membrane proteins. The Bax oligomers are found inserted into the mitochondrial membrane. Moreover, we show that in the presence of Bcl-2, Bax oligomer formation and insertion into the mitochondrial membrane is inhibited. The Superdex 200 (16/60) column was from Amersham Pharmacia Biotech, and 14% polyacrylamide gels and 10% NuPage gels were from Novex (San Diego, CA). CHAPS 1The abbreviations used are:CHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonatePAGEpolyacrylamide gel electrophoresisDTTdithiothreitolVDACvoltage-dependent anion channelANTadenosine nucleotide translocatorMBmitochondrial bufferHAhemagglutininMops4-morpholinepropanesulfonic acidrBaxrecombinant monomeric Bax was from Roche Molecular Biochemicals, and Triton X-100 was from Fluka. Polyclonal anti-Bax antibodies were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY), monoclonal anti-Bax antibodies were from R & D Systems (Minneapolis, MN), monoclonal anti-Bcl-2 antibodies were from Genosys (Cambridge, UK), polyclonal anti-Bcl-XL antibodies were from Transduction Laboratories (Lexington, KY), monoclonal anti-Bak antibodies were from Oncogene Research Products (Boston, MA), monoclonal anti-VDAC and polyclonal anti-catalase antibodies were from Calbiochem, monoclonal anti-HA antibodies were from Babco (Richmond, CA), monoclonal anti-Myc and polyclonal anti-His antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), monoclonal anti-COX antibodies were from Molecular Probes, Inc. (Eugene, OR), monoclonal anti-Golgi 58-kDa protein antibodies were from Sigma, and the monoclonal anti-calnexin and anti-Hsp70 antibodies were from Affinity Bioreagents (Golden, CO). The polyclonal rabbit anti-ANT antibody was a kind gift from Prof. Theo Wallimann (43Rojo M. Wallimann T. Biochim. Biophys. Acta. 1994; 1187: 360-367Crossref PubMed Scopus (14) Google Scholar). 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate polyacrylamide gel electrophoresis dithiothreitol voltage-dependent anion channel adenosine nucleotide translocator mitochondrial buffer hemagglutinin 4-morpholinepropanesulfonic acid recombinant monomeric Bax HEK cells, HeLa cells, and a stable HeLa cell line constitutively overexpressing Bcl-2 (HeLa-Bcl-2) (44Estoppey S. Rodriguez I. Sadoul R. Martinou J.-C. Cell Death Differ. 1997; 4: 34-38Crossref PubMed Scopus (34) Google Scholar) were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mml-glutamine, 50 μg/ml streptomycin, and 50 IU/ml penicillin. Apoptosis was induced by culturing the cells in medium containing 1 μm staurosporine for 16 h; alternatively, the cells were UV irradiated (280 mJ cm−2) and subsequently cultured for 16 h. Control cells were cultured in the absence of staurosporine or without UV irradiation. At the end of the treatment, the cells were harvested in phosphate-buffered saline containing 1 mm EDTA, recovered by centrifugation at 750 × g for 10 min, washed, and suspended in mitochondrial buffer (MB) (210 mmmanitol, 70 mm sucrose, 1 mm EDTA, 10 mm Hepes-NaOH, pH 7.5). The cells suspended in MB supplemented with complete protease inhibitor mixture (Roche Molecular Biochemicals) were disrupted by passage through a 25G1 0.5 × 25 needle. The sample was passed through the needle five times and subsequently centrifuged at 2000 × g for 3 min. The supernatant was saved, and the pellet was resuspended in MB. The breakage procedure was repeated four times. The combined supernatants were centrifuged at 2000 × g for 3 min to remove nuclei and unbroken cells. The supernatant was subsequently centrifuged at 13,000 × g for 10 min. This supernatant was kept and centrifuged at 100,000 × g for 30 min to give the cytosolic cell fraction. The pellet fraction, corresponding to the mitochondrial fraction, was suspended in MB and recentrifuged at 13,000 × g for 10 min. Alternatively, the first 13,000 × g mitochondrial pellet was suspended in 4 ml of MB, and 2-ml portions were layered on top of a discontinuous sucrose gradient consisting of 20 ml of 1.2 m sucrose, 10 mm Hepes-NaOH, 1 mm EDTA, pH 7.5, on top of 17 ml of 1.6 m sucrose, 10 mm Hepes-NaOH, 1 mm EDTA, pH 7.5. The sample was centrifuged at 27,000 rpm in a Beckman SW28 rotor for 2 h at 4 °C. Mitochondria were recovered at the interface of 1.2 and 1.6 m sucrose and washed in MB. All manipulations were carried out at 4 °C. Isolated mitochondria (3 mg/ml) suspended in 1 ml of KCl buffer (15 mm Hepes-NaOH, 125 mm KCl, 4 mm MgCl2, 5 mmNaH2PO4, 0.5 mm EGTA, 5 mm succinate, pH 7.5) were incubated with 50 nmrecombinant caspase 8-cleaved Bid for 30 min at 30 °C. At the end of the incubation, the sample was centrifuged at 13,000 ×g for 10 min, and the mitochondria were washed with MB buffer. The washed mitochondrial fraction was suspended in MB containing 2% CHAPS or 2% Triton X-100, incubated on ice for 1 h, sonicated, and centrifuged at 100,000 × g for 30 min. The supernatant corresponds to the solubilized mitochondrial fraction. Protein concentrations were determined using the Bio-Rad protein assay kit. The mitochondrial pellet was suspended in 0.1 mNa2CO3, pH 12, to a final protein concentration of 3 mg/ml and incubated on ice for 20 min. At the end of the incubation, the sample was centrifuged at 100,000 × gfor 1 h. The supernatant, which contains nonmembrane-integrated proteins, was saved, and CHAPS was added to a final concentration of 2%. The pellet was suspended in MB containing 2% CHAPS, incubated on ice for 1 h, sonicated, and centrifuged at 100,000 ×g for 30 min. The supernatant, which contains solubilized integral membrane proteins, was saved. Gel filtrations were performed at 4 °C on a Superdex 200 (16/60) column equilibrated in 25 mm Hepes-NaOH, 300 mm NaCl, 0.2 mmDTT, 2% (w/v) CHAPS, pH 7.5, and run at a flow rate of 1 ml/min. When Triton extracts were analyzed, CHAPS was replaced by 2% Triton X-100. The column was calibrated with gel filtration standard proteins from Amersham Pharmacia Biotech giving the following elution volumes: thyroglobulin (669,000 Da), 49.9 ml; ferritin (440,000 Da), 55.9 ml; catalase (232,000 Da), 65.5 ml; BSA (67,000 Da), 77.5 ml; ovalbumin (43,000 Da), 83.2 ml; chymotrypsinogen A (25,000 Da), 92.9 ml; ribonuclease A (13,700 Da), 97.4 ml. The void volume of the column was determined by blue Sepharose that eluted at 43.3 ml. A 500-μl sample was loaded onto the column, and the eluate was monitored at 280 nm. After 20 min elution (20 ml), fractions of 2 ml were collected, and aliquots from the fractions were analyzed by Western blotting. The samples were separated on 14% SDS-PAGE under reducing conditions and transferred to polyvinylidene difluoride membranes. The proteins were detected with the specific antibodies as indicated in the figures, and the blots were developed with the ECL system from Amersham Pharmacia Biotech. Bax was tagged with His, HA, and Myc at the N terminus, and the constructs were cloned into the pC1 vector (Stratagene). HEK cells were then transfected with the three different Bax constructs (Fugene6) (Roche Molecular Biochemicals). The transfection efficiency was between 50 and 60%. 100 μm ZVAD was added 60 min prior to the addition of 1.6 μg of each of the Bax plasmids, and the cells were cultured for 16 h. At the end of the incubation, the cells were harvested, and mitochondria were isolated by differential centrifugation as described above. Mitochondria isolated from transfected HEK cells corresponding to 0.5 mg of mitochondrial protein was incubated with 1 μm caspase 8-cleaved Bid in KCl buffer (15 mm Hepes-NaOH, 125 mm KCl, 4 mmMgCl2, 5 mm NaH2PO4, 0.5 mm EGTA, 5 mm succinate, pH 7.5) for 15 min at 30 °C. At the end of the incubation, the mitochondria were recovered by centrifugation and resuspended in 400 μl of MB containing 1 mm EGTA. The cross-linkers bis-(sulfosuccinimidyl)suberate (Pierce) and disuccinimidyl suberate (Pierce) were added to final concentrations of 2 mm, and the sample was incubated for 30 min at room temperature. The reaction was stopped by the addition of Tris-HCl, pH 8.0, to a final concentration of 20 mm and subsequent incubation for 15 min at room temperature. The mitochondria were recovered by centrifugation and solubilized by incubation in radioimmune precipitation buffer (19 mm Tris-HCl, 140 mm NaCl, 1% Nonidet P-40, 0.1% SDS, 1% sodium deoxycholate) for 30 min at 4 °C. The sample was centrifuged at 12,000 × g for 10 min, and the supernatant was treated with 50 μl of protein G-Sepharose for 3 h at 4 °C. Protein G-Sepharose was removed by centrifugation, and 4 μg of anti-His antibody and 50 μl of protein G-Sepharose were added and incubated on rotation for 16 h at 4 °C. Protein G-Sepharose was recovered by centrifugation and washed four times with 400 μl of radioimmune precipitation buffer. The washed Sepharose was suspended in sample buffer, and the samples were analyzed on 10% NuPage gels in a Mops running buffer system (Novex). Proteins were transferred to nitrocellulose membranes and analyzed by Western blot with four different antibodies, anti-HA, anti-Myc, anti-Bax, and anti-VDAL. Cytosolic samples were centrifuged at 100,000 × g for 5 min and then treated with cross-linker and analyzed as above. Full-length Bax with a His tag at the N terminus was expressed in Escherichia coli and purified as previously described (45Montessuit S. Mazzei G. Magnenat E. Antonsson B. Protein Expression Purif. 1999; 15: 202-206Crossref PubMed Scopus (39) Google Scholar). Oligomerization was induced by disrupting the bacteria in buffer containing 1% Triton X-100. During the purification, Triton was exchanged for 1% octyl glucoside. The purified protein was stored in 25 mm Hepes-NaOH, 0.2 mm DTT, 1% octyl glucoside, 30% glycerol, pH 7.5, at −80 °C. Full-length Bid with a His tag at the N terminus was expressed inE. coli and purified as previously described (46Desagher S. Osen-Sand A Nichols A. Eskes R. Montessuit S. Lauper S. Maundrell K. Antonsson B. Martinou J-C. J. Cell Biol. 1999; 144: 891-901Crossref PubMed Scopus (1093) Google Scholar). The purified Bid was stored in 25 mm Tris-HCl, 0.1 mm DTT, 30% glycerol, pH 7.5, at −80 °C. Caspase 8-cleaved Bid was generated by mixing 200 μl of Bid (12.4 mg/ml) with 200 μl of caspase cleavage buffer (50 mm Hepes-NaOH, 10 mm DTT, 100 mm NaCl, 10% sucrose, pH 7.5) and 1 μl of caspase 8 (3 mg/ml), and the sample was incubated for 2 h at 20 °C. Over 95% of Bid was cleaved as estimated by SDS-PAGE. Cleaved Bid was separated from caspase 8 by binding to Ni2+-nitrilotriacetic acid-agarose; step-eluted by 100 mm imidazole; dialyzed into 25 mm Tris-HCl, 0.1 mm DTT, 30% glycerol, pH 7.5; and stored at −80 °C. Cultured HeLa cells have been used to study the quaternary structure of Bax in apoptotic cells. Apoptosis was induced by culturing the cells in the presence of 1 μm staurosporine for 16 h or by UV irradiation. Consistent with published results, in untreated HeLa cells, Bax was found both in the cytosol and associated with the mitochondria (Fig. 1). In apoptotic cells, Bax almost completely disappeared from the cytosol. Although a small increase in Bax associated with the mitochondria was seen in the apoptotic cells, this does not fully account for the decreased cytosolic Bax concentration (Fig. 1), suggesting that cytosolic Bax that is not translocated to the mitochondria is eliminated in apoptotic cells, possibly through proteolysis (47Wood D.E. Thomas A. Devi L.A. Berman Y. Beavis R.C. Reed J.C. Newcomb E.W. Oncogene. 1998; 17: 1069-1078Crossref PubMed Scopus (308) Google Scholar). To study the quaternary structure of Bax associated with mitochondria from control and apoptotic HeLa cells, mitochondrial proteins were extracted with 2% Triton X-100 as described under "Materials and Methods." The soluble mitochondrial extracts were analyzed on gel filtration, and the fractions from the column were analyzed by Western blot with an anti-Bax antibody. Gel filtration separates molecules according to the Stokes radius and gives an estimate of their molecular weights and thus of their quaternary structure. Bax extracted from either control or apoptotic HeLa cell mitochondria eluted as a single peak at fractions 20–24, corresponding to a molecular mass of 260,000 Da (Fig.2). Monomeric Bax was found neither in control nor apoptotic mitochondrial extract. Furthermore, analysis of other mitochondrial membrane proteins that have been suggested to interact with Bax, including Bak, Bcl-XL, and VDAC, all coeluted with Bax as high molecular weight complexes in fractions 20–24. Again, no difference was observed between mitochondrial extracts from control and apoptotic cells (Fig. 2). However, the mitochondrial membrane protein Cox IV eluted with a main peak in fractions 28–30 and a minor peak in fractions 22–24. The void volume of the column was at fraction 12. Triton X-100 has been reported to induce dimerization of Bax and other Bcl-2 family members (48Hsu Y.-T. Youle R.J. J. Biol. Chem. 1998; 273: 10777-10783Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar). In a previous study, we reported that Triton X-100 was able to trigger oligomerization of soluble monomeric Bax (41Antonsson B. Montessuit S. Lauper S. Eskes R. Martinou J.-C. Biochem. J. 2000; 345: 271-278Crossref PubMed Scopus (563) Google Scholar). Therefore, Triton might have induced the oligomerization of Bax and possibly other proteins extracted from the mitochondrial membrane, masking any differences in protein quaternary structure between control and apoptotic cells. Previous studies on the ability of various detergents to trigger Bax oligomerization showed that in contrast to Triton X-100 and octyl glucoside, CHAPS had no effect on Bax quaternary structure (41Antonsson B. Montessuit S. Lauper S. Eskes R. Martinou J.-C. Biochem. J. 2000; 345: 271-278Crossref PubMed Scopus (563) Google Scholar). Therefore, we examined whether CHAPS could extract Bax from mitochondria. As shown in Fig. 3,2% CHAPS was as efficient as 2% Triton X-100 in extracting Bax from the mitochondrial membrane of both control and apoptotic cells. This is an important control to ensure that we were not extracting and analyzing only a subpopulation of Bax from the mitochondria. Mitochondria were isolated by differential centrifugation from control and apoptotic HeLa cells, proteins were extracted with 2% CHAPS as described under "Materials and Methods," and the soluble mitochondrial extracts were analyzed on gel filtration. In the mitochondrial extract from control cells, Bax eluted in fractions 36–40. This corresponds to a molecular mass of 20,000 Da, which is close to the theoretical calculated molecular mass of monomeric Bax, 22,000 Da (Fig.4 A). In contrast, in the mitochondrial extract from cells where apoptosis had been induced by staurosporine treatment, Bax eluted mainly as large oligomers in fractions 20–28. Two peaks with estimated molecular masses of 260,000 and 96,000 Da were consistently detected (n = 9) (Fig.4 A). The amount of monomeric Bax eluting in fractions 36–38 varied between preparations and could depend on the percentage of cells that had entered apoptosis at the time of harvest. Identical results were obtained with a monoclonal anti-Bax antibody, confirming that the protein detected was indeed Bax (results not shown). Western blot analysis with marker antibodies for various organelles showed that the mitochondria isolated by differential centrifugation was contaminated mainly by ER (Fig. 5). To ensure that the Bax oligomers were associated with the mitochondria, the mitochondrial fraction was further purified on a sucrose gradient; this purification step improved the purity of the preparation. The sucrose gradient-purified mitochondria confirmed the result; oligomeric Bax (fractions 20–28) was found in the mitochondrial extract from apoptotic cells, whereas monomeric Bax (fractions 36–40) was present in both apoptotic and control mitochondrial extracts (Fig.4 B). In addition, inducing apoptosis by exposing HeLa cells to UV irradiation also induced oligomerization of Bax associated with the mitochondria. Similar to mitochondrial extract from staurosporine-treated cells, two peaks of Bax oligomers at fractions 20–22 (M r 260,000) and fractions 26–28 (M r 96,000) were detected on gel filtration (Fig. 6). Furthermore, we treated isolated HeLa mitochondria with 50 nm caspase 8-cleaved Bid for 30 min. The extract from the Bid-treated mitochondria contained the same Bax oligomers as mitochondria from staurosporine- or UV-treated HeLa cells (Fig. 6). Thus, inducing apoptosis by three different stimuli all resulted in Bax oligomerization. In addition to its interactions with Bcl-2 family members, Bax has also been reported to interact with other mitochondrial membrane proteins. Interactions with the outer mitochondrial membrane protein VDAC and the inner membrane protein ANT, both part of the permeability transition pore, have been reported (49Shimizu S. Narita M. Tsujimoto Y. Nature. 1999; 399: 483-487Crossref PubMed Scopus (1928) Google Scholar, 50Marzo I. Brenner C. Zamzami N. Jurgensmeier J.M. Susin S.A. Vieira H.L.A. Prevost M.-C. Xie Z. Matsuyama S. Reed J.C. Kroemer G. Science. 1998; 281: 2027-2031Crossref PubMed Scopus (1058) Google Scholar). To investigate whether the Bax oligomers contained these proteins, the fractions from the gel filtration column were analyzed for Bak, Bcl-XL, VDAC, and ANT (Fig.4 A). Two major peaks of Bak were detected, eluting in fractions 24–26 and 30–32. A low amount was also present over a wide molecular weight range (fractions 14–32), which suggests various forms of oligomers and/or complexes. However, no difference in the distribution between control and apoptotic cells was detected. Thus, since the Bax oligomers are not present in the mitochondrial membrane of nonapoptotic cells, interactions between Bak and the Bax oligomers are unlikely. Bcl-XL was similarly detected over a large range (fractions 16–32) with a major peak in fractions 26–32. As for Bak, no difference between control and ap
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