Bax in Murine Thymus Is a Soluble Monomeric Protein That Displays Differential Detergent-induced Conformations
1998; Elsevier BV; Volume: 273; Issue: 17 Linguagem: Inglês
10.1074/jbc.273.17.10777
ISSN1083-351X
Autores Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoBcl-2, Bcl-XL, and Bax are members of the Bcl-2 family that play important roles in apoptosis regulation. These proteins are believed to be membrane-bound and to regulate apoptosis through formation of homo- and heterodimers. However, we recently found by subcellular fractionation that whereas Bcl-2 is predominantly a membrane protein as previously reported, Bax and a significant fraction of Bcl-XL are soluble in thymocyte and splenocyte extracts. In addition, we have demonstrated that the ability of Bax to form dimers appears to be a detergent-induced phenomenon that coincides with a detergent-induced conformational change. We have further investigated the tertiary and quaternary states of Bax in the presence of various detergents. Detergents such as Triton X-100 and Triton X-114 readily enable Bax hetero- and homodimerization. However, other detergents such as polydocanol, W-1, octyl glucoside, dodecyl maltoside, Tween 20, and sodium cholate allow varying degrees of Bax hetero- and homodimerization. Detergents such as 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (Chaps) and Brij 35 allow neither hetero- nor homodimer formation. Immunoprecipitation analysis with the conformation-sensitive antibody uBax 6A7 revealed that whereas Triton X-100 readily exposes the N-terminal Bax epitope (amino acid 13–19), only limited exposure of the epitope occurs in Triton X-114, polydocanol, dodecyl maltoside, and sodium cholate, and no exposure of this epitope was observed in W-1, Chaps, octyl glucoside, Tween 20, and Brij 35. Moreover, we could not detect any proteins associated with the cytosolic form of Bax based on immunopurification of this protein. Sephacryl S-100 gel filtration chromatography analysis of the cytosolic Bax indicated that this protein is monomeric and displays an apparent molecular mass of 25 kDa. Induction of apo-ptosis which causes the insertion of the soluble form of Bax into membranes did not result in appreciable Bax/Bcl-XL, Bax/Bcl-2 or Bax/Bax dimer formation as determined by cross-linking studies. Further analysis of Bax after apoptosis induction by immunoprecipitation in the presence of Chaps also revealed no significant heterodimer formation. In conclusion, Bax displays several distinct states in different detergents that expose defined regions of the protein. In addition, these results suggest that mechanisms other than the simple dimerization among members of the Bcl-2 family may be required for the regulation of apoptosis. Bcl-2, Bcl-XL, and Bax are members of the Bcl-2 family that play important roles in apoptosis regulation. These proteins are believed to be membrane-bound and to regulate apoptosis through formation of homo- and heterodimers. However, we recently found by subcellular fractionation that whereas Bcl-2 is predominantly a membrane protein as previously reported, Bax and a significant fraction of Bcl-XL are soluble in thymocyte and splenocyte extracts. In addition, we have demonstrated that the ability of Bax to form dimers appears to be a detergent-induced phenomenon that coincides with a detergent-induced conformational change. We have further investigated the tertiary and quaternary states of Bax in the presence of various detergents. Detergents such as Triton X-100 and Triton X-114 readily enable Bax hetero- and homodimerization. However, other detergents such as polydocanol, W-1, octyl glucoside, dodecyl maltoside, Tween 20, and sodium cholate allow varying degrees of Bax hetero- and homodimerization. Detergents such as 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (Chaps) and Brij 35 allow neither hetero- nor homodimer formation. Immunoprecipitation analysis with the conformation-sensitive antibody uBax 6A7 revealed that whereas Triton X-100 readily exposes the N-terminal Bax epitope (amino acid 13–19), only limited exposure of the epitope occurs in Triton X-114, polydocanol, dodecyl maltoside, and sodium cholate, and no exposure of this epitope was observed in W-1, Chaps, octyl glucoside, Tween 20, and Brij 35. Moreover, we could not detect any proteins associated with the cytosolic form of Bax based on immunopurification of this protein. Sephacryl S-100 gel filtration chromatography analysis of the cytosolic Bax indicated that this protein is monomeric and displays an apparent molecular mass of 25 kDa. Induction of apo-ptosis which causes the insertion of the soluble form of Bax into membranes did not result in appreciable Bax/Bcl-XL, Bax/Bcl-2 or Bax/Bax dimer formation as determined by cross-linking studies. Further analysis of Bax after apoptosis induction by immunoprecipitation in the presence of Chaps also revealed no significant heterodimer formation. In conclusion, Bax displays several distinct states in different detergents that expose defined regions of the protein. In addition, these results suggest that mechanisms other than the simple dimerization among members of the Bcl-2 family may be required for the regulation of apoptosis. Apoptosis is a natural cell elimination process that occurs widely among multicellular organisms. Members of the Bcl-2 family including Bcl-2, Bcl-XL, and Bax represent some of the most well known regulators of this process. Bax was first described as a pro-apoptotic protein that can bind and counteract the pro-survival function of Bcl-2 (1Oltvai Z.N. Milliman C.L. Korsmeyer S.J. Cell. 1993; 74: 609-619Abstract Full Text PDF PubMed Scopus (5878) Google Scholar). Overexpression of Bax in transfected mammalian cells in many cases makes the cells more susceptible to apoptosis induced by external stimuli (for reviews see refs. 2Yang E. Korsmeyer S.J. Blood. 1996; 88: 386-401Crossref PubMed Google Scholar and 3Kroemer G. Nat. Med. 1997; 3: 614-620Crossref PubMed Scopus (1717) Google Scholar), whereas only in some isolated cases it suppresses cell death (4Middleton G. Nuñez G. Davies A.M. Development. 1996; 122: 695-701Crossref PubMed Google Scholar, 5Oh J.H. O'Malley K.L. Krajewski S. Reed J.C. Oh Y.J. Neuroreport. 1997; 8: 1851-1856Crossref PubMed Scopus (27) Google Scholar). Expression of Bax in fission yeast, however, has been reported to directly induce either growth arrest or cell death (6Greenhalf W. 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Based on yeast two-hybrid select systems and/or immunoprecipitation studies, it has been reported that Bax can form homodimers (1Oltvai Z.N. Milliman C.L. Korsmeyer S.J. Cell. 1993; 74: 609-619Abstract Full Text PDF PubMed Scopus (5878) Google Scholar) or heterodimers with Bcl-2, Bcl-XL, and Bid (1Oltvai Z.N. Milliman C.L. Korsmeyer S.J. Cell. 1993; 74: 609-619Abstract Full Text PDF PubMed Scopus (5878) Google Scholar, 12Sato T. Hanada M. Bodrug S. Irie S. Iwama N. Boise L.H. Thompson C.B. Golemis E. Fong L. Wang H.-G. Reed J.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9238-9242Crossref PubMed Scopus (594) Google Scholar, 13Sedlak 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, 14Wang K. Yin X.-M. Chao D.T. Milliman C.L. Korsmeyer S.J. Genes Dev. 1996; 10: 2859-2869Crossref PubMed Scopus (808) Google Scholar). It can also form heterodimers with adenovirus-encoded Bcl-2 homolog E19 kD (15Han J. Sabbatini P. Perez D. Rao L. Modha D. White E. Gene Dev. 1996; : 461-477Crossref PubMed Scopus (318) Google Scholar, 16Chen G. Branton P.E. Yang E. Korsmeyer S.J. Shore G.C. J. Biol. Chem. 1996; 271: 24221-24225Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 17Huang D.C.S. Cory S. Strasser A. Oncogene. 1997; 14: 405-414Crossref PubMed Scopus (231) Google Scholar) and herpesvirus saimiri-encoded Bcl-2 homolog ORF16 (18Nava V.E. Cheng E.H.-Y. Veliuona M. Zou S. Clem R.J. Mayer M.L. Hardwick J.M. J. Virol. 1997; 71: 4118-4122Crossref PubMed Google Scholar) but not with a Bcl-2 homolog KSbcl-2 encoded by herpesvirus 8 (19Cheng E.H.-Y. Nicholas J. Bellows D.S. Hayward G.S. Guo H.-G. Reitz M.S. Hardwick J.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 690-694Crossref PubMed Scopus (399) Google Scholar). In addition to these dimerization domains, Bax also has a predicted membrane spanning segment at its C-terminal end. In Bcl-2, this C-terminal hydrophobic region is responsible for anchoring this protein to membranes of various organelles including mitochondria, endoplasmic reticulum, and nuclei (20Hockenbery D. Nuñez G. Milliman C. Schreiber R.D. Korsmeyer S.J. Nature. 1990; 348: 334-336Crossref PubMed Scopus (3544) Google Scholar, 21Krajewski S. Tanaka S. Takayama S. Schibler M.J. Fenton W. Reed J.C. Cancer Res. 1993; 53: 4701-4714PubMed Google Scholar, 22Akao Y. Otsuki Y. Kataoka S. Ito Y. Tsujimoto Y. Cancer Res. 1994; 54: 2468-2471PubMed Google Scholar). Based on the presence of this hydrophobic region and the propensity of Bax to form heterodimers with Bcl-2, it was believed that Bax co-localizes with Bcl-2 in membranes. However, recently, several studies have shown that Bax is predominantly a soluble protein in thymocytes, splenocytes, and HL-60 promyelocytic leukemia cells (23Hsu Y.-T. Wolter K.G. Youle R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3668-3672Crossref PubMed Scopus (1032) Google Scholar,24Hsu Y.-T. Youle R.J. J. Biol. Chem. 1997; 272: 13829-13834Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar). Functional analysis of Bax by knock-out studies indicate that this protein is essential for spermatogenesis (25Knudson C.M. Tung K.S.K. Tourtellotte W.G. Brown G.A.J. Korsmeyer S.J. Science. 1995; 270: 96-99Crossref PubMed Scopus (1314) Google Scholar, 26Rodriguez I. Ody C. Araki K. Garcia I. Vassalli P. EMBO J. 1997; 16: 2262-2270Crossref PubMed Scopus (495) Google Scholar). In a double knock-out system, a deficiency in Bax prevents an increased cell death in the immature neurons of Bcl-XL knock-out mice (27Shindler K.S. Latham C.B. Roth K.A. J. Neurosci. 1997; 17: 3112-3119Crossref PubMed Google Scholar). Bax has been also implicated in anti-viral defense in promoting cell death in virally infected cells (28Brauweiler A. Garrus J.E. Reed J.C. Nyborg J.K. Virology. 1997; 231: 135-140Crossref PubMed Scopus (126) Google Scholar). In addition, Bax has been described as a tumor suppressor (29Yin C. Knudson C.M. Korsmeyer S.J. Van Dyke T. Nature. 1997; 385: 637-640Crossref PubMed Scopus (595) Google Scholar), and in certain cases of human colorectal cancer, frameshift mutations were found in the gene encoding Bax (30Rampino N. Yamamoto H. Ionov Y. Li Y. Sawai H. Reed J.C. Perucho M. Science. 1997; 275: 967-969Crossref PubMed Scopus (1235) Google Scholar,31Yamamoto H. Sawai H. Perucho M. Cancer Res. 1997; 57: 4420-4426PubMed Google Scholar). Physiologically, Bax plays the role of sensitizing cells to apoptosis. However, little is known about the molecular basis by which Bax promotes cell death. One hypothesis, known as the dimer rheostat model, suggests that the formation of Bax homodimers promotes cell death, and in healthy living cells, the formation of Bax homodimers were prevented by Bax heterodimerization with the prosurvival factors Bcl-2 and Bcl-XL (1Oltvai Z.N. Milliman C.L. Korsmeyer S.J. Cell. 1993; 74: 609-619Abstract Full Text PDF PubMed Scopus (5878) Google Scholar, 32Yang E. Zha J. Jockel J. Boise L.H. Thompson C.B. Korsmeyer S.J. Cell. 1995; 80: 285-291Abstract Full Text PDF PubMed Scopus (1897) Google Scholar). However, several recent mutagenesis studies show that dimerization may not be essential for the regulation of apoptosis (33Cheng E.H.-Y. Levine B. Boise L.H. Thompson C.B. Hardwick J.M. Nature. 1996; 379: 554-556Crossref PubMed Scopus (444) Google Scholar, 34Simonian P.L. Grillot D.A.M. Merino R. Nuñez G. J. Biol. Chem. 1996; 271: 22764-22772Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 35Simonian P.L. Grillot D.A.M. Andrews D.W. Leber B. Nuñez G. J. Biol. Chem. 1996; 271: 32073-32077Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Furthermore, we found that the cytosolic Bax in murine thymocytes undergoes a detergent-induced conformational change that is associated with the propensity of this protein to form either homodimers or heterodimers with Bcl-2 and Bcl-XL (24Hsu Y.-T. Youle R.J. J. Biol. Chem. 1997; 272: 13829-13834Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar). In this paper we have explored the epitope exposure and dimerization of the cytosolic and membrane-bound Bax and identified several different conformational states of this protein in the presence of various types of detergents. Synthetic peptides were purchased from Peptide Technologies Corp. Imject maleimide-activated keyhole limpet hemocyanin was obtained from Pierce. PEG 4000, fetal bovine sera, and hypoxanthine/aminopterin/thymidine medium were from Life Technologies, Inc. Iscove's medium was from Biofluid. Disuccinimidyl glutarate (DSG) 1The abbreviations used are: DSG, disuccinimidyl glutarate; Chaps, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PBS, phosphate-buffered saline; DSP, dithiobis(succinimidyl propionate). and dithiobis(succinimidyl propionate) (DSP) cross-linkers were from Pierce. Fractogel EMD TMAE-650M and AF-heparin-650M beads were bought from EM Separations Technology and TosoHaas, respectively. SPOTs peptides were obtained from Genosys. Sheep anti-mouse immunoglobulin peroxidase conjugate and ECL Western blotting detection kit were purchased from Amersham Pharmacia Biotech. Bolton-Hunter reagent was from NEN Life Science Products. Immobilon membranes were from Millipore. All other reagents were obtained from Sigma. New anti-Bax monoclonal antibodies were generated by immunizing mice with keyhole limpet hemocyanin conjugated to peptides corresponding to amino acids 3–16 of rat Bax (CGSGDHLGGGGPTSS) and amino acids 43–62 of mouse Bax (PELTLEQPPQDASTKKLSEC). Splenocytes from immunoreactive mice were fused by PEG 4000 to murine NS-1 myeloma cells and selected with hypoxanthine/aminopterin/thymidine medium (24Hsu Y.-T. Youle R.J. J. Biol. Chem. 1997; 272: 13829-13834Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar, 36Molday L.L. Cook N.J. Kaupp U.B. Molday R.S. J. Biol. Chem. 1990; 265: 18690-18695Abstract Full Text PDF PubMed Google Scholar). The anti-rat and species-independent Bax antibodies were designated as α rBax 1D1 and α uBax 2C8, respectively. Monoclonal antibodies α mBax 5B7, α hBax 1F6, and α uBax 6A7 were purified from ascites fluids by ammonium sulfate precipitation and DEAE fractionation (24Hsu Y.-T. Youle R.J. J. Biol. Chem. 1997; 272: 13829-13834Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar). The purified antibodies were immobilized onto CNBr-activated Sepharose 4B at 2.5 mg of protein/ml packed beads (24Hsu Y.-T. Youle R.J. J. Biol. Chem. 1997; 272: 13829-13834Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar, 37Hsu Y.-T. Molday R.S. Nature. 1993; 361: 76-79Crossref PubMed Scopus (312) Google Scholar). For the detergent-dependent Bax heterodimerization and uBax 6A7 antibody binding studies, murine thymocytes were subjected to hypotonic lysis and Dounce homogenization at a cell density of 5 × 107/ml essentially as described previously (24Hsu Y.-T. Youle R.J. J. Biol. Chem. 1997; 272: 13829-13834Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar). Soluble protein extracts prepared from high speed centrifugation (130,000 × g) were adjusted to 150 mm NaCl either in the absence of detergent or in the presence of 0.2% Triton X-100, Triton X-114, polydocanol, W-1, Chaps, octyl glucoside, dodecyl maltoside, Tween 20, Brij 35, or sodium cholate. The soluble extract (4.5 ml) was mixed with 150 μl of α mBax 5B7 or α uBax 6A7 antibody beads either in the absence or in the presence of appropriate detergents and allowed to incubate for 2 h. The unbound proteins were removed by washing the beads twice with 10 ml of 10 mm Hepes, pH 7.4, and 150 mm NaCl or with the same buffer containing 0.2% of the appropriate detergent. The bound proteins were then eluted off the beads with 180 μl of 0.1 m acetic acid containing 0.2% Triton X-100. The acid eluants were neutralized with 30 μl of 1m Tris, pH 8.0. For the study of Bax homodimerization, murine and rat thymocytes were hypotonically lysed at a cell density of 1 × 108/ml. The soluble proteins from the two cell types were prepared as described above. The soluble extracts were mixed 1:1 and used for the immunoprecipitation studies using the α mBax 5B7 antibody Sepharose beads in the presence of various detergents as described above. Alternatively, immunoprecipitation of Bax was carried out from detergent-solubilized whole cell lysate. Murine thymocytes were solubilized in 10 ml of 10 mm Hepes, pH 7.4, 150 mm NaCl, and in the presence of 1% Triton X-100, Triton X-114, polydocanol, Chaps, octyl glucoside, or dodecyl maltoside or 0.5% W-1 at a cell density of 5 × 107/ml. The lysate was spun at 14,000 rpm in a Sorvall SA 600 rotor for 15 min to pellet the nuclei and unsolubilized material. Bax complex was then immunoprecipitated from 4.5 ml of the detergent-solubilized lysate with 150 μl of the α mBax 5B7 antibody beads as described above. For the study of Bax heterodimerization in dexamethasone-treated thymocytes, murine thymocytes (7.5 × 107/ml) in Iscove's medium were subjected to treatment with 2 μmdexamethasone for 4 h. The cells were then collected, spun down, and solubilized in 10 mm Hepes, pH 7.4, 150 mmNaCl containing 1% Triton X-100 or Chaps. Immunoprecipitation of Bax was carried out as described above. For the above immunoprecipitation studies, all lytic, solubilization, and washing buffers contain proteolytic inhibitors (25 μg/ml phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, and 1 μg/ml aprotinin) as described previously (24Hsu Y.-T. Youle R.J. J. Biol. Chem. 1997; 272: 13829-13834Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar). Soluble protein extracts from murine thymocytes were prepared as described above except that the lysate was prepared at a cell density of 1 × 108/ml. One hundred and eighty ml of the extract in the presence of 38 mm NaCl was loaded onto a 20-ml AF-heparin-650M column. Flow-through from the column was collected and then loaded onto a 15-ml Fractogel EMD TMAE-650M anion exchange column equilibrated in the same buffer. After washing the column with 3 column volumes of the lysis buffer, Bax was eluted off the column with 3 column volumes of the elution buffer (10 mm Hepes, pH 7.4, and 125 mm NaCl). The eluant was then incubated with 0.5 ml of α mBax 5B7 antibody beads for 3 h at 4 °C. The beads were then washed, and the bound Bax was eluted off the beads with 0.1 m acetic acid containing 0.1% Triton X-100. Six fractions of 0.3-ml samples were collected and neutralized with 40 μl of 1 m Tris, pH 8.0. The relative molecular weight of the cytosolic Bax was determined by gel filtration over a Sephacryl S-100 column. Murine thymic soluble extract (1 × 108/ml) was loaded onto a TMAE 650M anion exchange column as described above to concentrate the Bax. Half a ml of the 0.125m NaCl eluant was then loaded onto the gel filtration column (74 ml) equilibrated in 10 mm Hepes, pH 7.4, and 150 mm NaCl. One-ml fractions were collected and analyzed by Western blotting with α uBax 2C8 antibody. The peak fraction containing murine Bax was assigned as the elution volume (V e) for the purpose of calculating its molecular weight. Blue dextran (2 × 103 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), and ribonuclease A (13.7 kDa) were run separately as molecular weight standards. For whole cell cross-linking of dexamethasone-treated thymocytes using membrane-permeable cross-linkers, thymocytes were treated with 2 μmdexamethasone for 4 h (23Hsu Y.-T. Wolter K.G. Youle R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3668-3672Crossref PubMed Scopus (1032) Google Scholar). The cells were washed once in PBS and resuspended in PBS at a cell density of 2.5 × 107/ml. Ten ml of the cell suspension were then incubated with 1 mmDSP or DSG for 30 min on ice. The reaction was quenched with 10 mm Tris, pH 7.4, and 100 mm glycine buffer. The cells were then spun down, and the cell pellet was resuspended in 2 ml of 1% SDS. The samples were run on a 10% SDS-polyacrylamide gel in the absence of β-mercaptoethanol and analyzed by Western blotting. Ten micrograms of anti-murine Bax 5B7, anti-human Bax 1F6, and anti-universal Bax 6A7 monoclonal antibodies were iodinated with Bolton-Hunter reagent according to the manufacturer's protocol. Murine thymocytes were treated with dexamethasone for 4 h and subjected to hypotonic lysis and Dounce homogenization at a cell density of 5 × 107/ml as described above. The lysate (0.5 ml) was adjusted to 150 mm NaCl and incubated with 50 ng (2 μCi/μg) of iodinated antibodies either with or without the presence of 50 μg of cold antibodies for 30 min on ice. The samples were then subjected to ultracentrifugation at 61,000 rpm for 30 min in a TLA 120.1 rotor. The radioactivity of the membrane pellets was then determined with a gamma counter. SDS-polyacrylamide gel electrophoresis (12% polyacrylamide gel unless specified) and Western blotting were carried out as described previously (24Hsu Y.-T. Youle R.J. J. Biol. Chem. 1997; 272: 13829-13834Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar). For immunoblotting analysis, the blots were probed with either α mBax 5B7 (1:10 diluted culture fluid), α uBcl-XL 2H12 (1:10 diluted culture fluid), α mBcl-2 10C4 (1:10 diluted culture fluid), α rBax 1D1 (1:10 diluted culture fluid), or α uBax 2C8 (1:20 diluted culture fluid) diluted in the blocking buffer for 45 min. The blots were then washed in PBS, 0.05% Tween 20 and incubated in blocking buffer containing 1:7000 diluted sheep anti-mouse immunoglobulin peroxidase for an additional 30 min. The blots were again washed in PBS, 0.05% Tween 20 and then in PBS and visualized by ECL Western blotting detection kit. The N-terminal epitopes of several anti-Bax monoclonal antibodies were determined by peptide mapping (SPOTs) analysis of a nested set of peptides corresponding to the N-terminal segments of Bax used for the generation of antibodies. As shown in Fig. 1, the binding specificity of α mBax 5B7, α hBax 1F6, and α rBax 1D1 monoclonal antibodies lies within amino acids 7–14. The first 4 amino acids within this particular region are distinct between mouse, human, and rat and appear to account for the species specificity of these antibodies. The epitope for the α uBax 6A7 monoclonal antibody, produced against a peptide sequence (amino acids 12–24) common to murine, human, and rat Bax, was found within amino acids 13–19 which partially overlaps with epitopes of the above-described species-specific antibodies. The propensity of Bax to form Bax/Bcl-2 and Bax/Bcl-XL heterodimers in the presence of nonionic detergents Triton X-100 and Nonidet P-40 (24Hsu Y.-T. Youle R.J. J. Biol. Chem. 1997; 272: 13829-13834Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar) led us to investigate the effect of other detergents in the induction of this process. Murine thymocytes were solubilized in either Triton X-100, Triton X-114, polydocanol, W-1, Chaps, octyl glucoside, or dodecyl maltoside. Immunoprecipitation of Bax was then carried out in the presence of these detergents using α mBax 5B7 monoclonal antibody (Fig. 2). Detergents such as Tween 20, Brij 35, or sodium cholate failed to efficiently solubilize the thymocytes, and therefore they were excluded from this experiment. The immunoprecipitated samples were analyzed by Western blotting with α uBax 2C8, α mBcl-2 10C4, and α uBcl-XL 2H12 monoclonal antibodies for the detection of murine Bax, Bcl-2, and Bcl-XL, respectively. As shown in Fig. 2, Triton X-100 and its related homolog Triton X-114 readily enable Bax/Bcl-2 and Bax/Bcl-XL heterodimer formation. Other detergents such as octyl glucoside mediate Bax/Bcl-XL heterodimer formation but allow much less Bax/Bcl-2 heterodimer. Meanwhile, detergents such as polydocanol, W-1, and dodecyl maltoside enable only Bax/Bcl-XL formation with a clear absence of Bax/Bcl-2 heterodimer. Finally the zwitterionic detergent Chaps allows neither Bax/Bcl-2 nor Bax/Bcl-XL heterodimer formation. Addition of 0.1% SDS to the Triton X-100 solubilization buffer disrupts Bax/Bcl-2 heterodimer formation but does not affect Bax heterodimerization to Bcl-XL (data not shown), suggesting that the interaction between Bax and Bcl-XL is comparably stronger than that of Bax and Bcl-2. Based on this study, it appears that different detergents may induce different sets of conformational changes in Bax, and perhaps in Bcl-2 and Bcl-XL as well, to facilitate the differential heterodimerization of Bax with Bcl-2 or Bcl-XL. We have previously reported that Bax homodimerization, like heterodimerization, appears to be a detergent-dependent process (24Hsu Y.-T. Youle R.J. J. Biol. Chem. 1997; 272: 13829-13834Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar). Since we find Bax heterodimerization to be dependent upon the type of detergent present, we set out to examine the effect of different detergents on Bax homodimerization. Bax was immunoprecipitated from a mixture of the soluble protein extracts of murine and rat thymocytes either in the absence of detergent or in the presence of Triton X-100, Triton X-114, polydocanol, W-1, Chaps, octyl glucoside, dodecyl maltoside, Tween 20, Brij 35, or sodium cholate. Murine Bax was immunoprecipitated from the mixture by α mBax 5B7 monoclonal antibody to examine its propensity to dimerize with rat Bax. The immunoprecipitated samples were analyzed by Western blotting with anti-murine Bax 5B7 and anti-rat Bax 1D1 monoclonal antibodies (Fig. 3,top and middle). As revealed by Western blotting, α mBax 5B7 antibody immunoprecipitated murine Bax under all conditions. Homodimerization of murine Bax to rat Bax, as determined by Western blotting with anti-rat Bax 1D1 antibody, occurs most readily in Triton X-100 and Triton X-114 (Fig. 3, middle, lanes e and f) and is reduced in the presence of polydocanol and dodecyl maltoside (Fig. 3, middle, lanes g and k). Bax homodimers do not form in the absence of detergent (Fig. 3,middle, lane d), as expected, nor in the presence of W-1, Chaps, octyl glucoside, Tween 20, Brij 35, and sodium cholate (Fig. 3,middle, lanes h, i, j, l, m, and n). To determine if Bax heterodimerization with Bcl-XL occurs under similar conditions, the above-described immunoprecipitated samples were analyzed by Western blotting with α uBcl-XL 2H12 antibody. The results indicate that Bax can differentially form heterodimers with Bcl-XL in the presence of most of these detergents with the exception of Chaps and Brij 35 (Fig. 3,bottom, lanes i and m). In the absence of detergent, as previously reported (24Hsu Y.-T. Youle R.J. J. Biol. Chem. 1997; 272: 13829-13834Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar), Bax does not heterodimerize with Bcl-XL (Fig. 3, bottom, lane d). These results suggest that the Bax homodimerization state is not only dependent upon the type of detergent but also appears to be distinct from its heterodimerization state. We have previously described the exposure of an N-terminal epitope of Bax (amino acids 12–24), which contains the binding site for the α uBax 6A7 antibody, in the presence of Triton X-100 and Nonidet P-40 (24Hsu Y.-T. Youle R.J. J. Biol. Chem. 1997; 272: 13829-13834Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar). In order to further investigate the conformation-dependent exposure of this epitope that correlates with Bax hetero- and homodimerization and competes for Bax-Bcl-XL heterodimer formation, we extended the immunoprecipitation study with α uBax 6A7 antibody in the presence of various detergents. The immunoprecipitated samples were analyzed by Western blotting with α mBax 5B7 monoclonal antibody for the detection of murine Bax bound to the 6A7 antibody. Whereas the α uBax 6A7 antibody binds strongly to Bax in the presence of Triton X-100 (Fig. 4, top, lane c), a decreased affinity was observed in the presence of Triton X-114, polydocanol, and sodium cholate (Fig. 4, top, lanes d, e and l), and only trace binding was observed in the presence of dodecyl maltoside (Fig. 4, top, lane i). Bax did not bind to the uBax 6A7 antibody either in the absence of detergent or in the presence of W-1, Chaps, octyl glucoside, Tween 20, and Brij 35 (Fig. 4,top, lanes b, f, g, h, j, and k). Thus, the epitope for the 6A7 antibody (now mapped to amino acids 13–19), which is normally buried, apparently adapts the exposed conformation only in the presence of selected detergents. We have also previously reported that the 6A7 antibody binding site competes with heterodimer formation (24Hsu Y.-T. Youle R.J. J. Biol. Chem.
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