Bax and Adenine Nucleotide Translocator Cooperate in the Mitochondrial Control of Apoptosis

Xiang J., Chao D. T., Korsmeyer S. J., Proc. Natl. Acad. Sci. U.S.A. 93, 14559 (1996).

Zamzami N., et al., J. Exp. Med. 183, 1533 (1996);

Susin S. A., et al., ibid. 184, 1331 (1996).

10.1126/science.275.5303.1129

; R. M. Kluck E. Bossy-Wetzel D. R. Green D. D. Newmeyer ibid. p. 1132.

Shimizu S., et al., Proc. Natl. Acad. Sci. U.S.A. 95, 1455 (1998).

M. G. vander Heiden

Chandal N. S., Williamson E. K., Schumacker P. T., Thompson C. B., Cell 91, 627 (1997).

Wolter K. G., et al., J. Cell Biol. 139, 1281 (1997);

Rossé T., et al., Nature 391, 496 (1998);

Gross A., Jockel J., Wei M. C., Korsmeyer S., EMBO J. 17, 3878 (1998).

Pastorino J. G., Chen S.-T., Tafani M., Snyder J. W., Farber J. L., J. Biol. Chem. 273, 7770 (1998).

Jürgensmeier J. M., et al., Proc. Natl. Acad. Sci. U.S.A. 95, 4997 (1998).

Sato T., et al., ibid. 91, 9238 (1994);

Manon S., Chaudhuri B., Guerin M., FEBS Lett. 415, 29 (1998).

Recombinant human Bcl-2(1–218) or mouse Bax(1–171) proteins lacking the hydrophobic transmembrane (TM) domain [deletion of amino acids 219 through 239 (Δ219–239) in the case of Bcl-2; Δ172–192 for Bax] Bcl-2Δα5/6[Bcl-2(Δ143–184)] Bax ΔIGDE (Δ66–69) and Bax Δα5/6 (Δ106–153) were created with a two-step polymerase chain reaction (PCR) method [

Zha H., et al., Mol. Cell. Biol. 16, 6494 (1996);

]. These five proteins were then tagged with His 6 or glutathione S -transferase (in the case of Bax Δα5/6) and purified as described [(8);

Schendel S., et al., Proc. Natl. Acad. Sci. U.S.A. 94, 5113 (1997);

Xie Z. H., Schendel S., Matsuyama S., Reed J. C., Biochemistry 37, 6410 (1998)].

Nicolli A., Basso E., Petronilli V., Wenger R. M., Bernardi P., J. Biol. Chem. 271, 2185 (1996);

Halestrap A. P., Woodfield K. Y., Connern C. P., ibid. 272, 3346 (1997) .

Zoratti M., Szabò I., Biochem. Biophys. Acta Rev. Biomembranes 1241, 139 (1995).

Klingenberg M., J. Membrane Biol. 56, 97 (1980).

Marzo I., et al., J. Exp. Med. 187, 1261 (1998).

Zamzami N., et al., Oncogene 16, 1055 (1998).

PTPC was prepared from Wistar rat brains or from Bax −/− [

10.1126/science.270.5233.96

] or Bax +/+ C57Bl/6 mouse brains (14). For Bax immunodepletion the PTPC preparation was incubated with polyclonal antibody to Bax (anti-Bax; 50 μg/ml) (Δ21 Santa-Cruz) for 2 hours at room temperature. Sham immunodepletion was done with a preimmune rabbit antiserum. A 0.1 volume of protein A and protein G agarose beads was added for 30 min and removed by centrifugation (10 min 2000 g ). PTPC in the supernatant (∼1 μg of protein per milligram of lipid) was reconstituted into lipid vesicles (optionally supplemented with 0.5 μCi of [ 14 C]cholesterol per 100 mg of lipids) by overnight dialysis. Recombinant proteins were added during the dialysis step at a dose corresponding to 1% (Bax or Bax mutants) or 5% (Bcl-2) of total PTPC proteins. Liposomes were ultrasonicated in 5 mM malate and 10 mM KCl (optionally supplemented with 5 μCi of [ 3 H]glucose per milliliter or 5 μCi of [ 3 H]inulin per milliliter) and separated on Sephadex G50 columns (14). Proteoliposomes containing maximum hexokinase activity (14) were incubated (60 min room temperature) with PT pore–opening agents in 125 mM sucrose plus 10 mM Hepes (pH 7.4). When the release of [ 3 H]glucose or [ 3 H]inulin was assessed liposomes were separated on Sephadex G50 Nick columns (Pharmacia). Alternatively liposomes were equilibrated for 30 min with 80 nM 3 3′-dihexylocarbocyanine [DiOC 6 (3)] and then analyzed in a cytofluorometer (14).

I. Marzo C. Brenner H. L. A. Vieira G. Kroemer unpublished data.

Cheng E. H.-Y., Levine B., Boise L. H., Thompson C. B., Hardwick J. M., Nature 379, 554 (1996);

Knudson C. M., Korsmeyer S. J., Nature Genet. 16, 358 (1997) .

Zha H., Reed J., J. Biol. Chem. 272, 31482 (1997).

The S. cerevisiae W301-1B control strain ( MATα ade2 trp1 leu2 ura3 his3 ) and the triple ANT mutant JL-1-3 strain ( MATα LEU::aac1 HIS::aac2 URA::aac3 ade2 trp1 leu2 ura3 his3 ) [

Drgon T., Sabova L., Nelson N., Kolarov J., FEBS Lett. 289, 159 (1991);

] were transfected with a pJG-4-5 vector containing the Trp1 selection marker and murine Bax under the control of the Gal promoter and the lethal effect of Bax overexpression (controlled by immunoblot) was assessed [

Matsuyama S., Xu Q., Velours J., Reed J. C., Mol. Cell 1, 327 (1998);

]. The pJG-4-5 vector containing v-Ras instead of Bax served as a negative control.

Cells or organs were homogenized in H buffer (150 μM MgCl 2 10 mM KCl 10 mM tris-HCl pH 7.6) followed by elimination of nuclei (3 min at 900 g at 4°C recovery of supernatant) elimination of soluble proteins (10 min at 6800 g ) resuspension of the mitochondria-containing pellet in H buffer supplemented with 0.5% Triton (v:v final concentration; 0.5 mg of protein per milliliter) addition of polyclonal rabbit antiserum (1 μg of antibody per 10 μg of protein in sample) specific for Bcl-2 Bax (Santa Cruz Biotechnology) human interleukin-4 (IL-4) (Pharmingen) or ANT [

Gironcalle J., Schmid H. H. O., Biochemistry 35, 15440 (1996);

] incubation for 90 min at 37°C addition of 10% protein A and G agarose beads (Santa Cruz Biotechnology) for 30 min at 37°C recovery (10 min 2000 g ) of the washed (two times in phosphate-buffered saline pH 7.4) beads in SDS–polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and immunodetection with monoclonal antibody (mAb) to Bax (P19 Santa Cruz) mAb to Bcl-2 (clone 124 Dako) or antiserum to ANT.

A human Bak cDNA without TM [

Jürgensmeier J. M., et al., Mol. Biol. Cell 8, 325 (1997);

] human Bcl-2 lacking the TM murine Bax cDNA lacking the TM Ras Fas Traf-3 or a human ANT2 cDNA fragment (amino acids 105 to 156) were subcloned in the Eco R1–Xho I sites of pGilda or pJG4-5. EGY48 cells were used for lacZ reporter gene assays in conjunction with pGilda (LexA DNA–binding domain) and pJG4-5 (B42 transactivation domain) and psH 18-34 reporter plasmids. Filter assays were performed for galactosidase measurement with cells plated on either galactose- or glucose-containing minimal medium (19) [

Hanada M., Aime-Sempé C., Sato T., Reed J. C., J. Biol. Chem. 270, 11962 (1995);

Zha H., Aimé-Sempé C., Sato T., Reed J. C., ibid. 271, 7440 (1996)].

Rück A., Dolder M., Wallimann T., Brdiczka D., FEBS Lett. 426, 97 (1998).

Antonsson B., et al., Science 277, 370 (1997);

Schlesinger P. H., et al., Proc. Natl. Acad. Sci. U.S.A. 94, 11357 (1997).

Brustovetsky N., Klingenberg M., Biochemistry 35, 8483 (1996).

Bargou R. C., et al., J. Clin. Invest. 97, 2651 (1996).

Simbula G., Glascott P. A., Akita S., Hoek J. B., Farber J. L., Am. J. Physiol. 42, C479 (1997).

Beutner G., Rück A., Riede B., Welte W., Brdiczka D., FEBS Lett. 396, 189 (1996).

Lohret T. A., Jensen R. E., Kinnally K. W., J. Cell Biol. 137, 377 (1997);

González Barroso M. M., et al., Biochemistry 36, 10930 (1997);

Negre Salvayre A., et al., FASEB J. 11, 809 (1997).

Bisaccia F., et al., Biochim. Biophys. Acta 1292, 281 (1996).

Mitochondria were purified on a percoll gradient [

Reinhart P. H., Taylor W. M., Bygrave F. L., Biochem. J. 204, 731 (1982);

Petit P. X., et al., FEBS Lett. 426, 111 (1998);

; S. A. Susin N. Larochette M. Geuskens G. Kroemer Methods Enzymol. in press] and were functional in standard oxymetric tests (respiratory control ratio >5).

Susin S. A., et al., Exp. Cell Res. 236, 397 (1997).

10.1016/S0092-8674(00)80085-9

Greenawalt J. W., Methods Enzymol. 31, 310 (1974).

We thank D. Andrews for Rat-1 cells; D. Brdiczka for help in ANT purification; T. Drgon for ANT-deficient yeast; H. J. Duine for BA; S. Korsmeyer for Bax −/− mice; N. Roy for ANT plasmid; H. Schmid for the ANT-specific antibody; A. Zweibaum for HT29 cells; and N. Larochette and D. Métivier for technical assistance. Supported by Agence Nationale pour la Recherche sur le SIDA Association pour la Recherche sur le Cancer CNRS Fondation pour la Recherche Médicale and Ligue Française contre le Cancer (to G.K.) and University of California Breast Cancer Research Program (grant number IRB-009B) and CapCure Incorporated (to J.C.R.). S.A.S. and E.L.A.V. receive fellowships from the European Commission I.M. from the Spanish Ministry of Science and J.M.J. from the Deutsche Forschungsgemeinschaft.