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J. Biol. Chem., Vol. 281, Issue 47, 35802-35811, November 24, 2006

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tBid Elicits a Conformational Alteration in Membrane-bound Bcl-2 Such That It Inhibits Bax Pore Formation^*

Jun Peng, Chibing Tan, G. Jane Roberts, Olga Nikolaeva, Zhi Zhang, Suzanne M. Lapolla, Steve Primorac, David W. Andrews¹, and Jialing Lin²

From the Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190 and the Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario L8N 3Z5, Canada

During initiation of apoptosis, Bcl-2 family proteins regulate the permeability of mitochondrial outer membrane. BH3-only protein, tBid, activates pro-apoptotic Bax to release cytochrome c from mitochondria. tBid also activates anti-apoptotic Bcl-2 in the mitochondrial outer membrane, changing it from a single-spanning to a multispanning conformation that binds the active Bax and inhibits cytochrome c release. However, it is not known whether other mitochondrial proteins are required to elicit the tBid-induced Bcl-2 conformational alteration. To define the minimal components that are required for the functionally important Bcl-2 conformational alteration, we reconstituted the reaction using purified proteins and liposomes. We found that purified tBid was sufficient to induce a conformational alteration in the liposome-tethered, but not cytosolic Bcl-2, resulting in a multispanning form that is similar to the one found in the mitochondrial outer membrane of drug-treated cells. Mutations that abolished tBid/Bcl-2 interaction also abolished the conformational alteration, demonstrating that a direct tBid/Bcl-2 interaction at the membrane is both required and sufficient to elicit the conformational alteration. Furthermore, active Bax also elicited the Bcl-2 conformational alteration. Bcl-2 mutants that displayed increased or decreased activity in the conformational alteration assay showed corresponding activities in inhibiting pore formation by Bax in vitro and in preventing apoptosis in vivo. Thus, there is a strong correlation between the direct interaction of membrane-bound Bcl-2 and tBid with activation of Bcl-2 in vitro and in vivo.

Received for publication, August 30, 2006

^* This work was supported in part by American Heart Association Postdoctoral Fellowship 0420070Z (to O. N.), National Institutes of Health Grant GM062964 (to J. L.), and Canadian Institutes of Health Research Grant FRN 12517 (to D. W. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Holds the Canada Research Chair in Membrane Biogenesis.

² To whom correspondence should be addressed: 940 Stanton L. Young Boulevard, BMSB 935, P.O. Box 26901, Oklahoma City, OK 73190. Tel.: 405-271-2227 (ext. 1216); Fax: 405-271-3092; E-mail: jialing-lin{at}ouhsc.edu.

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