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Vol. 20, Issue 8, 2276-2285, April 15, 2009

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Mitochondrial Outer Membrane Proteins Assist Bid in Bax-mediated Lipidic Pore Formation

Blanca Schafer^*, Joel Quispe, Vineet Choudhary, Jerry E. Chipuk, Teddy G. Ajero, Han Du^*, Roger Schneiter, and Tomomi Kuwana^*

*Department of Pathology, University of Iowa Carver College of Medicine, Iowa City, IA 52242 National Resource for Automated Molecular Microscopy, Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037 Division of Biochemistry, Department of Medicine, University of Fribourg, Fribourg CH1700, Switzerland Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN 38105

Submitted October 22, 2008; Revised February 11, 2009; Accepted February 13, 2009
Monitoring Editor: Thomas D. Fox

Mitochondrial outer membrane permeabilization (MOMP) is a critical step in apoptosis and is regulated by Bcl-2 family proteins. In vitro systems using cardiolipin-containing liposomes have demonstrated the key features of MOMP induced by Bax and cleaved Bid; however, the nature of the "pores" and how they are formed remain obscure. We found that mitochondrial outer membranes contained very little cardiolipin, far less than that required for liposome permeabilization, despite their responsiveness to Bcl-2 family proteins. Strikingly, the incorporation of isolated mitochondrial outer membrane (MOM) proteins into liposomes lacking cardiolipin conferred responsiveness to cleaved Bid and Bax. Cardiolipin dependence was observed only when permeabilization was induced with cleaved Bid but not with Bid or Bim BH3 peptide or oligomerized Bax. Therefore, we conclude that MOM proteins specifically assist cleaved Bid in Bax-mediated permeabilization. Cryoelectron microscopy of cardiolipin-liposomes revealed that cleaved Bid and Bax produced large round holes with diameters of 25–100 nm, suggestive of lipidic pores. In sum, we propose that activated Bax induces lipidic pore formation and that MOM proteins assist cleaved Bid in this process in the absence of cardiolipin.

Address correspondence to: Tomomi Kuwana (t-kuwana{at}uiowa.edu)

Abbreviations used: ExOMV, proteo-liposomes composed of lipid-extracted outer membrane vesicle; LMV, light membrane vesicle; LUV, large unilamellar vesicle; MOM, mitochondrial outer membrane; MOMP, mitochondrial outer membrane permeabilization; OG, octylglucoside; OMV, outer membrane vesicle; ReOMV, reformed outer membrane vesicle.