tBid elicits a conformational alteration in membrane-bound Bcl-2 such that it inhibits Bax pore formation.
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.
Peng
Jun
J
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190, USA.
Tan
Chibing
C
Roberts
G Jane
GJ
Nikolaeva
Olga
O
Zhang
Zhi
Z
Lapolla
Suzanne M
SM
Primorac
Steve
S
Andrews
David W
DW
Lin
Jialing
J
D000818:Animals; D017209:Apoptosis; D051023:BH3 Interacting Domain Death Agonist Protein / Q000737:chemistry / Q000502:physiology*; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005456:Fluorescent Dyes / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000378:metabolism; D008081:Liposomes / Q000737:chemistry; D008928:Mitochondria / Q000378:metabolism; D009154:Mutation; D011487:Protein Conformation; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D051028:bcl-2-Associated X Protein / Q000737:chemistry / Q000502:physiology*
D051023:BH3 Interacting Domain Death Agonist Protein; D005456:Fluorescent Dyes; D008081:Liposomes; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein