title: Bax activation initiates the assembly of a multimeric catalyst that facilitates Bax pore formation in mitochondrial outer membranes.
abstract: Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP) is essential for "intrinsic" apoptotic cell death. Published studies used synthetic liposomes to reveal an intrinsic pore-forming activity of Bax, but it is unclear how other mitochondrial outer membrane (MOM) proteins might facilitate this function. We carefully analyzed the kinetics of Bax-mediated pore formation in isolated MOMs, with some unexpected results. Native MOMs were more sensitive than liposomes to added Bax, and MOMs displayed a lag phase not observed with liposomes. Heat-labile MOM proteins were required for this enhanced response. A two-tiered mathematical model closely fit the kinetic data: first, Bax activation promotes the assembly of a multimeric complex, which then catalyzes the second reaction, Bax-dependent pore formation. Bax insertion occurred immediately upon Bax addition, prior to the end of the lag phase. Permeabilization kinetics were affected in a reciprocal manner by [cBid] and [Bax], confirming the "hit-and-run" hypothesis of cBid-induced direct Bax activation. Surprisingly, MOMP rate constants were linearly related to [Bax], implying that Bax acts non-cooperatively. Thus, the oligomeric catalyst is distinct from Bax. Moreover, contrary to common assumption, pore formation kinetics depend on Bax monomers, not oligomers. Catalyst formation exhibited a sharp transition in activation energy at ∼28°C, suggesting a role for membrane lipid packing. Furthermore, catalyst formation was strongly inhibited by chemical antagonists of the yeast mitochondrial fission protein, Dnm1. However, the mammalian ortholog, Drp1, was undetectable in mitochondrial outer membranes. Moreover, ATP and GTP were dispensable for MOMP. Thus, the data argue that oligomerization of a catalyst protein, distinct from Bax and Drp1, facilitates MOMP, possibly through a membrane-remodeling event.
authors.affiliation: La Jolla Institute for Allergy and Immunology, La Jolla, California, United States of America.
authors.forename: Yulia
authors.initials: Y
authors.lastname: Kushnareva
authors.forename: Alexander Y
authors.initials: AY
authors.lastname: Andreyev
authors.forename: Tomomi
authors.initials: T
authors.lastname: Kuwana
authors.forename: Donald D
authors.initials: DD
authors.lastname: Newmeyer
mesh_terms: D000818:Animals; D051023:BH3 Interacting Domain Death Agonist Protein / Q000378:metabolism; D055162:Biocatalysis / Q000187:drug effects; D034284:Dynamin I / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008297:Male; D008930:Mitochondria, Liver / Q000187:drug effects / Q000378:metabolism; D033681:Mitochondrial Membrane Transport Proteins / Q000378:metabolism*; D051336:Mitochondrial Membranes / Q000378:metabolism*; D000083162:Mitochondrial Permeability Transition Pore; D008954:Models, Biological; D010539:Permeability / Q000187:drug effects; D055503:Protein Multimerization / Q000187:drug effects; D052999:Quinazolinones / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013816:Thermodynamics; D051028:bcl-2-Associated X Protein / Q000378:metabolism*
chemical_list: C549041:3-(2,4-dichloro-5-methoxyphenyl)-2-sulfanyl-4(3H)-quinazolinone; D051023:BH3 Interacting Domain Death Agonist Protein; D008081:Liposomes; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D052999:Quinazolinones; D051028:bcl-2-Associated X Protein; D034284:Dynamin I