Research Article: Bax Activation Initiates the Assembly of a Multimeric Catalyst that Facilitates Bax Pore Formation in Mitochondrial Outer Membranes

Date Published: September 25, 2012

Publisher: Public Library of Science

Author(s): Yulia Kushnareva, Alexander Y. Andreyev, Tomomi Kuwana, Donald D. Newmeyer, J. Marie Hardwick

Abstract: Bax promotes mitochondrial permeabilization during apoptosis via a phase-transition-like event in the membrane and oligomerization of a catalyst molecule that facilitates Bax pore formation.

Partial Text: Mitochondria are well known to be essential for cell life, as they produce ATP and other products of key biosynthetic pathways. Intriguingly, mitochondria are also often critical for cell death [1]–[4]. In vertebrates, apoptotic cell death typically involves a canonical “intrinsic” apoptotic pathway that depends on mitochondrial outer membrane permeabilization (MOMP). MOMP is induced by the pro-apoptotic Bcl-2 family proteins Bax and/or Bak [5]–[12].

To uncover features of Bax-dependent pore formation in a near-physiological but experimentally tractable system, we used isolated mitochondrial outer membranes (MOMs). Earlier we showed that isolated MOMs from Xenopus laevis eggs spontaneously reseal to form “Outer Membrane Vesicles” (OMVs) that can entrap fluorescent dextrans [9]. Xenopus OMVs become permeabilized upon the addition of recombinant BH3-only proteins such as cleaved Bid (cBid), and this permeabilization is inhibited by the anti-apoptotic Bcl-xL protein. Here, we prepared OMVs from rat liver mitochondria (Figure 1A), which were advantageous for our studies because they do not respond to added cBid alone (as shown by others; [32],[33]), but become permeabilized by a combination of cBid and Bax. Immunoblot analysis showed that rat liver OMVs lack detectable amounts of Bax ([32],[34] and Figure S1C). They contain some Bak (albeit less than the amount in mouse liver mitochondria; not shown), but this resident Bak protein is apparently insufficient to permeabilize mitochondria, or is perhaps inhibited by another MOM protein such as VDAC2 [35]. Therefore, we could measure the quantitative response to recombinant Bax in the absence of endogenous Bax-like activity. Purified rat liver OMVs were largely devoid of inner mitochondrial membrane proteins, as shown by immunoblots (Figure 1B). Contamination with the inner membrane was ≈5% based on succinate dehydrogenase activity (Figure 1C). As expected, OMVs were highly enriched in the outer membrane proteins VDAC and monoaminooxidase (Figure 1B,C). We also detected the ER protein Calnexin in OMV preparations (Figure 1B), consistent with the physical association of mitochondria and ER [36].

Source:

http://doi.org/PBIOLOGY-D-12-01000