Assembly of the Mitochondrial Apoptosis-induced Channel,
MAC |
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Authors: | Sonia Martinez-Caballero Laurent M Dejean Michael S Kinnally Kyoung Joon Oh Carmen A Mannella and Kathleen W Kinnally |
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Abstract: | Although Bcl-2 family proteins control intrinsic apoptosis, the mechanisms
underlying this regulation are incompletely understood. Patch clamp studies of
mitochondria isolated from cells deficient in one or both of the pro-apoptotic
proteins Bax and Bak show that at least one of the proteins must be present
for formation of the cytochrome c-translocating channel,
mitochondrial apoptosis-induced channel (MAC), and that the single channel
behaviors of MACs containing exclusively Bax or Bak are similar. Truncated Bid
catalyzes MAC formation in isolated mitochondria containing Bax and/or Bak
with a time course of minutes and does not require VDAC1 or VDAC3.
Mathematical analysis of the stepwise changes in conductance associated with
MAC formation is consistent with pore assembly by a barrel-stave model.
Assuming the staves are two transmembrane α-helices in Bax and Bak,
mature MAC pores would typically contain ∼9 monomers and have diameters of
5.5–6 nm. The mitochondrial permeability data are inconsistent with
formation of lipidic pores capable of transporting megadalton-sized
macromolecules as observed with recombinant Bax in liposomes.Permeabilization of the mitochondrial outer membrane is the commitment step
in intrinsic apoptosis. This process is tightly regulated by Bcl-2 family
proteins that control formation of the megachannel mitochondrial
apoptosis-induced channel
(MAC)2 in this
membrane. MAC formation correlates with release of pro-apoptotic factors,
including cytochrome c from the intermembrane space into the cytosol,
and initiates apoptosis
(1–7).MAC is absent from normal mitochondria but forms in the outer membrane
early in apoptosis, reaching peak conductances of 1.5–5 nS. This channel
is formed in the presence of the multidomain pro-apoptotic proteins Bax and/or
Bak
(8–13),
and may be composed of these proteins along with other components
(14,
15). Unlike Bax, Bak is
normally a resident of the mitochondrial outer membrane and is bound to VDAC2,
another outer membrane protein
(16). However, Bak is not
available for oligomerization until another pro-apoptotic protein, like t-Bid,
disrupts the interaction of Bak with VDAC2. In contrast, most Bax is located
in the cytoplasm until an apoptotic signal induces the translocation of Bax to
the outer membrane of mitochondria and eventual Bax oligomerization in this
same membrane (14,
17).Bax and Bak have multiple putative transmembrane domains; the amphipathic
helices 5 and 6 of Bax are predicted to form, at least in part, the pore of
the cytochrome c release channel
(18). Bax lacking helices 5
and 6 does not translocate to mitochondria nor cause cytochrome c
release (19,
20). Given the structural
similarities between Bax and Bak, the same helices may be important in
formation of the MAC pore by both proteins
(21). Although Bax and Bak are
certainly involved in MAC formation, the exact molecular composition of this
channel remains unknown.In this study we report that Bax and Bak are functionally redundant with
regard to MAC formation and cytochrome c release in mouse embryonic
fibroblasts (MEF). This is true despite the fact that Bak normally resides in
the outer membrane, whereas Bax is generally translocated to this membrane to
induce MAC formation. Our experimental design bypasses Bax translocation and
any underlying autocatalytic mechanism that might be involved
(22). Instead, it focuses on
formation of the MAC pore. Early MAC-associated conductance increments are
relatively small, suggesting that Bax-dependent formation of the cytochrome
c-permeable pore does not occur prior to membrane insertion of Bax.
Mathematical modeling of the conductance changes indicates that, if MAC is a
circular pore assembled by sequential addition of helices 5 and 6 from Bax
and/or Bak monomers, the mature, cytochrome c transport-competent
pore is likely a 9–10-mer of these proteins. |
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