Bax forms multispanning monomers that oligomerize to permeabilize membranes during apoptosis

Abstract

Bax promotes cell death by permeabilizing mitochondrial outer membranes by an unresolved mechanism. However, in cells lacking the gene c-myc, membrane permeabilization by Bax is blocked by changes in the mitochondria that prevent Bax oligomerization. Drug-treated c-myc null cells and cells expressing Myc were used to map the topology of Bax in membranes prior to and after mitochondrial permeabilization. Chemical labeling of single cysteine mutants of Bax using a membrane bilayer impermeant cysteine-specific modifying agent revealed that Bax inserted both the 'pore domain' (helices alpha5-alpha6), and the tail-anchor (helix alpha9) into membranes prior to oligomerization and membrane permeabilization. Additional topology changes for Bax were not required in Myc-expressing cells to promote oligomerization and cytochrome c release. Our results suggest that unlike most pore-forming proteins, Bax membrane permeabilization results from oligomerization of transmembrane monomers rather than concerted insertion of the pore domains of a preformed oligomer.

Bak apoptotic pores involve a flexible C-terminal region and juxtaposition of the C-terminal transmembrane domains

“…To test whether an α9:α9 interface also occurs in Bax after it becomes oligomerized, a single cysteine was placed at six positions in α9 of a mitochondrial form of Bax, S184L 53 ( Figure 3c). Each variant was able to mediate apoptosis after etoposide, even the G179C variant which, as observed previously, 20 expressed at very low levels (Supplementary Figure S1c). When membrane fractions were incubated with tBid, CuPhe was able to link α9 at two positions (I175C and α178C) (Figures 3c and d).…”

“…To identify α9 residues buried in the hydrophobic interior of the MOM, cysteine variants were labeled with the membrane-impermeable sulfhydryl reagent 4-acetamido-4ʹ-((iodoacetyl)amino)stilbene-2,2ʹ-disulfonic acid (IASD). 20,23,49 The two negative charges on IASD prevent its entry into hydrophobic regions, including membranes, protein interior and protein interfaces. 23 IASD efficiently labeled cysteine residues on the cytoplasmic side of the MOM (Figures 2a, e and g; G186C).…”

“…23 IASD efficiently labeled cysteine residues on the cytoplasmic side of the MOM (Figures 2a, e and g; G186C). IASD also strongly labeled cysteine placed at the far C terminus (Figure 2a, BakGGCK), probably by passing through channels 20,50 such as VDAC, which allows passage of metabolites up to 4000 Da. 51 Quantification of IASD labeling before and after tBid is shown in Figure 2b.…”

“…As protein pores and ion channels often have α-helices facing the pore lumen, 62,63 the same may be the case for pores formed by Bak and Bax. Although the Bax α5 and α6 helices were thought to insert into and traverse the membrane after Bax activation, 20 recent evidence that these helices in activated Bak and Bax lie in-plane on the membrane surface 22,23 leaves α9 as the only region likely to traverse the MOM either before or after apoptosis. Despite this, we found that Bak α9 did not line a pore, as IASD labeling did not increase along one side of the helix that would become accessible to IASD present within the pore lumen.…”

“…6,7,14,17,18 In addition to dimerizing, parts of activated Bak and Bax associate with the lipid bilayer. 19 In Bax, the α5 and α6 helices may insert into the MOM, 20 although recent studies indicate that they lie in-plane on the membrane surface, with the hydrophobic α5 sandwiched between the membrane and a BH3:groove dimer interface. 7,[21][22][23] The dimers can be linked via cysteine residues placed in α6, 18,24,25 and more recently via cysteine residues in either α3 or α5, 6,21 allowing detection of the higherorder oligomers associated with pore formation.…”