Lipids modulate the BH3-independent membrane targeting and activation of BAX and Bcl-xL

Abstract

Regulation of apoptosis is tightly linked with the targeting of numerous Bcl-2 proteins to the mitochondrial outer membrane (MOM), where their activation or inhibition dictates cell death or survival. According to the traditional view of apoptotic regulation, BH3-effector proteins are indispensable for the cytosol-to-MOM targeting and activation of proapoptotic and antiapoptotic members of the Bcl-2 protein family. This view is challenged by recent studies showing that these processes can occur in cells lacking BH3 effectors by as yet to be determined mechanism(s). Here, we exploit a model membrane system that recapitulates key features of MOM to demonstrate that the proapoptotic Bcl-2 protein BAX and antiapoptotic Bcl-xL have an inherent ability to interact with membranes in the absence of BH3 effectors, but only in the presence of cellular concentrations of Mg²⁺/Ca²⁺ Under these conditions, BAX and Bcl-xL are selectively targeted to membranes, refolded, and activated in the presence of anionic lipids especially the mitochondrial-specific lipid cardiolipin. These results provide a mechanistic explanation for the mitochondrial targeting and activation of Bcl-2 proteins in cells lacking BH3 effectors. At cytosolic Mg²⁺ levels, the BH3-independent activation of BAX could provide localized amplification of apoptotic signaling at regions enriched in cardiolipin (e.g., contact sites between MOM and mitochondrial inner membrane). Increases in MOM cardiolipin, as well as cytosolic [Ca²⁺] during apoptosis could further contribute to its MOM targeting and activity. Meanwhile, the BH3-independent targeting and activation of Bcl-xL to the MOM is expected to counter the action of proapoptotic BAX, thereby preventing premature commitment to apoptosis.

Keywords: apoptosis; divalent cations; membrane protein folding; mitochondria permeabilization; protein–lipid interactions.

Fig. 1.

BH3-independent membrane interactions of BAX and Bcl-xL. (A) Schematic representations of the BH3-dependent and BH3-independent membrane targeting and activation of BAX and Bcl-xL. The former requires activation by BH3-only effector proteins [e.g., Bid or Bim (8, 9)], while the latter is observed in the absence of these proteins. Cytosolic [Mg²⁺] and [Ca²⁺] ([Mg²⁺]_c, [Ca²⁺]_c) as well as mitochondrial [Ca²⁺] ([Ca²⁺]_M) are indicated as reference (61, 62). The latter represents the upper bound under stimulus induced [Ca²⁺] accumulation, while resting [Ca²⁺]_M is ∼0.0001 mM (61). (B–D) The BH3-independent membrane interactions of BAX and Bcl-xL are induced under physiological-like conditions (37 °C, pH 7.5) in a lipid-dependent manner (brown, 1TOCL:2POPC; green, 1TOCL:6POPC; cyan, 1POPS:2POPC; yellow, POPC). (B) BAX-mediated permeabilization of ANTS/DPX containing LUV, triggered by the addition of 1 mM Mg²⁺. (C) BAX-mediated leakage of 1TOCL:6POPC LUV in the presence of various Mg²⁺ (blue) or Ca²⁺ (red) concentrations. (D) Membrane insertion of full-length Bcl-xL (Bcl-xL FL) measured with the environmental-sensitive fluorophore NBD selectively attached to a single Cys at W169C mutant in α6.

Fig. 2.

Membrane anchoring of full-length Bcl-xL via C-terminal α8 helix in 1TOCL:2POPC LUV (pH 7.5, 37 °C). The transmembrane insertion of the anchoring α8 helix was tested by measuring the protection of the NBD fluorophore attached in the middle of α8 at G222C from the soluble quencher dithionite. Addition of dithionite results in a rapid and complete decrease in fluorescence in the absence of membrane interactions (gray, black). The formation of nanodiscs in the presence of Bcl-xL provides strong protection from quenching (green), consistent with the transmembrane orientation of α8 under such conditions (–14). The insertion of full-length Bcl-xL into vesicles in the presence of divalent cations results in intermediate quenching (Mg²⁺, blue; Ca²⁺, red).

Fig. 3.

BH3-independent membrane insertion of Bcl-xL in 1TOCL:2POPC LUV. The membrane insertion of Bcl-xL α6 helix was tested using NBD-labeled Bcl-xL W169C. (A) Representative NBD spectrum before and after the addition of Mg²⁺ at 25 °C. The Mg²⁺-dependent insertion Bcl-xL was reversed by the addition of EDTA (SI Appendix, Fig. S6A). (B) Relative membrane insertion of Bcl-xL quantified using the increases in NBD fluorescence intensity at 510 nm. The membrane-inserted populations were proportional to [Mg²⁺]/[Ca²⁺].

Fig. 4.

BH3-independent refolding of membrane-inserted Bcl-xL in 1TOCL:2POPC LUV at pH 7.5 at 25 °C. FRET measurements between mCherry conjugated at the N terminus of Bcl-xL and an Alexa 488 fluorophore at Bcl-xL D189C, as previously described (15, 16). (A) Representative emission spectra showing FRET between both fluorophores. The addition of either Mg²⁺ (blue) or Ca²⁺ (red) leads to loss of FRET in the presence of LUV, indicative of BH4 release. Bcl-xL refolding was reversible by the addition of EDTA at 37 °C (SI Appendix, Fig. S6B). (B) Representative smFRET distributions of Bcl-xL measured using fluorescence correlation spectroscopy (FCS) at different [Mg²⁺] concentrations: 0 mM Mg²⁺ (black) soluble Bcl-xL, 2 mM Mg²⁺ (purple) membrane-inserted Bcl-xL with BH4 released, and 0.5 mM Mg²⁺ (orange) membrane-inserted Bcl-xL with intermediate compactness. (C) FRET efficiencies calculated for ensemble (pink) and FCS (green). (D) Lipid-dependent modulation of Bcl-xL refolding in membranes with increasing cardiolipin content.

Fig. 5.

Kinetics of Bcl-xL membrane insertion and refolding in 1TOCL:2POPC LUV at pH 7.5. Time dependence of Bcl-xL membrane insertion (triangles) and BH4 release (circles) in the presence of 1 mM Mg²⁺ (blue) or 1 mM Ca²⁺ (red). (A) The presence of Mg²⁺ or Ca²⁺ did not affect the kinetics of either event, consistent with our steady-state measurements. Insertion and refolding do not occur simultaneously; instead, Bcl-xL refolding in the bilayer is delayed relative to its membrane insertion. (B) Arrhenius plot of Bcl-xL BH3-independent refolding kinetics measured between 25 and 42 °C in the presence of 1 mM Mg²⁺ or Ca²⁺. The shaded area represents a 95% confidence band for the slope estimate.

Fig. 6.

Pore-inhibiting action of Bcl-xL in the absence of BH3 effectors (pH 7.5, 37 °C). Inhibition of BH3-independent BAX mediated leakage by Bcl-xL FL and Bcl-xL in the absence of BH3-effectors in the presence of 1 mM Mg²⁺. Measurements were performed in LUV with high (1TOCL:2POPC) and low (1TOCL:6POPC) cardiolipin to capture the refolded and compact conformations, respectively, of membrane inserted Bcl-xL (Fig. 4D). (A) The refolded form of Bcl-xL (BH4 released) in 1TOCL:2POPC LUV is active (blue) and able to inhibit BAX. The presence or absence of the C-terminal membrane anchor helix did not affect inhibition of BAX leakage (red vs. blue). (B) Measurements at various BAX:Bcl-xL ratios in 1TOCL:2POPC LUV revealed that at least three Bcl-xL units are required per one BAX to achieve complete inhibition (red). (C) The compact conformation of membrane inserted Bcl-xL in low cardiolipin was also active and able to inhibit BAX-mediated leakage.

Fig. 7.

Schematic representation of the proposed mechanism of BH3-independent MOM targeting and activation of Bcl-2 proteins. The results presented here demonstrate that changes in membrane lipid composition, coupled with cellular levels of divalent cations, result in the membrane targeting of BAX and Bcl-xL. This targeting leads to the activation of both proapoptotic BAX and antiapoptotic Bcl-xL in the absence of BH3-only effector proteins, which explains the results reported for cell lines lacking BH3-only effectors (19, 20). At normal low concentrations of cardiolipin, Bcl-xL is targeted to the bilayer in a compact conformation, consistent with its canonic inhibition of apoptosis. Increases in cardiolipin content at the MOM [such as those observed during apoptosis (27, 28)] or local enrichment at mitochondrial contact sites (–26) results in the insertion of Bcl-xL in a refolded conformation, which leads to the release of its BH4 helix, involved in noncanonic apoptotic inhibition (55). Both Bcl-xL conformations, unreleased BH4 at low cardiolipin and BH4 released at high cardiolipin, are active and able to block BAX-mediated leakage (Fig. 6).