The N-Terminal Domain of Bcl-x_L Reversibly Binds Membranes in a pH-Dependent Manner

Abstract

Bcl-x L regulates apoptosis by maintaining the integrity of the mitochondrial outer membrane by adopting both soluble and membrane-associated forms. The membrane-associated conformation does not require a conserved, C-terminal transmembrane domain and appears to be inserted into the bilayer of synthetic membranes as assessed by membrane permeabilization and critical surface pressure measurements. Membrane association is reversible and is regulated by the cooperative binding of approximately two protons to the protein. Two acidic residues, Glu153 and Asp156, that lie in a conserved hairpin of Bcl-x L ΔTM appear to be important in this process on the basis of a 16% increase in the level of membrane association of the double mutant E153Q/D156N. Contrary to that for the wild type, membrane permeabilization for the mutant is not correlated with membrane association. Monolayer surface pressure measurements suggest that this effect is primarily due to less membrane penetration. These results suggest that E153 and D156 are important for the Bclx L ΔTM conformational change and that membrane binding can be distinct from membrane permeabilization. Taken together, these studies support a model in which Bcl-x L activity is controlled by reversible insertion of its N-terminal domain into the mitochondrial outer membrane. Future studies with Bcl-x L mutants such as E153Q/D156N should allow determination of the relative contributions of membrane binding, insertion, and permeabilization to the regulation of apoptosis.Bcl-2 proteins act as checkpoints in the regulation of apoptosis by integrating intracellular signals to control the permeability and possibly the morphology of the mitochon-drion (1-9). For many Bcl-2 proteins, this checkpoint involves a change in localization from the cytosol to the mitochondrion (10-13). For example, Bcl-x L displays mixed localization to the cytosol and to organellar membranes, including the mitochondrion. After an apoptotic stimulus, Bcl-x L appears to localize primarily to the mitochondrial outer membrane where it may bind other apoptotic factors such as Bad (10,11,14) or form an ion channel thought to maintain the integrity of the mitochondrial membrane (11,(15)(16)(17). While mixed localization of Bcl-x L is well-established, the relative importance of cytosolic-and membranous-localized protein to the regulation of apoptosis is not. Intriguingly, the membrane activity of Bcl-x L may contribute more to its anti-apoptotic activity than its ability to sequester pro-apoptotic proteins: a mutant

Ligand Binding and Membrane Insertion Compete with Oligomerization of the BclXL Apoptotic Repressor

“…Importantly, our data also suggest that although the BclXL_dTM construct is predominantly monomeric in solution, it undergoes oligomerization within bicelles to such an extent that it is stable up to a temperature of 120°C. This observation is in agreement with the view that the TM domain is not critical for the insertion of apoptotic repressors into membranes 33; 42; 44 . However, the observation that the BclXL_dTM oligomers within membranes are more stable than the BclXL_FL oligomers is being reported here for the first time.…”

Membrane promotes tBID interaction with BCLXL

“…2e), but bound to anionic vesicles at acidic pH (Fig. 2f) 22 . Moreover, we tethered BCL XL Ct green to GUVs at neutral pH via its histidine tag when small amounts of the lipid derivative DOGS-NTA-Ni (Ni-lipid) were incorporated into GUVs (Fig.…”

Anti-apoptotic Bcl-2 Family Proteins Disassemble Ceramide Channels

“…Full-length human Bcl-x L was purified using two different protocols as described (40,41), except omitting all detergents. Bcl-x L mutants (⌬N76, lacking amino acids 2-76; ⌬loop, lacking amino acids 26 -83; ⌬TM, lacking C-terminal amino acids 213-233 (42) and full-length Bcl-x L (aa1-233)) were produced as N-terminal glutathione S-transferase fusion proteins from pGEX vectors using Escherichia coli DH1␣ as the host strain and isolated as described previously (40), except omitting all detergents.…”

“…Bcl-x L mutants (⌬N76, lacking amino acids 2-76; ⌬loop, lacking amino acids 26 -83; ⌬TM, lacking C-terminal amino acids 213-233 (42) and full-length Bcl-x L (aa1-233)) were produced as N-terminal glutathione S-transferase fusion proteins from pGEX vectors using Escherichia coli DH1␣ as the host strain and isolated as described previously (40), except omitting all detergents. pHis-GB1-CED-9-(1-251) fusion protein was expressed and purified in a manner similar to Bcl-x L using Ni ϩ2 affinity and size-exclusion chromatographies (41).…”