Modulation of Membrane Interactions of Anti-Apoptotic Regulator Bcl-xL by Lipids

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The Bcl-2 family of proteins (e.g., pro-apoptotic Bax and anti-apoptotic Bcl-xL) regulates the mitochondrial outer membrane permeabilization during the early stages of apoptosis. The prevalent Embedded Together Model of Bcl-2 action suggests that the membrane environment is critical for their proper functional interactions, consistent with the increasing evidence of lipids being involved in the regulation of apoptotic response. In this study, we apply a collection of fluorescence-based methods to investigate the effect of various lipids on the pH-triggered membrane interactions of Bcl-xL. The initial membrane association was studied using a FRET assay with donor-labeled Bcl-xL and acceptor-labeled vesicles, while the insertion/refolding of Bcl-xL into the membrane was monitored using the environment-sensitive probe NBD selectively attached in the middle of hydrophobic helix α6. Our results demonstrate that the lipid composition affects the pH-dependence of both initial membrane association and subsequent insertion/refolding of Bcl-xL. We found that a linear correlation exists between the membrane surface potential created by anionic lipids and the pKa of membrane binding, suggesting that the initial step is controlled by an electrostatic mechanism. The effect of lipids on the membrane insertion/refolding step is more complex and appears to be influenced by the size of the lipid headgroup. The kinetics of both the membrane association and membrane insertion/refolding is affected by the presence of non-bilayer forming lipids commonly found in mitochondria. While the presence of phosphatidylethanolamine accelerated the process, addition of lysophosphatidylcholine had the opposite effect, suggesting that mechanical properties of the bilayer also play a role. Taken together our results indicate that lipids can modulate the membrane interactions of Bcl-xL in multiple ways, providing an additional regulatory mechanism that ensures proper control of a complex cascade of apoptotic reactions leading to cell death or survival. NIHGM-069783, Fulbright-CONICYT, BRTP.