FCS Studies of the Pore Formation by Protein BAX in Lipid Membranes

Abstract

BAX is a pro-apoptotic member of the BCL-2 protein family. At the onset of apoptosis, monomeric, cytoplasmic BAX is activated and translocates to the outer mitochondrial membrane, where it forms oligomeric pores. The biophysical mechanism of BAX pore formation and the structure of the BAX pore are not clear. To study the mechanism of BAX pore formation in lipid membranes we designed an in vitro system employing giant unilamellar vesicles (GUVs) and fluorescently labeled BAX combined with the single-molecule sensitivity technique, dual-focus scanning FCS. Use of scanning FCS in experiments, where two spectrally different populations of BAX molecules interact with a GUV membrane, allowed correlation of the pore formation by BAX with the ability of BAX to oligomerize in lipid membranes. As a result, we show that BAX binds lipid membranes as a monomer and then undergoes oligomerization to form BAX pore protein-lipid complexes. FCS analysis of the populations of GUVs over a period of time showed that BAX pore complexes grow in size and increase in number with time. Analysis of the diffusion coefficients of these BAX complexes using Saffman-Delbruck theory estimates that the in-membrane hydrodynamic radius of a BAX pore complex ranges from 1 to 31 nm. Formation of BAX pore complexes in a lipid membrane is inhibited in the presence of BCL-XL (in-membrane BAX is 100% monomeric) and can be rescued by the addition of cut BID. We also show confocal 3D reconstructions of a giant BAX pore with fluorescent BAX accumulating in the edge of the pore. Lifetime of the giant BAX pore (5-10 min) together with the accumulation of BAX at the edge of the pore and the loss of surface tension in a GUV support the toroidal BAX pore model.