Detergent-activated BAX Protein Is a Monomer

Olena Ivashyna,Ana J García-Sáez,Jonas Ries,Eric T Christenson,Petra Schwille,Paul H Schlesinger

doi:10.1074/jbc.m109.023853

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 an oligomeric pore. The chemical mechanism of BAX activation is controversial, and several in vitro and in vivo methods of its activation are known. One of the most commonly used in vitro methods is activation with detergents, such as n-octyl glucoside. During BAX activation with n-octyl glucoside, it has been shown that BAX forms high molecular weight complexes that are larger than the combined molecular weight of BAX monomer and one detergent micelle. These large complexes have been ascribed to the oligomerization of BAX prior to its membrane insertion and pore formation. This is in contrast to the in vivo studies that suggest that active BAX inserts into the outer mitochondrial membrane as a monomer and then undergoes oligomerization. Here, to simultaneously determine the molecular weight and the number of BAX proteins per BAX-detergent micelle during detergent activation, we have used an approach that combines two single-molecule sensitivity technique, fluorescence correlation spectroscopy, and fluorescence-intensity distribution analysis. We have tested a range of detergents as follows: n-octyl glucoside, dodecyl maltoside, Triton X-100, Tween 20, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, and cholic acid. With these detergents we observe that BAX is a monomer before, during, and after interaction with micelles. We conclude that detergent activation of BAX is not congruent with oligomerization and that in physiologic buffer conditions BAX can assume two stable monomeric conformations, one inactive and one active.

Highlights

Localized in the cytoplasm of healthy nondying cells [1]
During apoptosis BAX is activated and translocates to the outer mitochondrial membrane [2] where it inserts as a monomer [3], undergoes oligomerization [4], and forms a pore through which cytochrome c and other apoptotic factors are released into the cytoplasm
Detergent-activated Fluorescently Labeled BAX⌬C Can Release Cytochrome c from Isolated Mitochondria—For the fluorescence correlation spectroscopy (FCS) and fluorescence-intensity distribution analysis (FIDA) experiments, we prepared recombinant, fluorescently labeled human BAX⌬C containing a fluorophore at a single cysteine residue (Fig. 1, A and B)

Summary

Introduction

Localized in the cytoplasm of healthy nondying cells [1]. During apoptosis BAX is activated and translocates to the outer mitochondrial membrane [2] where it inserts as a monomer [3], undergoes oligomerization [4], and forms a pore through which cytochrome c and other apoptotic factors are released into the cytoplasm. These apoptotic factors induce the activation of the effector caspases that execute the cell death process This mechanism, which is generally correct, requires that soluble BAX becomes integrated into the mitochondrial membrane where it forms a functional oligomeric pore capable of cytochrome c release. During activation by nonionic detergents, to gain the ability to form pores in a bilayer membrane, BAX needs to undergo a major conformational transition from a globular protein with two pore-forming ␣-helices 5 and 6 hidden in the protein core [8] to a conformation in which these two helices are exposed and inserted into a lipid membrane [3, 5, 9] The nature of this active conformation of BAX is important for the understanding of the death decision in cells. The ease of dialysis is why, in most cases, OG is used to activate BAX in vitro

Results

Discussion

Conclusion