Detergent-activated BAX protein is a monomer.
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.
Ivashyna
Olena
O
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
García-Sáez
Ana J
AJ
Ries
Jonas
J
Christenson
Eric T
ET
Schwille
Petra
P
Schlesinger
Paul H
PH
D003902:Detergents / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D008823:Micelles; D051336:Mitochondrial Membranes / Q000737:chemistry* / Q000378:metabolism; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000502:physiology; D051028:bcl-2-Associated X Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
C494826:BAX protein, human; D003902:Detergents; D008823:Micelles; D051028:bcl-2-Associated X Protein