Nonionic detergents induce dimerization among members of the Bcl-2 family

Abstract

Members of the Bcl-2 family (including Bcl-2, Bcl-XL, and Bax) play key roles in the regulation of apoptosis. These proteins are believed to be membrane-associated and have been proposed to regulate apoptosis through both homodimerization and heterodimerization. We have found that whereas Bcl-2 is predominantly membrane-associated as previously reported, significant amounts of Bcl-XL and most of the Bax proteins are not membrane-associated and thus appear in the cytosolic fraction of thymocyte and splenocyte extracts. This finding allows the study of the dimerization properties and conformation of these proteins in the absence of detergent perturbation. For this analysis, we have produced monoclonal antibodies that are specific for known epitopes of Bax, Bcl-2, and Bcl-XL. An antibody to an N-terminal epitope (alpha uBax 6A7) between amino acids 12 and 24 fails to bind the soluble cytosolic form of Bax, indicating that this epitope is normally buried. Nonionic detergents alter the Bax conformation to expose this epitope. In the presence of nonionic detergent, the 6A7 antibody avidly binds the monomeric form of Bax, but not Bax complexed with either Bcl-XL or Bcl-2. In contrast, a monoclonal antibody to an adjacent epitope of Bax (alpha mBax 5B7) within amino acids 3-16 binds the soluble and detergent-altered forms of Bax and also binds the Bax.Bcl-XL or the Bax.Bcl-2 complex. Surprisingly, in the absence of detergent Bax fails to form homodimers or heterodimers with Bcl-XL. These results demonstrate a novel conformational state of members of the Bcl-2 family under a physiological condition that is distinct from the detergent-altered state that forms dimers and is currently believed to regulate apoptosis.