Bax in Murine Thymus Is a Soluble Monomeric Protein That Displays Differential Detergent-induced Conformations

Abstract

Bcl-2, Bcl-XL, and Bax are members of the Bcl-2 family that play important roles in apoptosis regulation. These proteins are believed to be membrane-bound and to regulate apoptosis through formation of homo- and heterodimers. However, we recently found by subcellular fractionation that whereas Bcl-2 is predominantly a membrane protein as previously reported, Bax and a significant fraction of Bcl-XL are soluble in thymocyte and splenocyte extracts. In addition, we have demonstrated that the ability of Bax to form dimers appears to be a detergent-induced phenomenon that coincides with a detergent-induced conformational change. We have further investigated the tertiary and quaternary states of Bax in the presence of various detergents. Detergents such as Triton X-100 and Triton X-114 readily enable Bax hetero- and homodimerization. However, other detergents such as polydocanol, W-1, octyl glucoside, dodecyl maltoside, Tween 20, and sodium cholate allow varying degrees of Bax hetero- and homodimerization. Detergents such as 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (Chaps) and Brij 35 allow neither hetero- nor homodimer formation. Immunoprecipitation analysis with the conformation-sensitive antibody uBax 6A7 revealed that whereas Triton X-100 readily exposes the N-terminal Bax epitope (amino acid 13-19), only limited exposure of the epitope occurs in Triton X-114, polydocanol, dodecyl maltoside, and sodium cholate, and no exposure of this epitope was observed in W-1, Chaps, octyl glucoside, Tween 20, and Brij 35. Moreover, we could not detect any proteins associated with the cytosolic form of Bax based on immunopurification of this protein. Sephacryl S-100 gel filtration chromatography analysis of the cytosolic Bax indicated that this protein is monomeric and displays an apparent molecular mass of 25 kDa. Induction of apo-ptosis which causes the insertion of the soluble form of Bax into membranes did not result in appreciable Bax/Bcl-XL, Bax/Bcl-2 or Bax/Bax dimer formation as determined by cross-linking studies. Further analysis of Bax after apoptosis induction by immunoprecipitation in the presence of Chaps also revealed no significant heterodimer formation. In conclusion, Bax displays several distinct states in different detergents that expose defined regions of the protein. In addition, these results suggest that mechanisms other than the simple dimerization among members of the Bcl-2 family may be required for the regulation of apoptosis.