BAX IN MURINE THYMUS IS A SOLUBLE MONOMERIC PROTEIN THAT DISPLAYS DIFFERENTIAL DETERGENT-INDUCED CONFORMATIONS

Bcl-2, Bcl-X-L, and Bar are members of the Bcl-2 family that play impo rtant roles in apoptosis regulation. These proteins are believed to be membrane-bound and to regulate apoptosis through formation of homo-an d heterodimers. However, we recently found by subcellular fractionatio n that whereas Bcl-2 is predominantly a membrane protein as previously reported, Bar and a significant fraction of Bcl-X-L are soluble in th ymocyte and splenocyte extracts. In addition, we have demonstrated tha t the ability of Bar to form dimers appears to be a detergent-induced phenomenon that coincides with a detergent-induced conformational chan ge. We have further investigated the tertiary and quaternary states of Bar in the presence of various detergents. Detergents such as Triton X-100 and Triton X-114 readily enable Bar hetero-and homodimerization. However, other detergents such as polydocanol, W-l, octyl glucoside, dodecyl maltoside, Tween 20, and sodium cholate allow varying degrees of Bar hetero-and homodimerization. Detergents such as cholamidopropyl )dimethylammonio]-1-propanesulfonic acid (Chaps) and Brij 35 allow nei ther hetero-nor homodimer formation, Immunoprecipitation analysis with the conformation-sensitive antibody uBax 6A7 revealed that whereas Tr iton X-100 readily exposes the N-terminal Bax epitope (amino acid 13-1 9), only limited exposure of the epitope occurs in Triton X-114, polyd ocanol, dodecyl maltoside, and sodium cholate, and no exposure of this epitope was observed in W-1, Chaps, octyl glucoside, Between 20, and Brij 35. Moreover, we could not detect any proteins associated with th e cytosolic form of Bar based on immunopurification of this protein. S ephacryl S-100 gel filtration chromatography analysis of the cytosolic Bar indicated that this protein is monomeric and displays an apparent molecular mass of 25 kDa. Induction of apoptosis which causes the ins ertion of the soluble form of Bar into membranes did not result in app reciable Bax/Bcl-X-L, Bax/Bcl-2 or Bax/Bax dimer formation as determin ed by cross-linking studies, Further analysis of Bar after apoptosis i nduction by immunoprecipitation in the presence of Chaps also revealed no significant heterodimer formation. In conclusion, Bar displays sev eral distinct states in different detergents that expose defined regio ns of the protein. In addition, these results suggest that mechanisms other than the simple dimerization among members of the Bcl-2 family m ay be required for the regulation of apoptosis.