CED-4, a pro-apoptotic factor in Caenorhabditis elegans, activates the cell death protease CED-3. CED-9 directly binds to CED-4 and represses this. However, it has remained unclear whether a mammalian CED-9 homologue, Bcl-X(L), inhibits the function of the mammalian CED-4 homologue, Apaf-1, by direct binding. To analyze the interaction, we adopted a yeast two-hybrid system. Since Bcl-X(L) and the CED-4-like portion of Apaf-1 failed to exhibit a positive result in the assay, we prepared "fragment libraries" of bcl-X(L) or apaf-1
cDNA. By screening of the apaf-1
"fragment library," we obtained nine clones interacting with Bcl-X(L), all containing the same region within the ATPase domain, designated BBR: the Bcl-X(L) binding region. Binding of BBR to Bcl-X(L) was also confirmed by immunoprecipitation assays. Bcl-2, Bcl-w, A1/Bfl-1, and Boo/Diva failed to show the same capacity for binding to BBR as Bcl-X(L). These results indicate that Bcl-X(L) directly binds to a specific region in Apaf-1.