The C-elegans orthologue ceBNIP3 interacts with CED-9 and CED-3 but kills through a BH3-and caspase-independent mechanism

We have studied ceBNIP3, the orthologue of BNIP3 in C, elegans. Sequence an alysis reveals that the different domains of BNIP3 have been conserved thro ughout evolution. ceBNIP3 contains a C-terminal transmembrane (TM) domain, a conserved domain (CD) of 19 amino acids, a BCL-2 homology-3 (BH3)-like do main and a PEST sequence. ceBNIP3 is expressed primarily as a 25 kDa monome r and a 50 kDa homodimer, After transfection, ceBNIP3 protein is rapidly de graded through a ubiquitin-dependent pathway by the proteasome. Like BNIP3, the TM domain of ceBNIP3 mediates the localization of the protein to mitoc hondria and is also necessary for homodimerization and cell death in mammal ian cells. Neither the putative BH3 domain nor conserved domain is necessar y for killing. ceBNIP3 protein interacts with CED-9 and BCL-X-L, but unlike other pro-apoptotic BCL-2 family members, the BH3-like domain does not par ticipate in dimerization, The ceBNIP3 TM domain mediates interaction with b oth CED-9 and BCL-XL. ceBNIP3 interacts with CED-3 but co-expression of CED -3 and ceBNIP3 does not significantly enhance induction of cell death in th e presence or absence of CED-4, ceBNIP3 kills mammalian cells by a caspase- independent mechanism. In conclusion, we find that although ceBNIP3 interac ts with CED-9 and CED-3 it kills by a BH3- and caspase-independent mechanis m.