BNIP3 Heterodimerizes with Bcl-2/Bcl-XL and Induces Cell Death Independent of a Bcl-2 Homology 3 (BH3) Domain at Both Mitochondrial and Nonmitochondrial Sites

Abstract

BNIP3 (formerly NIP3) is a pro-apoptotic, mitochondrial protein classified in the Bcl-2 family based on limited sequence homology to the Bcl-2 homology 3 (BH3) domain and COOH-terminal transmembrane (TM) domain. BNIP3 expressed in yeast and mammalian cells interacts with survival promoting proteins Bcl-2, Bcl-X_L, and CED-9. Typically, the BH3 domain of pro-apoptotic Bcl-2 homologues mediates Bcl-2/Bcl-X_Lheterodimerization and confers pro-apoptotic activity. Deletion mapping of BNIP3 excluded its BH3-like domain and identified the NH₂ terminus (residues 1–49) and TM domain as critical for Bcl-2 heterodimerization, and either region was sufficient for Bcl-X_L interaction. Additionally, the removal of the BH3-like domain in BNIP3 did not diminish its killing activity. The TM domain of BNIP3 is critical for homodimerization, pro-apoptotic function, and mitochondrial targeting. Several TM domain mutants were found to disrupt SDS-resistant BNIP3 homodimerization but did not interfere with its killing activity or mitochondrial localization. Substitution of the BNIP3 TM domain with that of cytochromeb ₅ directed protein expression to nonmitochondrial sites and still promoted apoptosis and heterodimerization with Bcl-2 and Bcl-X_L. We propose that BNIP3 represents a subfamily of Bcl-2-related proteins that functions without a typical BH3 domain to regulate apoptosis from both mitochondrial and nonmitochondrial sites by selective Bcl-2/Bcl-X_L interactions.