BAX and BAK are "multidomain" proapoptotic proteins that initiate mitochondrial dysfunction but also localize to the endoplasmic reticulum (ER). Mouse embryonic fibroblasts deficient for BAX and BAK (DKO cells) were found to have a reduced resting concentration of calcium in the ER ([Ca2+]er) that results in decreased uptake of Ca2+ by mitochondria after Ca2+ release from the ER. Expression of SERCA (sarcoplasmic-endoplasmic reticulum Ca2+ adenosine triphosphatase) corrected [Ca2+]er and mitochondrial Ca2+ uptake in DKO cells, restoring apoptotic death in response to agents that release Ca2+ from intracellular stores (such as arachidonic acid, C2-ceramide, and oxidative stress). In contrast, targeting of BAX to mitochondria selectively restored apoptosis to "BH3-only" signals. A third set of stimuli, including many intrinsic signals, required both ER-released Ca2+ and the presence of mitochondrial BAX or BAK to fully restore apoptosis. Thus, BAX and BAK operate in both the ER and mitochondria as an essential gateway for selected apoptotic signals.

The regulated clearance of mitochondria is a well recognized but poorly understood aspect of cellular homeostasis, and defects in this process have been linked to aging, degenerative diseases, and cancer. Mitochondria are recycled through an autophagy-related process, and reticulocytes, which completely eliminate their mitochondria during maturation, provide a physiological model to study this phenomenon. Here, we show that mitochondrial clearance in reticulocytes requires the BCL2-related protein NIX (BNIP3L). Mitochondrial clearance does not require BAX, BAK, BCL-X L, BIM, or PUMA, indicating that NIX does not function through established proapoptotic pathways. Similarly, NIX is not required for the induction of autophagy during terminal erythroid differentiation. NIX is required for the selective elimination of mitochondria, however, because mitochondrial clearance, in the absence of NIX, is arrested at the stage of mitochondrial incorporation into autophagosomes and autophagosome maturation. These results yield insight into the mechanism of mitochondrial clearance in higher eukaryotes. Furthermore, they show a BAX-and BAK-independent role for a BCL2-related protein in development.autophagy ͉ mitochondria ͉ BCL2 family B CL2-related proteins play essential roles in the regulation of programmed cell death. Members of the BCL2 family are divided into subgroups based on the presence of one or more BCL2 homology domains (BH1-BH4) (1). BCL2-related proteins possessing a single BH3 domain (BH3-only proteins) are activated by diverse death-inducing stimuli including DNA damage, glucocorticoids, and growth factor deprivation, and their signals are integrated at the mitochondria by the multidomain proapoptotic proteins BAX and BAK (2). BH3-only proteins activate BAX and BAK either directly or indirectly, through binding to and inhibiting the function of antiapoptotic BCL2-related proteins (3-5). BAX or BAK activation in turn causes cytochrome c release, caspase activation, and apoptosis (6, 7). BNIP3 and NIX (also known as BNIP3L) are related proteins with limited homology to BH3-only proteins in a BH3-like domain (8-10). BNIP3 and NIX have uncertain biological function. BNIP3 and NIX localize to the mitochondria when overexpressed, induce cytochrome c release, and cause apoptosis (11-13), however, BNIP3 also causes necrosis-like cell death (14). Hypoxia induces and retinoblastoma protein represses BNIP3 expression through HIF-1␣ and E2F binding sites in the BNIP3 promoter, respectively (15-17). In contrast, NIX is induced by G q -coupled hypertrophic agonists in neonatal rat cardiomyocytes, by p53 in U2OS osteosarcoma cells, and by differentiation of human erythroid cells (18)(19)(20). Accordingly, NIX functions as an effector of G q -dependent cardiomyopathy and negatively regulates tumor growth in nude mice injected with U2OS osteosarcoma cells (19,21). NIX has a role in erythroid development, because Nix Ϫ/Ϫ mice exhibit anemia and erythroid hyperplasia (22). Results and DiscussionDefective Erythropoiesis in Nix ؊...