Apaf1/CED4 family members play central roles in apoptosis regulation as activators of caspase family cell death proteases. These proteins contain a nucleotidebinding (NB) self-oligomerization domain and a caspase recruitment domain (CARD). A novel human protein was identified, NAC, that contains an NB domain and CARD. The CARD of NAC interacts selectively with the CARD domain of Apaf1, a caspase-activating protein that couples mitochondria-released cytochrome c (cyt-c) to activation of cytosolic caspases. Cyt-c-mediated activation of caspases in cytosolic extracts and in cells is enhanced by overexpressing NAC and inhibited by reducing NAC using antisense/DNAzymes. Furthermore, association of NAC with Apaf1 is cyt c-inducible, resulting in a mega-complex (>1 MDa) containing both NAC and Apaf1 and correlating with enhanced recruitment and proteolytic processing of pro-caspase-9. NAC also collaborates with Apaf1 in inducing caspase activation and apoptosis in intact cells, whereas fragments of NAC representing only the CARD or NB domain suppress Apaf1-dependent apoptosis induction. NAC expression in vivo is associated with terminal differentiation of short lived cells in epithelia and some other tissues. The ability of NAC to enhance Apaf1-apoptosome function reveals a novel paradigm for apoptosis regulation.CED4 family proteins constitute a unique family of caspaseactivating molecules. The founding member of this family, CED4, was discovered in the nematode Caenorhabditis elegans in screens for genes that are essential for developmental programmed cell death (1). CED4 contains an N-terminal CARD 1 followed by an NB domain, the later containing classical Walker A and B box motifs recognized as important in binding nucleotide triphosphates. CED4 functions as an activator of the caspase, CED3, in vitro and in vivo (2, 3). The NB domain of CED4 oligomerizes in an ATP-dependent manner (4, 5), whereas the CARD binds a complementary N-terminal CARD found in the zymogen proform of CED3 (6). Protease activation is thought to result from the induced proximity of CED3 zymogens bound to oligomerized CED4, where the weak intrinsic protease activity of the proenzymes is sufficient for trans-proteolysis of closely juxtaposed pro-caspases (4, 7). Proteolytic cleavage of pro-CED3 then produces the large and small subunits of the heterotetrameric, autonomously active enzyme.The closest homologue of CED4 identified in humans and other mammals thus far is Apaf1 (apoptosis protease-activating factor-1) (8). Similar to CED4, the Apaf1 protein contains a CARD, followed by an NB domain that shares significant amino acid sequence identity with the NB domains of CED4 and a family of ATPases associated with pathogen resistance (R genes) in plants (3, 5, 9), thus constituting the NB-ARC (Apaf-1/R gene/CED4) domain family (also known as NACHT domain). Unlike CED4, however, the NB-ARC domain of Apaf1 is followed by multiple WD repeats. These WD domains participate in auto-repression of Apaf1, locking it into an inactive, unoligomerized state ...