3 and ?and4)4) is most likely produced by cytosolic homodimeric Smac60, not a heterodimer of Smac60 with endogenous wild type Smac, because we were unable to detect dimerization of cytosolic ectopic Smac56 with mitochondrial Smac56 (Fig. Smac is a recognition point for a posttranslational modification(s) that blocks homodimerization and IAP interaction, BAY 80-6946 (Copanlisib) and that amino acids 62C105 are required for the proapoptotic function of Smac. Introduction Human Smac/DIABLO is a cytoplasmically translated protein composed of 239 amino acids, the first 55 of which are required for mitochondrial import [1], [2]. The Smac gene consists of seven exons that can produce four isoforms: wild type Smac, Smac, Smac, and Smac [3], [4]. Wild type Smac lacks exon 2. Smac lacks exons 1 and 3, and translation of initiates at an alternative start codon within exon 2, rendering it incompetent for mitochondrial translocation. Smac lacks exons 2 and 3, while Smac lacks exons 2 and 4. Upon translocation to the mitochondrial intermembrane space, an inner membrane peptidase complex removes the first 55 amino acids to produce mature Smac56 (Smac56-239) [5]. The first four amino acids of mature Smac (A56VPI59) are an IBM, which complexes with the BIR3 (baculovirus IAP repeat) domain of X-linked IAP (XIAP) [6], [7]. Homologous IBM sequences occur in mitochondrial proteins BAY 80-6946 (Copanlisib) Grim, Reaper, and Hid [7], the mitochondrial serine protease Omi/HtrA2 [8], and the p12 subunit of caspase-9 [9]. Structural studies of Smac complexed with the third BIR domain of XIAP suggested that the IBM may be essential for the interaction with IAPs [6], [10]. The Smac monomer is a double hairpin bundle of three -helices [6]. Purified recombinant mature Smac forms an extraordinarily stable homodimer (half-life 20,000 years) [11]. The predominantly hydrophobic dimer interface forms an antiparallel four-helix bundle which has an arch shape [6]. The IBM of each Smac protomer can simultaneously interact with the second and third BIR domains of a single XIAP molecule [12]. Specific amino acid substitutions within the hydrophobic interface, such as F88D (also called F33D by subtraction of the first 55 residues), prevent Smac homodimerization [6]. The aforementioned Grim and Reaper have a GH3-like amphipathic helix, which is crucial to a proapoptotic function that is independent of IAP antagonism [13], [14]. Smac (also called Smac-S) and a truncated Smac76-239 mutant, both of which lack the IBM and localize to the cytosol, potentiated apoptosis evoked by chemotherapeutic agents [3], [15]. Importantly Smac, but not the truncated Smac76-239 mutant, complexed with XIAP, cIAP1, and cIAP2. While the IBM of Smac is not essential for the interaction with the IAPs, the segment close to the amino-terminus of mature Smac is necessary for IAP interaction [3]. There are eight human IAP family members, each of which has at least one BAY 80-6946 (Copanlisib) BIR domain. The BIR domain, which is the defining feature of IAPs, is responsible for binding caspases. BIR2 and BIR3 of XIAP directly bind and inhibit processed capase-3 and processed caspase-9, respectively [reviewed in [16], [17]]. While XIAP binds and inhibits caspases, other IAPs seem not to directly inhibit the catalytic activity of caspases [17]. However, cIAP1 can potently prevent caspase-9 activation of procaspase-3 via interaction with the IBM of the p12 subunit of processed caspase-9 [18]. In addition to three BIR domains, XIAP has a RING domain with ubiquitin (Ub) ligase activity. Livin (also called ML-IAP) has a single BIR of the BIR3 type and a RING domain [19]. Survivin, the smallest member of the IAP family, has a lone BIR domain which may not bind IBMs [19]. cIAPs 1 and 2, have a IFI16 CARD domain, which mediates protein interactions, three BIRs, and a RING domain. Apollon/BRUCE, the largest of the IAPs, has a single N-terminal BIR domain and a UBC (Ub conjugation domain) at the C-terminus [20]. The two remaining IAPs, testis specific IAP (Ts-IAP) and neuronal apoptosis inhibitory protein (NAIP), were not included in the present study. Transcripts of all the IAPs, except Ts-IAP and NAIP, were detected by RT-PCR in the cell model used here, namely the 911 line of human.