Cloning and characterization of Xenopus laevis Smac/DIABLO

Mitochondria-mediated apoptosis plays a central role in animal development and tissue homeostasis, and mitochondria contain several pro-apoptotic proteins that have key roles in apoptosis. Smac/DIABLO was identified as a mitochondrial protein that is released into the cytosol following apoptotic stimuli, subsequently blocking the anti-apoptotic activity of inhibitor of apoptosis proteins. Through expressed sequence tag (EST) analysis we detected evidence for the presence of a number of Xenopus counterparts to mammalian mitochondrial pro-apoptotic proteins. EST and genome sequencing provides evidence for the presence of endonuclease G, AIF, HtrA/Omi and Smac/DIABLO in Xenopus laevis and tropicalis. Here we report the cloning and characterization of X. laevis Smac/DIABLO (XSmac/DIABLO). In this study degenerate primers based on conserved regions of human, mouse and an EST predicted Smac from X. tropicalis were used to amplify cDNA templates from X. laevis. The full length cDNA of Xenopus Smac contained a complete open reading frame of 732 bp, encoding 244 amino acids, that when expressed is observed to be approximately 27 kDa in size. The protein sequence is 49% identical and 71% similar to human Smac, and includes the motifs involved in mitochondrial targeting, and IAP-binding (AIPV). Smac expression was detected throughout early development with multiple transcripts being detected by Northern blot analysis, suggesting the presence of alternatively spliced isoforms. Exogenous expression of Xenopus Smac enhances γ-irradiation-induced apoptosis in HeLa cells, demonstrating its functional equivalence with mammalian forms. Our study has identified the third vertebrate homologue of Smac/DIABLO, with its structural and functional similarities to mammalian Smac/DIABLO further illustrating the evolutionary conservation of apoptotic pathways across vertebrate species.