Dimerization of Smac is crucial for its mitochondrial retention by XIAP subsequent to mitochondrial outer membrane permeabilization

Following the apoptotic permeabilization of the outer mitochondrial membrane, the inter-membrane space protein second mitochondria-derived activator of caspases (Smac) is released into the cytosol. Smac efficiently promotes apoptosis by antagonizing x-linked inhibitor of apoptosis protein (XIAP), an inhibitor of caspases-9, -3, and -7, via a short NH₂-terminal inhibitor of apoptosis protein (IAP) binding motif (AVPI). Native Smac dimerizes to form a highly stable and inflexible elongated arch, however, a functional role for this outstretched structure so far remained unknown. Using time-lapse single-cell imaging of DLD-1 and HCT-116 colon cancer cells, we here demonstrate that upon mitochondrial outer membrane permeabilization physiological expression levels of XIAP are sufficient to selectively prolong the release of dimeric but not monomeric Smac. Elevating the expression of XIAP further extended the release duration of dimeric Smac and resulted in the mitochondrial retention of a significant proportion of the Smac pool. In contrast, monomeric Smac was always fully released and the release kinetics were not affected by altered XIAP expression. Our findings therefore indicate that the dimerization of Smac is critical for the XIAP-mediated retention of Smac at or inside the mitochondria.