An increase in intracellular Ca2+ is required for the activation of mitochondrial calpain to release AIF during cell death

Apoptosis-inducing factor (AIF), a flavoprotein with NADH oxidase activity anchored to the mitochondrial inner membrane, is known to be involved in complex I maintenance. During apoptosis, AIF can be released from mitochondria and translocate to the nucleus, where it participates in chromatin condensation and large-scale DNA fragmentation. The mechanism of AIF release is not fully understood. Here, we show that a prolonged (similar to 10 min) increase in intracellular Ca2+ level is a prerequisite step for AIF processing and release during cell death. In contrast, a transient ATP-induced Ca2+ increase, followed by rapid normalization of the Ca2+ level, was not sufficient to trigger the proteolysis of AIF. Hence, import of extracellular Ca2+ into staurosporine-treated cells caused the activation of a calpain, located in the intermembrane space of mitochondria. The activated calpain, in turn, cleaved membrane-bound AIF, and the soluble fragment was released from the mitochondria upon outer membrane permeabilization through Bax/Bak-mediated pores or by the induction of Ca2+-dependent mitochondrial permeability transition. Inhibition of calpain, or chelation of Ca2+, but not the suppression of caspase activity, prevented processing and release of AIF. Combined, these results provide novel insights into the mechanism of AIF release during cell death.