1. ¹Institute of Environmental Medicine, Division of Toxicology, Karolinska Institutet, Stockholm, Sweden
2. ²Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden

Correspondence: B Zhivotovsky, Division of Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Box 210, Stockholm SE-17177, Sweden. Tel: +46 8 524 875 88; Fax: +46 8 32 90 41; E-mail: boris.zhivotovsky@ki.se

Received 17 April 2008; Revised 1 July 2008; Accepted 21 July 2008; Published online 19 September 2008.

Abstract

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 (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.