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 ( approximately 10 min) increase in intracellular Ca(2+) level is a prerequisite step for AIF processing and release during cell death. In contrast, a transient ATP-induced Ca(2+) increase, followed by rapid normalization of the Ca(2+) level, was not sufficient to trigger the proteolysis of AIF. Hence, import of extracellular Ca(2+) 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 Ca(2+)-dependent mitochondrial permeability transition. Inhibition of calpain, or chelation of Ca(2+), 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.
Norberg
E
E
Institute of Environmental Medicine, Division of Toxicology, Karolinska Institutet, Stockholm, Sweden.
Gogvadze
V
V
Ott
M
M
Horn
M
M
Uhlén
P
P
Orrenius
S
S
Zhivotovsky
B
B
D000179:Acrylates / Q000494:pharmacology; D000818:Animals; D051033:Apoptosis Inducing Factor / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D045304:Cytochromes c / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D008954:Models, Biological; D019311:Staurosporine / Q000494:pharmacology
D000179:Acrylates; D051033:Apoptosis Inducing Factor; C100442:PD 150606; D045304:Cytochromes c; D002154:Calpain; D019311:Staurosporine; D002118:Calcium