Mitochondrial m-calpain plays a role in the release of truncated apoptosis-inducing factor from the mitochondria

Calpains, calcium-dependent cysteine proteases, are involved in a variety of cellular processes. We have reported on the characteristics of mitochondrial μ-calpain and have shown that ERp57-associated mitochondrial μ-calpain cleaves the apoptosis-inducing factor (AIF) to a truncated form (tAIF). In addition, we found an unknown mitochondrial calpain. In this study, we identified and characterized this undescribed mitochondrial calpain in rat liver mitochondrial intermembrane space. The mitochondrial μ- and unknown calpains were separated by DEAE-Sepharose column chromatography. We immunoprecipitated the unknown calpain with anti-calpain small subunit and identified it as calpain 2 (m-calpain large subunit) by nanoflow-LC-MS/MS analysis and database searching. Because the identified mitochondrial calpain was stained with anti-m-calpain large subunit antibody, we named it mitochondrial m-calpain. The Ca2+ dependency of mitochondrial m-calpain was similar to that of cytosolic m-calpain. Immunoprecipitation analyses showed that mitochondrial m-calpain is associated with a 75-kDa glucose-regulated protein, a member of the heat shock protein 70 family. We also investigated the involvement of mitochondrial m-calpain in the release of tAIF from mitochondria. Calpain inhibitor, PD150606, an anti-voltage-dependent anion channel (VDAC), and anti-Bax antibodies prevented the release of tAIF from mitochondria. In addition, we found that mitochondrial m-calpain truncated VDAC in Ca2+-dependent manner. This cleavage of VDAC promotes the mitochondrial accumulation of Bax and the release of tAIF from mitochondria. The accumulated Bax in mitochondrial outer membrane was co-immunoprecipitated with VDAC. Our results demonstrated that mitochondrial m-calpain plays a role in the release of tAIF from mitochondria by cleaving VDAC, and tAIF is released through VDAC-Bax pores.