ERp57-associated mitochondrial μ-calpain truncates apoptosis-inducing factor

Abstract

Calpains, calcium-dependent neutral cystein proteases, are involved in a variety of cellular processes. We have previously shown the characteristics of mitochondrial μ-calpain even though calpastatin, a specific endogenous inhibitor of cytosolic calpains, was not present in the mitochondria. This suggested that the regulatory system of mitochondrial calpains differs from that of cytosolic calpains, and endogenous regulatory molecule(s) must exist in the mitochondria. In this study, we have identified ERp57 in partially purified mitochondrial μ-calpain using peptide mass fingerprinting based on MALDI-TOFMS. ERp57 is a member of the protein-disulfide isomerase (PDI) family and functions as a molecular chaperone within the ER. We showed that ERp57 was present in the mitochondria and was associated with mitochondrial μ-calpain. PDI inhibitors, such as DTNB and PAO, caused a degradation of the mitochondrial μ-calpain large subunit. The release of apoptosis-inducing factor (AIF) from the mitochondrial inner membrane was inhibited by treatment of the isolated mitochondria with DTNB and immunoprecipitation of ERp57-associated mitochondrial μ-calpain. Mitochondrial μ-calpain band in casein zymography disappeared by treatment with anti-ERp57 antibody. Our results demonstrate that ERp57 forms complexes with mitochondrial μ-calpain, and ERp57-associated mitochondrial μ-calpain cleaves AIF to a truncated form.