Submitochondrial localization of associated mu-calpain and calpastatin.

Recently, we have reported the presence of calpain-calpastatin system in mitochondria of bovine pulmonary smooth muscle [P. Kar, T. Chakraborti, S. Roy, R. Choudhury, S. Chakraborti, Arch. Biochem. Biophys. 466 (2007) 290-299]. Herein, we report its localization in the mitochondria. Immunoblot, immunoelectron microscopy and casein zymographic studies suggest that mu-calpain and calpastatin are present in the inner mitochondrial membrane; but not in the outer mitochondrial membrane or in the inter membrane space or in the matrix of the mitochondria. Co-immunoprecipitation studies suggest that mu-calpain-calpastatin is associated in the inner mitochondrial membrane. Additionally, the proteinase K and sodium carbonate treatments of the mitoplasts revealed that mu-calpain is integrally and calpastatin is peripherally embedded to the outer surface of inner mitochondrial membrane. These studies indicate that an association between mu-calpain and calpastatin occurs in the inner membrane towards the inter membrane space of the mitochondria, which provides better insight about the protease regulation towards initiation of apoptotic processes mediated by mitochondria.