title: Ca²+-induced release of mitochondrial m-calpain from outer membrane with binding of calpain small subunit and Grp75.
abstract: Although mitochondrial μ- and m-calpains play significant roles in apoptotic cell death, their activating mechanisms have not been determined. The purpose of this study was to determine the core factors that are involved in activating mitochondrial outer membrane (OM)-bound calpains. To accomplish this, we solubilized OM-bound calpains and separated them by DEAE-Sepharose column chromatography, and identified them by immunoblots. We also determined the core factors that activated the OM-bound calpains and release them from the OM by calpain assays, immunoprecipitations, and immunoblots. The OM-bound m-calpain large subunit was not associated with the small subunit or with Grp75 chaperone. Free calpain small subunit was located in the IMS and caused the release of the OM-bound m-calpain large subunit from the OM together with Grp75, ATP, and Ca²+. Our results showed that the activating mechanism of mitochondrial OM-bound m-calpain and the release of mitochondrial m-calpain from the OM have important implications in facilitating apoptotic cell death.
authors.affiliation: Department of Biochemistry and Biotechnology, Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki 036-8561, Japan.
authors.forename: Taku
authors.initials: T
authors.lastname: Ozaki
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Yamashita
authors.forename: Sei-ichi
authors.initials: S
authors.lastname: Ishiguro
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002154:Calpain / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism*; D051336:Mitochondrial Membranes / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D019704:Protein Disulfide-Isomerases / Q000737:chemistry / Q000378:metabolism; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D018840:HSP70 Heat-Shock Proteins; D008565:Membrane Proteins; D021122:Protein Subunits; C018551:glucose-regulated proteins; D002154:Calpain; C449000:m-calpain; C449002:mu-calpain; D019704:Protein Disulfide-Isomerases; C085868:PDIA3 protein, human; D002118:Calcium