N Terminus of Calpain 1 Is a Mitochondrial Targeting Sequence
RamaKrishna Badugu, Matthew Garcia, Vimala Bondada, Aashish Joshi, James W. Geddes
2007
Journal of Biological Chemistry
The ubiquitous m-and -calpains are thought to be localized in the cytosolic compartment, as is their endogenous inhibitor calpastatin. Previously, -calpain was found to be enriched in mitochondrial fractions isolated from rat cerebral cortex and SH-SY5Y neuroblastoma cells, but the submitochondrial localization of -calpain was not determined. In the present study, submitochondrial fractionation and digitonin permeabilization studies indicated that both calpain 1 and calpain small subunit 1,
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... h together form -calpain, are present in the mitochondrial intermembrane space. The N terminus of calpain 1 contains an amphipathic ␣-helical domain, and is distinct from the N terminus of calpain 2. Calpain 1, but not calpain 2, was imported into mitochondria. Removal of the N-terminal 22 amino acids of calpain 1 blocked the mitochondrial calpain import, while addition of this N-terminal region to calpain 2 or green fluorescent protein enabled mitochondrial import. The N terminus of calpain 1 was not processed following mitochondrial import, but was removed by autolysis following calpain activation. Calpain small subunit 1 was not directly imported into mitochondria, but was imported in the presence of calpain 1. The presence of a mitochondrial targeting sequence in the N-terminal region of calpain 1 is consistent with the localization of -calpain to the mitochondrial intermembrane space and provides new insight into the possible functions of this cysteine protease. Calpains (EC 3.4.22.17) are a family of Ca 2ϩ -activated cysteine proteases, including both ubiquitous and tissue-specific isoforms, that cleave their substrate proteins at discrete sites to modulate activity (1-3). The best characterized, and the predominant calpains in the central nervous system, are the classical m-and -calpains. Their physiological roles have not been fully elucidated but include cell motility, cell differentiation, membrane fusion, platelet activation, and signal transduction (3). Also extensively investigated have been the pathological roles of calpains in cell death, where calpains can cleave key structural proteins and contribute to the release of death-related proteins such as apoptosis-inducing factor (AIF) 3 (4 -9). At present, it is unclear whether theand m-calpains have distinct or overlapping functions. They are each heterodimers consisting of a unique 80-kDa large catalytic subunit (calpain 1 or 2) and a common 28-kDa small regulatory subunit (calpain small subunit 1 or 2) (2). In vitro, the substrates of m-and -calpains are similar, if not identical (10). Knock-out of the -calpain large subunit, calpain 1, results in viable mice with reduced platelet aggregation and impaired tyrosine phosphorylation in platelets, but not overt phenotype (11). Knock-out of the m-calpain large subunit, calpain 2, or of calpain small subunit 1 (CSS1) is embryonically lethal (12-14) . Both m-and -calpains are considered to be cytosolic enzymes (2, 3, 15, 16 ). An association of m-and -calpains with subcellular organelles including endoplasmic reticulum and Golgi apparatus has been observed, but this association is hydrophobic, and the calpains are largely localized to the cytoplasmic surface of the organelle membranes (17-19). Previously, calpain-like activity was observed in both the mitochondrial matrix and intermembrane space of rat liver mitochondria (20). We recently observed an association between -calpain and mitochondria (21), but whether this represented a hydrophobic association on the external mitochondrial surface or a submitochondrial localization of -calpain was not determined. An atypical calpain, calpain 10, was recently localized to the mitochondrial matrix (22). The results of the present study demonstrate that -calpain, including both the large and small subunits, is present in the mitochondrial intermembrane space. The results further demonstrate that the -calpain large subunit, calpain 1, contains a mitochondrial targeting sequence in its N terminus, and that CSS1 is imported into mitochondria by attaching to the calpain 1 large subunit. The identification of a mitochondrial targeting sequence in calpain 1 and localization of -calpain to the mitochondrial intermembrane space provides new insight into its possible functions. EXPERIMENTAL PROCEDURES Reagents-Bicinchoninic acid protein assay kit and the Supersignal West Pico chemiluminescent substrate were
doi:10.1074/jbc.m706851200
pmid:18070881
fatcat:qw5gdm6f2bb5zgpjyvhcjsgywe