N Terminus of Calpain 1 Is a Mitochondrial Targeting Sequence

Abstract

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, which 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 the -and 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)(13)(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 ret...

An increase in intracellular Ca2+ is required for the activation of mitochondrial calpain to release AIF during cell death

“…Similarly, we have recently observed that stimulation of mitochondrial ROS production promotes Ca 2 þ -mediated AIF processing (data not shown). Hence, the results obtained in earlier studies, 23,33 along with our current observations, suggest a scenario in which the processing of AIF might be regulated by a three-step mechanism. First, a prolonged increase in Ca 2 þ level activates mitochondrial calpain in the IMS.…”

“…It has recently been shown that calpain I contains a mitochondrial localization signal and that the small subunit of calpain I can be coimported into the IMS together with the calpain I large subunit. 23 In addition, the mitochondrial localization of calpain I was also demonstrated by live imaging of cells transfected with a GFP construct containing the N terminal of calpain I. The authors suggested that the mitochondrial outer membrane represents a barrier separating calpain I from its endogenous inhibitor calpastatin.…”

“…Although both m-and m-calpains are generally considered to be cytosolic, mitochondria were also shown to contain Ca 2 þ -dependent calpain 1. 23 To investigate whether AIF can be processed by a mitochondrial protease, we used several independent approaches. First, a sucrose gradient purification of mitochondria from U1810 cells was performed to exclude the presence of non-mitochondrial proteases.…”

“…[28][29][30][31] However, recent findings indicate that calpain 1 is also present within the mitochondria. 23 It has been suggested that OMM permeabilization should precede AIF processing during apoptosis, as the overexpression of Bcl-2 and Bcl-X L prevented both AIF cleavage and release. 18 In addition, based on the results obtained using recombinant proteins and permeabilized, isolated mitochondria, it has been reported that cytosolic calpain I can cleave AIF.…”

“…The authors suggested that the mitochondrial outer membrane represents a barrier separating calpain I from its endogenous inhibitor calpastatin. 23 If so, the only trigger needed for the activation of mitochondrial calpain I would be a Ca 2 þ signal. Our findings support this hypothesis, as we could detect calpain I in purified mitochondria and the addition of Ca 2 þ led to the generation of the soluble 57 kDa AIF fragment (cf.…”