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J. Biol. Chem., Vol. 283, Issue 6, 3409-3417, February 8, 2008

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N Terminus of Calpain 1 Is a Mitochondrial Targeting Sequence^*

RamaKrishna Badugu¹, Matthew Garcia¹, Vimala Bondada, Aashish Joshi, and James W. Geddes²

From the Spinal Cord and Brain Injury Research Center and the Department of Anatomy and Neurobiology, University of Kentucky, Lexington, Kentucky

The ubiquitous m- and µ-calpains are thought to be localized in the cytosolic compartment, as is their endogenous inhibitor calpastatin. Previously, µ-calpain was found to be enriched in mitochondrial fractions isolated from rat cerebral cortex and SH-SY5Y neuroblastoma cells, but the submitochondrial localization of µ-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 µ-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 µ-calpain to the mitochondrial intermembrane space and provides new insight into the possible functions of this cysteine protease.

Received for publication, August 16, 2007 , and in revised form, December 7, 2007.

^* This work was supported in part by National Institutes of Health, U. S. Public Health Service Grants P01NS058484, P01AG010836, and R01NS045726, and by the Kentucky Spinal Cord and Head Injury Research Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed: B477 BBSRB, University of Kentucky, Lexington, KY 40536-0509. Tel.: 859-323-5135; Fax: 859-257-5737; E-mail: jgeddes{at}uky.edu.

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