N terminus of calpain 1 is a mitochondrial targeting sequence.
The ubiquitous m- and mu-calpains are thought to be localized in the cytosolic compartment, as is their endogenous inhibitor calpastatin. Previously, mu-calpain was found to be enriched in mitochondrial fractions isolated from rat cerebral cortex and SH-SY5Y neuroblastoma cells, but the submitochondrial localization of mu-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 mu-calpain, are present in the mitochondrial intermembrane space. The N terminus of calpain 1 contains an amphipathic alpha-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 mu-calpain to the mitochondrial intermembrane space and provides new insight into the possible functions of this cysteine protease.
Badugu
RamaKrishna
R
Spinal Cord and Brain Injury Research Center, University of Kentucky, Lexington, Kentucky.
Garcia
Matthew
M
Spinal Cord and Brain Injury Research Center, University of Kentucky, Lexington, Kentucky; Department of Anatomy and Neurobiology, University of Kentucky, Lexington, Kentucky.
Bondada
Vimala
V
Spinal Cord and Brain Injury Research Center, University of Kentucky, Lexington, Kentucky.
Joshi
Aashish
A
Spinal Cord and Brain Injury Research Center, University of Kentucky, Lexington, Kentucky; Department of Anatomy and Neurobiology, University of Kentucky, Lexington, Kentucky.
Geddes
James W
JW
Spinal Cord and Brain Injury Research Center, University of Kentucky, Lexington, Kentucky; Department of Anatomy and Neurobiology, University of Kentucky, Lexington, Kentucky. Electronic address: jgeddes@uky.edu.
D000595:Amino Acid Sequence; D000818:Animals; D002154:Calpain / Q000737:chemistry*; D045744:Cell Line, Tumor; D002540:Cerebral Cortex / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000378:metabolism
D002154:Calpain; C484806:CAPN1 protein, human