Signal Transduction
Deregulation of Mitochondrial Membrane Potential by Mitochondrial Insertion of Granzyme B and Direct Hax-1 Cleavage*

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The cytoplasm and the nucleus have been identified as activity sites for granzyme B (GrB) following its delivery from cytotoxic lymphocyte granules into target cells. Here we report on the ability of exogenous GrB to insert into and function within a proteinase K-resistant mitochondrial compartment. We identified Hax-1 (HS-1-associated protein X-1), a mitochondrial protein involved in the maintenance of mitochondrial membrane potential, as a GrB substrate within the mitochondrion. GrB cleaves Hax-1 into two major fragments: an N-terminal fragment that localizes to mitochondria and a C-terminal fragment that localizes to the cytosol after being released from GrB-treated mitochondria. The N-terminal Hax-1 fragment major cellular impact is on the regulation of mitochondrial polarization. Overexpression of wild-type Hax-1 or its uncleavable mutant form protects the mitochondria against GrB or valinomycin-mediated depolarization. The N-terminal Hax-1 fragment functions as a dominant negative form of Hax-1, mediating mitochondrial depolarization in a cyclophilin D-dependent manner. Thus, induced expression of the N-terminal Hax-1 fragment results in mitochondrial depolarization and subsequent lysosomal degradation of such altered mitochondria. This study is the first to demonstrate GrB activity within the mitochondrion and to identify Hax-1 cleavage as a novel mechanism for GrB-mediated mitochondrial depolarization.

Cell Death
Cytochrome c
Death Protease
Immunology
Mitochondria
Mitochondrial Apoptosis

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*

This work was supported, in whole or in part, by National Institutes of Health Grants RO1 CA109285 and RO1 CA111786 (to H. R.).

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S6.

1

Both authors contributed equally to this work.