Overexpressed Mitochondrial Hinge Protein, a Cytochromec-Binding Protein, Accelerates Apoptosis by Enhancing the Release of Cytochromecfrom Mitochondria

Abstract Holocytochrome c released from mitochondria has been revealed to be one of the contributors of apoptosis. To investigate how the cytochrome c protein is released from mitochondria, we examined the effects of overexpression of the hinge protein, a cytochrome c -binding protein, or cytochrome c on apoptosis by introducing their cDNAs under a constitutive promoter. Overexpression of the cytochrome c and hinge protein mRNAs was confirmed by Northern blotting, although marked accumulation of the cytochrome c protein was not observed. In transfectants of the hinge protein gene as well as cytochrome c gene, apoptosis was accelerated as judged by FITC-conjugated Annexin V binding to the cell surface and DNA fragmentation. In addition, enhancement of the release of cytochrome c into cytosol was demonstrated in these transfectants by a subcellular fractionation experiment, followed by Western blotting. These findings suggest that the release of the cytochrome c protein from mitochondria is regulated by the hinge protein involved in the respiratory chain in the apoptotic process.