OVEREXPRESSED MITOCHONDRIAL HINGE PROTEIN, A CYTOCHROME C-BINDING PROTEIN, ACCELERATES APOPTOSIS BY ENHANCING THE RELEASE OF CYTOCHROME-C FROM MITOCHONDRIA

Holocytochrome c released from mitochondria has been revealed to be on e of the contributors of apoptosis. To investigate how the cytochrome c protein is released from mitochondria, we examined the effects of ov erexpression of the hinge protein, a cytochrome c-binding protein, or cytochrome c on apoptosis by introducing their cDNAs under a constitut ive promoter. Overexpression of the cytochrome c and hinge protein mRN As was confirmed by Northern blotting, although marked accumulation of the cytochrome c protein was not observed. In transfectants of the hi nge protein gene as well as cytochrome c gene, apoptosis was accelerat ed as judged by FITC-conjugated Annexin V binding to the cell surface and DNA fragmentation. In addition, enhancement of the release of cyto chrome c into cytosol was demonstrated in these transfectants by a sub cellular fractionation experiment, followed by Western blotting. These findings suggest that the release of the cytochrome c protein from mi tochondria is regulated by the hinge protein involved in the respirato ry chain in the apoptotic process. (C) 1998 Academic Press.