Dominant cell death induction by extramitochondrially targeted apoptosis-inducing factor

The complete AIF cDNA comprising the amino-terminal mitochondrial localizat ion sequence (MLS) and the oxidoreductase domain has been fused in its carb oxyl terminus to enhanced green fluorescent protein (GFP), thereby engineer ing an AIF-GFP fusion protein that is selectively targeted to the mitochond rial intermembrane space. Upon induction of apoptosis, the AIF-GFP protein translocates together with cytochrome c (Cyt-c) to the extramitochondrial c ompartment. Microinjection of recombinant AIF leads to the release of AIF-G FP and Cyt-c-GFP, indicating that ectopic AIF can favor permeabilization of the outer mitochondrial membrane. These mitochondrial effects of AIF are c aspase independent, whereas the Cyt-c-microinjection induced translocation of AIF-GFP and Cyt-c-GFP is suppressed by the pan-caspase inhibitor Z-VAD.f mk. Upon prolonged culture, transfection-enforced overexpression of AIF res ults in spontaneous translocation of AIF-GFP from mitochondria, nuclear chr omatin condensation, and cell death. These effects are caspase independent and do not rely on the oxidoreductase function of AIF. Spontaneous AIF-GFP translocation and subsequent nuclear apoptosis can be retarded by overexpre ssion of a Bcl-2 protein selectively targeted to mitochondria, but not by a Bcl-2 protein targeted to the endoplasmic reticulum. Overexpression of a m utant AIF protein in which the MLS has been deleted (AIF Delta 1-100) resul ts in the primary cytosolic accumulation of AIF. AIF Delta 1-100-induced ce ll death is suppressed by neither Z-VAD.fmk or by Bcl-2. Thus, extramitocho ndrially targeted AIF is a dominant cell death inducer.