Dominant cell death induction by extramitochondrially targeted apoptosis-inducing factor.
The complete AIF cDNA comprising the amino-terminal mitochondrial localization sequence (MLS) and the oxidoreductase domain has been fused in its carboxyl terminus to enhanced green fluorescent protein (GFP), thereby engineering an AIF-GFP fusion protein that is selectively targeted to the mitochondrial intermembrane space. Upon induction of apoptosis, the AIF-GFP protein translocates together with cytochrome c (Cyt-c) to the extramitochondrial compartment. Microinjection of recombinant AIF leads to the release of AIF-GFP and Cyt-c-GFP, indicating that ectopic AIF can favor permeabilization of the outer mitochondrial membrane. These mitochondrial effects of AIF are caspase independent, whereas the Cyt-c-microinjection induced translocation of AIF-GFP and Cyt-c-GFP is suppressed by the pan-caspase inhibitor Z-VAD.fmk. Upon prolonged culture, transfection-enforced overexpression of AIF results in spontaneous translocation of AIF-GFP from mitochondria, nuclear chromatin 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 overexpression of a Bcl-2 protein selectively targeted to mitochondria, but not by a Bcl-2 protein targeted to the endoplasmic reticulum. Overexpression of a mutant AIF protein in which the MLS has been deleted (AIF Delta 1-100) results in the primary cytosolic accumulation of AIF. AIF Delta 1-100-induced cell death is suppressed by neither Z-VAD.fmk or by Bcl-2. Thus, extramitochondrially targeted AIF is a dominant cell death inducer.
Centre National de la Recherche Scientifique, UMR1599, Institut Gustave Roussy, F-94805 Villejuif, France.
M
M
Loeffler
E
E
Daugas
S A
SA
Susin
N
N
Zamzami
D
D
Metivier
A L
AL
Nieminen
G
G
Brothers
J M
JM
Penninger
G
G
Kroemer
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D051033:Apoptosis Inducing Factor; D019556:COS Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D003574:Cytochrome c Group / Q000235:genetics / Q000378:metabolism; D005420:Flavoproteins / Q000235:genetics / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D017930:Genes, Reporter / Q000235:genetics; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008845:Microinjections; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals / Q000235:genetics*; D021381:Protein Transport / Q000502:physiology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013997:Time Factors; D014162:Transfection
C494829:AIFM1 protein, human; C494831:Aifm1 protein, rat; D051033:Apoptosis Inducing Factor; D003574:Cytochrome c Group; D005420:Flavoproteins; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins