Loeffler M, Daugas E, Susin S A, Zamzami N, Metivier D, Nieminen A L, Brothers G, Penninger J M, Kroemer G
Centre National de la Recherche Scientifique, UMR1599, Institut Gustave Roussy, F-94805 Villejuif, France.
FASEB J. 2001 Mar;15(3):758-67. doi: 10.1096/fj.00-0388com.
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.
包含氨基末端线粒体定位序列（MLS）和氧化还原酶结构域的完整AIF cDNA在其羧基末端与增强型绿色荧光蛋白（GFP）融合，从而构建了一种选择性靶向线粒体膜间隙的AIF-GFP融合蛋白。在诱导凋亡时，AIF-GFP蛋白与细胞色素c（Cyt-c）一起转运到线粒体外区室。显微注射重组AIF导致AIF-GFP和Cyt-c-GFP的释放，表明异位AIF可促进线粒体外膜的通透性。AIF的这些线粒体效应不依赖于半胱天冬酶，而Cyt-c显微注射诱导的AIF-GFP和Cyt-c-GFP的转运被泛半胱天冬酶抑制剂Z-VAD.fmk抑制。长时间培养后，转染强制过表达AIF导致AIF-GFP从线粒体自发转运、核染色质浓缩和细胞死亡。这些效应不依赖于半胱天冬酶，也不依赖于AIF的氧化还原酶功能。选择性靶向线粒体的Bcl-2蛋白过表达可延缓AIF-GFP的自发转运和随后的核凋亡，但靶向内质网的Bcl-2蛋白则不能。缺失MLS的突变AIF蛋白（AIF Delta 1-100）过表达导致AIF主要在细胞质中积累。Z-VAD.fmk或Bcl-2均不能抑制AIF Delta 1-100诱导的细胞死亡。因此，线粒体外靶向的AIF是一种主要的细胞死亡诱导剂。
