Kim Jong-Tae, Kim Kwang Dong, Song Eun Young, Lee Hee Gu, Kim Jae Wha, Kim Jung Woo, Chae Suhn-Kee, Kim Eunhee, Lee Myeong-Sok, Yang Young, Lim Jong-Seok
Department of Biological Sciences, Research Center for Women's Diseases, Sookmyung Women's University, Chungpa-Dong, Yongsan-Gu, Seoul 140-742, Republic of Korea.
FEBS Lett. 2006 Nov 27;580(27):6375-83. doi: 10.1016/j.febslet.2006.10.049. Epub 2006 Nov 3.
Apoptosis-inducing factor (AIF) is a ubiquitous FAD-binding flavoprotein comprised of 613 amino acids and plays an important role in caspase-independent apoptosis. During apoptotic induction, AIF is translocated from the mitochondrial intermembrane space to the nucleus, where it interacts with DNA and activates a nuclear endonuclease. By performing a yeast two-hybrid screen with mature AIF, we have isolated the eukaryotic translation initiation factor 3 subunit p44 (eIF3g). Our deletion mutant analysis revealed that the eIF3g N-terminus interacts with the C-terminal region of AIF. The direct interaction between AIF and eIF3g was confirmed in a GST pull-down assay and also verified by the results of co-immunoprecipitation and confocal microscopy studies. Using an in vitro TNT coupled transcription-translation system, we found that mature AIF could inhibit newly-translated protein synthesis and this inhibition was significantly blocked by eIF3g competitively. These results were also confirmed in cells. In addition, mature AIF overexpression specifically resulted in the activation of caspase-7, thereby amplifying the inhibition of protein synthesis including eIF3g cleavage. Our data suggest that eIF3g is one of the cytosolic targets that interacts with mature AIF, and provide insight into the AIF's cellular functions of the inhibition of protein synthesis during apoptosis.
凋亡诱导因子（AIF）是一种普遍存在的结合黄素腺嘌呤二核苷酸（FAD）的黄素蛋白，由613个氨基酸组成，在不依赖半胱天冬酶的凋亡过程中发挥重要作用。在凋亡诱导过程中，AIF从线粒体膜间隙转移至细胞核，在细胞核中它与DNA相互作用并激活一种核酸内切酶。通过用成熟的AIF进行酵母双杂交筛选，我们分离出了真核生物翻译起始因子3亚基p44（eIF3g）。我们的缺失突变分析表明，eIF3g的N端与AIF的C端区域相互作用。AIF与eIF3g之间的直接相互作用在谷胱甘肽S-转移酶（GST）下拉实验中得到证实，并且共免疫沉淀和共聚焦显微镜研究的结果也验证了这一点。使用体外TNT偶联转录-翻译系统，我们发现成熟的AIF可以抑制新翻译的蛋白质合成，并且这种抑制作用被eIF3g竞争性地显著阻断。这些结果在细胞中也得到了证实。此外，成熟AIF的过表达特异性地导致半胱天冬酶-7的激活，从而放大了对包括eIF3g裂解在内的蛋白质合成的抑制作用。我们的数据表明，eIF3g是与成熟AIF相互作用的胞质靶标之一，并为AIF在凋亡过程中抑制蛋白质合成的细胞功能提供了深入了解。
