A high-throughput screening for mammalian cell death effectors identifies the mitochondrial phosphate carrier as a regulator of cytochrome c release
A high-throughput screening for mammalian cell death effectors identifies the mitochondrial phosphate carrier as a regulator of cytochrome c release
复制标题
DOI：
10.1038/sj.onc.1210600
复制
发表时间：
2008-01-03
期刊：
影响因子：
8
通讯作者：
Pimental-Muinos, Fx
中科院分区：
文献类型：
--
作者：
Alcala, S.;Klee, M.;Pimental-Muinos, Fx
复杂基因组的功能注释需要开发容量更大的新型实验平台。在这里，我们描述了一个高通量系统，旨在识别其过度表达诱导形态上不同的细胞死亡模式的 cDNA。该方法结合了两个自动化步骤，并依靠文库克隆与 GFP 的共表达来揭示垂死细胞呈现的形态特征。通过使用该系统，我们筛选了135 000个cDNA克隆并获得了90个独立分子。有趣的是，确定了三种死亡类别，即：凋亡、空泡化和自噬。在促凋亡克隆中，我们发现了线粒体载体家族的四个成员：磷酸盐和腺嘌呤核苷酸（3 型）转运蛋白，以及线粒体载体同源物 (MTCH) 1 和 2。这些分子的表达诱导细胞色素 c 释放和 caspase-9 依赖性死亡。其中一种磷酸盐载体能够与通透性转换孔复合物 ANT1 和 VDAC1 的成员相互作用，并且在凋亡激活剂存在的情况下其与 ANT1 的结合得以稳定。 siRNA 消耗该载体可延迟细胞色素 c 动员和细胞凋亡。这些结果将先前未描述的细胞凋亡功能归因于磷酸盐载体，并且更普遍地表明，在进化过程中利用了各种线粒体转运蛋白的共同特性来调节细胞凋亡。
Functional annotation of complex genomes requires the development of novel experimental platforms with increased capacity. Here, we describe a high-throughput system designed to identify cDNAs whose overexpression induces morphologically distinct cell death modalities. The methodology incorporates two robotized steps, and relies on coexpression of library clones with GFP to reveal the morphological features presented by the dying cells. By using this system we screened 135 000 cDNA clones and obtained 90 independent molecules. Interestingly, three death categories were identified, namely; apoptotic, vacuolated and autophagic. Among the pro-apoptotic clones, we found four members of the mitochondrial carrier family: the phosphate and adenine nucleotide (type 3) transporters, and the mitochondrial carrier homologs (MTCHs) 1 and 2. Expression of these molecules induced cytochrome c release and caspase-9-dependent death. One of them, the phosphate carrier, was able to interact with members of the permeability transition pore complex ANT1 and VDAC1, and its binding to ANT1 was stabilized in the presence of apoptotic activators. Depletion of this carrier by siRNA delayed cytochrome c mobilization and apoptosis. These results attribute a previously undescribed apoptotic function to the phosphate carrier and, more generally, suggest that a common property of various mitochondrial transporters was exploited during evolution to regulate apoptosis.