A co-translational model to explain the in vivo import of proteins into HeLa cell mitochondria.
A co-translational model to explain the in vivo import of proteins into HeLa cell mitochondria.
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DOI：
10.1042/bj20040065
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发表时间：
2004-08
期刊：
影响因子：
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通讯作者：
A. Mukhopadhyay;L. Ni;H. Weiner
中科院分区：
文献类型：
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作者：
A. Mukhopadhyay;L. Ni;H. Weiner
在转染的HeLa细胞中采用双信号方法，即在N末端的线粒体信号和在EGFP（增强型绿色荧光蛋白）C末端的ER（内质网）或过氧化物酶体信号，以测试共翻译输入模型。将来自OTC（鸟氨酸转氨甲酰酶）或腺苷酸酶II的信号肽融合到EGFP的N-末端，并且将ER或过氧化物酶体信号融合到其C-末端。其理论基础是，如果游离前蛋白保留在胞质溶胶中，它可以通过翻译后途径分布在两个细胞器之间。所得到的融合蛋白被专门输入到线粒体中，这表明发生了共翻译输入。天然前ALDH（大鼠肝线粒体醛脱氢酶的前体），前OTC和rhodanese，每一个都添加了C-末端ER或过氧化物酶体信号，也仅易位到线粒体，再次表明这些天然蛋白存在共翻译输入途径。在氨甲喋呤（DHFR的底物类似物）存在下研究了前ALDH（sp）-DHFR（由前ALDH的前导序列（信号肽）与DHFR（二氢叶酸还原酶）融合组成的融合蛋白）的导入。发现在甲氨蝶呤存在下，70%的preALDH（sp）-DHFR被输入到线粒体中，这意味着70%的蛋白质利用共翻译输入途径，30%利用翻译后输入途径。因此，共翻译输入是线粒体蛋白输入的主要途径。提出了一个模型来解释如何结合因子之间的竞争可能会影响是否胞质载体蛋白，如DHFR，使用的共同或翻译后输入途径。
The dual signal approach, i.e. a mitochondrial signal at the N-terminus and an ER (endoplasmic reticulum) or a peroxisomal signal at the C-terminus of EGFP (enhanced green fluorescent protein), was employed in transfected HeLa cells to test for a co-translational import model. The signal peptide from OTC (ornithine transcarbamylase) or arginase II was fused to the N-terminus of EGFP, and an ER or peroxisomal signal was fused to its C-terminus. The rationale was that if the free preprotein remained in the cytosol, it could be distributed between the two organelles by using a post-translational pathway. The resulting fusion proteins were imported exclusively into mitochondria, suggesting that co-translational import occurred. Native preALDH (precursor of rat liver mitochondrial aldehyde dehydrogenase), preOTC and rhodanese, each with the addition of a C-terminal ER or peroxisomal signal, were also translocated only to the mitochondria, again showing that a co-translational import pathway exists for these native proteins. Import of preALDH(sp)-DHFR, a fusion protein consisting of the leader sequence (signal peptide) of preALDH fused to DHFR (dihydrofolate reductase), was studied in the presence of methotrexate, a substrate analogue for DHFR. It was found that 70% of the preALDH(sp)-DHFR was imported into mitochondria in the presence of methotrexate, implying that 70% of the protein utilized the co-translational import pathway and 30% used the post-translational import pathway. Thus it appears that co-translational import is a major pathway for mitochondrial protein import. A model is proposed to explain how competition between binding factors could influence whether or not a cytosolic carrier protein, such as DHFR, uses the co- or post-translational import pathway.